Quaking RNA binding protein (QKI) is essential for oligodendrocyte development as myelination requires myelin basic protein mRNA regulation and localization by the cytoplasmic isoforms . QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we define the targets of QKI in the mouse brain via CLIPseq and we show that QKI-6 binds 3′UTRs of a subset of astrocytic mRNAs. Binding is also enriched near stop codons, mediated partially by QKI-binding motifs (QBMs), yet spreads to adjacent sequences. Using a viral approach for mosaic, astrocyte-specific gene mutation with simultaneous translating RNA sequencing (CRISPR-TRAPseq), we profile ribosome associated mRNA from QKI-null astrocytes in the mouse brain. This demonstrates a role for QKI in stabilizing CLIP-defined direct targets in astrocytes in vivo and further shows that QKI mutation disrupts the transcriptional changes for a discrete subset of genes associated with astrocyte maturation. Quaking RNA binding protein (QKI) is known for its role in oligodendrocyte maturation. Here, the authors define the QKI targets in the mouse brain and show that loss of QKI disrupts the expression of cell maturation-associated genes in astrocytes in vivo. These proteins may directly transport RNAs to distal processes2, alter transcript stability, or control translation4 to prevent ectopic expression during mRNA transport or to activate translation in response to a molecular cue, for example. While these processes have been widely studied in other cell-types, it has recently become apparent that astrocytes also must have substantial cytoplasmic post-transcriptional regulation: for example, a set of mRNAs enriched on ribosomes in peripheral astrocyte processes (PAPs) have been defined, consistent with sequence-regulated local translation5. Likewise, there are clear examples of post-transcriptional regulation, via expression of specific cytoplasmic RBPs, being an essential aspect of differentiation and maturation of neurons6, microglia7, and oligodendrocytes8. However, aside from elegant work on the Fragile-X Mental Retardation Protein (FMRP)10, post-transcriptional regulators of such processes in astrocytes largely remain unknown.Post-transcriptional regulation of mRNA by RNA binding proteins (RBPs) is pervasive in the Central Nervous System (CNS). RBPs serve as the C. elegans GLD-1 and D. melanogaster HOW, and part of the signal transduction and activation of RNA (STAR) family of post-transcriptional RBPs. Members of this evolutionarily conserved family of proteins are characterized by conserved domains which are necessary for homodimerization and RNA binding11. STAR family proteins are important early in development as their expression regulates smooth muscle development, cell-fate, and germ-line development13. However, QKI expression in mice remains high in the brain throughout the lifespan15, suggesting ongoing regulatory roles in the CNS. QKI is predominantly expressed in glia in the brain16 and its function in oligodendrocytes has been well-studied: QKI plays a role in oligodendrocyte differentiation and development by regulating the stability of mRNAs that inhibit cell cycle progression and promote differentiation19. Further, cytoplasmic isoforms of QKI control the nuclear export, stability, and localization of myelin basic protein (MBP) mRNA to oligodendrocyte processes20.Quaking RNA binding protein (QKI) is the mammalian homolog of 22. In an astroglioma cell line, it has been shown that one QKI isoform stabilizes transcripts, particularly those induced by interferons23. Moreover, examination of PAP-localized transcripts from maturing astrocytes revealed a preponderance of QKI-binding motifs (QBMs) in their UTRs5. However, we still do not understand to which cytoplasmic targets QKI binds in vivo nor what the downstream effects on these targets are in the maturing postnatal brain. Furthermore, the study of this gene is complicated as full knockouts are embryonic lethal12, making it challenging to disentangle direct cellular function of QKI from indirect effects of disrupted development.Although most of our knowledge of QKI’s function in vivo comes from studies focused on oligodendrocytes, this protein is also expressed in astrocytes in both mice and humansHere, we focus on the cytoplasmic isoform QKI-6 to comprehensively define its targets in the maturing brain. We test the hypothesis that QKI-6 disproportionately binds PAP-localized transcripts and define the consequences of QKI mutation on mRNAs in astrocytes in vivo. We find, by Cross-Linking and Immunoprecipitation (CLIP)-seq, many QKI-6 targets are indeed astrocytic and overlap with a subset of the transcripts enriched on PAP ribosomes. Further, we apply an approach using CRISPR technology coupled to cell-type-specific profiling to define roles for this gene on mRNAs in astrocytes in vivo. Finally, we validate a role for QKI in transcript stability for the CLIP-defined targets.24, and 6–10 mRNAs have been shown to be altered in cultured astrocytes and related tumors after siRNA knockdown26, a compendium of all in vivo postnatal QKI targets has yet to be compiled. QKI has three major isoforms in humans and in mice, designated by their unique C-termini27, all of which are present in astrocytes16 as shown by colocalization with RPL10a-eGFP in Aldh1L1 transgenic bacTRAP mice  or IgG IP (background control) were performed on CL lysates. QKI-6 IP clearly yielded more end-labeled RNA than controls, and a shift in QKI-6/mRNA complexes was detected with decreasing RNAse concentrations, as expected . We processed the 6789 most abundant peaks to identify high-confidence QKI targets: we utilized differential expression pipelines to identify the subset of peaks that had significantly more counts in the QKI-6 CLIP replicates  than both IgG and Input samples. This intersectional analysis conservatively identified 437 peaks from 120 unique transcripts that were used in downstream analyses , which contains predicted QKI response elements (QREs): a full QBM (ACUAAY) that is within 20 bases of a second QBM half site (YAAY). Further, unbiased motif discovery30 in all peaks identified a motif that matched QKI’s QBM31  protein-RNA complexes Fig. . We chosMbp Fig. , as wellM31 Fig. . We furtM31 Fig. . Thus, w32. Despite this previous study being conducted in cultured myoblasts and using a pan-QKI antibody that will bind all isoforms (not just QKI-6), we found that 13%  of our QKI-6 peaks overlapped with the pan-QKI peaks. Thus, in different cell types which have different mRNAs transcribed, QKI will bind distinct but overlapping target sets.Lastly, we found that our data had significant overlap with the only publically available QKI CLIPseq study33. We found that the majority (72%) of QKI-6 peaks are in the 3′UTR, consistent with a potential role in post-splicing regulation, and only a small fraction (8%) were in introns  QKI-6 peaks in the 5′UTR of mouse brain transcripts 36, we mapped the distribution of QKI-6 targets to cells in the brain  p < 2.2E−16). Together, these data define QKI-6 targets, in vivo, and indicate a significant proportion of these targets are found in 3′UTRs of astrocyte-expressed transcripts. Furthermore, the significant overlap with PAP-enriched transcripts suggests a role for QKI in regulation of a subset of these transcripts in astrocytes.Within astrocytes, we have previously defined a set of transcripts enriched on ribosomes in the peripheral astrocyte process (PAP) and thus represent transcripts likely undergoing translational regulation either to control translation efficiency or localization12, and the most commonly used mouse model  is an enhancer deletion which impacts only oligodendrocytes16, we developed a strategy, CRISPR-TRAPseq, to delete QKI in astrocytes and assess changes in ribosomal association in these cells. We designed the deletion to be mosaic – impacting a subset of GFAP+ cells in otherwise normally developing brains - to mitigate most non-cell-autonomous effects from mutation. Specifically, we generated knock-in mice containing a loxP-stop-loxP (LSL) Cas9 allele in the Rosa locus that can be expressed under a Cre-inducible promoter , especially those found in astrocytes (OR = 8.8 FET p < .0016). Both up and down regulated transcripts had significant overlap with CLIP targets and those previously described as being translated in PAPs  , had a similar effect . In contrast, rare targets with QKI-6 binding in introns did not show a consistent direction of effect. It is worth noting the resultant impact on the final protein and mRNA levels following QKI deletion could be mixed as QKI may have distinct roles across its varied targets, or that deletion of all nuclear and cytoplasmic QKI isoforms across postnatal development results in a complicated mix of splicing, translational, and/other indirect effects within the cells. For example, Pbk and Ube2c genes do not appear to be QKI-6 CLIP targets in normal P21 brain, but are upregulated on QKI loss  Fig. , validatoss Fig. .As our gRNA targets all isoforms, QKI deletion could also feasibly impact splicing. In a transcript isoform level analysis, we detected 949 transcripts with differential expression arising from 833 genes, including all of those found with the gene level of analysis , and a variety of mitochondrial proteins involved in electron transport chain   vs. mature (P32) astrocytes15 and compared them to transcripts altered by QKI mutation. We found that indeed deleting QKI resulted in prolonged P7 gene expression and decreased P32 gene expression  as a proxy for morphological maturation. Interestingly, we found no significant difference in astrocytic NIV between QKI+ and QKI− astrocytes  Fig. . The relets Fig. . We saw ion Fig. , consist32, that QKI-6 target peaks generally reside in 3′UTRs, supporting a role of QKI in translational control44 and mRNA stability45. This finding is in contrast to previous pan-QKI CLIPseq studies in which introns represented a large percentage of signal32, thus highlighting a significant difference across QKI isoform binding. Consistent with binding having important biological roles, bound regions were more conserved and contained enrichment of QBMs. Second, peaks were enriched near stop codons and PolyA signals, and while enriched in QBMs, many sites did not contain them. Further binding was elevated even hundreds of nucleotides from the QBM/peak of highest binding. As QKI is known to homodimerize35, this spreading binding could be consistent with a cooperativity model where a high-affinity site recruits QKI, which then facilitates additional binding at lower-affinity sites nearby, either through the additional domains in the same molecule, or across dimerized QKI complexes. Of particular interest is the pattern of dual peaks flanking stop codons , neurotransmitter uptake , and astrocytic cytoskeleton (Gfap). Interestingly, Gfap is a known QKI target in human astrocytes25, and other cytoskeletal genes are QKI-regulated in oligodendrocytes18, leading to a hypothesis that QKI controls astrocyte development and/or astrogliosis in injury and disease.Motivated by curiosity about translational regulation in astrocytes, we next focused on the significant subset of CLIP targets known to be expressed in them. Our data showed an enrichment of QKI-6 targets in PAP-enriched transcripts47. While the Cre-Lox system is sometimes used to circumvent these issues, during the course of these studies floxed QKI mice were not available. Even then early postnatal studies remain challenging – constitutive astrocyte Cre lines are also expressed in radial glia in embryonic brain48, where QKI has clear roles in neural stem cells28. It is likely brain development would be substantially perturbed obscuring any of the later cell-autonomous effects in maturing astroglia. Some inducible Cre lines for astrocytes exist49, however, perinatal tamoxifen injection both dramatically alters sexual differentiation – an effect previously reported in rats that we replicated in mouse - and leads to substantial rates of delayed lethality: we saw 43% mortality before weaning across 57 pups injected with >100 µg of tamoxifen before P7. Furthermore, an ideal approach would perturb relatively few cells to limit non-cell-autonomous effects on development overall, and would be coupled to a method to specifically yet comprehensively profile cell-autonomous effects. To this end, we applied a CRISPR-TRAPseq approach, wherein sparse gene deletion in specific cell-types, mediated by postnatal gene editing, is coupled to TRAP for specific profiling of targeted cells. We utilized this method to define the consequences of QKI mutation on transcriptome-wide ribosomal association in astrocyte development. We saw alterations due to both changes in splicing as well as transcript levels on ribosomes, and these consequences included both direct (CLIP-target) and indirect effects within astrocytes. Generally, there was a tendency for QKI loss to decrease ribosomal association of QKI-6 CLIP targets, perhaps reflecting a loss of transcript stability as seen in cell line QKI mutant studies35. This result could be consistent with QKI stabilizing these mRNAs, increasing translation initiation, or perhaps stalling mRNAs on ribosomes. Nonetheless, gestalt analysis across altered transcripts argues that QKI is required for a specific aspect of astrocyte transcriptional maturation. This is consistent with a very recent report showing QKI deletion in nestin-positive stem cells disrupts specification of stem cells to glial and astrocyte lineages50. Thus, across the two studies, it appears QKI might be necessary both for early differentiation of glial stem cells prior to GFAP expression, as well as later transcriptional maturation of astrocytes as shown here.Testing this idea, however, represented a challenge. The most elegant approaches for defining gene function are deletion studies. However, astrocyte maturation and synaptogenesis are in vivo processes occurring over the first weeks of postnatal life. Yet, germline deletion of QKI is embryonic lethalp < 2.5E−6, p < 0.02 after FDR correction), with trends in lipid metabolic processes and signal peptide-containing genes. This suggests QKI is regulating translation of proteins with a role at or near the cell surface, consistent with a hypothesized role in regulating some locally translated transcripts29. Indeed, we noted an enrichment among QK targets of our previously PAP-defined transcripts, and this included some of the maturation regulated genes . Of particular interest among these is the GM2 ganglioside activator (GM2a), which activates lysosomal breakdown by beta-hexomaminidase A of GM2 gangliosides, the toxic byproduct that aggregates in Tays-Sachs and other gangliosidoses. Indeed, loss of GM2a itself can lead to the AB variant of this disease51. As these gangliosides are prevalent on neuronal membrane, the upregulation of this key lysosomal regulator during a period of astrocyte maturation that covers synaptic pruning, suggests QKI might dynamically regulate aspects of local lysosomal function related to synaptic pruning by astrocytes and the subsequent clearance of this breakdown product. Indeed, a very recent report defined a similar role for QKI in microglia in clearance of myelin debris after experimentally induced demyelination52.What is the specific function of the genes targeted by QKI during astrocyte maturation? A Gene Ontologies analysis of the specific subset of maturing astrocyte genes that are lost with QKI knockdown revealed an enrichment of glycoproteins  enabling access to other species. Likewise, existing TRAP and Cas9 FLEX cassettes would enable access to the catalog of available Cre mice, further broadening applicability of the approach. Here, focusing on astrocyte translation, we used this approach as a follow up and validation to a comprehensive CLIP analysis, allowing us to confirm impact on CLIP targets in vivo, and describe a role for QKI in transcriptional astrocyte maturation at a subset of transcripts.Finally, the CRISPR-TRAPseq approach proved useful in systematically defining the consequences of QKI deletion in sparse astroglia in a normally developing brain, and we believe it will be similarly useful for other contexts. For example, swapping promoters will allow targeting of different cell-types, as could electroporation at defined time points, provided yields were sufficient. Likewise, gRNAs could target any gene, though RBPs and transcription factors will be especially amenable to TRAP-based readouts. It was also encouraging that TRAP RNA appeared similar in purity with standard bacTRAP  and ROSA26 TALENs binding sites as follows:5′ ROSA26 TALEN binding site: 5′ tccctcgtgatctgcaactcc 3′3′ ROSA26 TALEN binding site: 5′ gggcgggagtcttctgggca 3′To generate a Cre-dependent Cas9-expressing mouse line, we engineered Cas9 into a targeting vector containing Rosa26 homology arms , flanking a CAG enhancer, Lox-Stop-Lox cassette , with 3xFlag-NLS-Cas9-NLS cloned downstream of the stop cassette. The Flag tag is in frame to facilitate confirmation of protein expression. Targeting vector was sequence confirmed then purified for injection into C57BL/6 X CBA F1 hybrid oocytes in conjunction with mRNA coding for a pair of custom TALENs designed to target the mouse ROSA26 locus between the homology arms using the ZiFit targeter (The TALEN kit used for TALE assembly was a gift from Keith Joung (Addgene kit # 1000000017). DNA fragments encoding ROSA TALEN repeat arrays were cloned into plasmid pJDS71. ROSA TALENs plasmids were linearized for in vitro transcription with EcoRI and TALENs RNA was synthesized using the mMessage mMachine T7 Ultra kit (Ambion) and purified with Megaclear columns (Ambion). The cassette of interest was introduced into the mouse genome via pronuclear injection of in vitro transcribed TALENs RNA and ROSA donor DNA. Founders with correctly targeting mutant alleles were identified using long-range PCR with ROSA specific oligos outside of the homology arms and insert specific oligos. Founders may have been mosaic with multiple mutant alleles and thus were bred to WT to generate heterozygous F1 offspring. Analysis of F1 offspring via long-range PCR confirmed germline transmission of the correctly targeted allele.Such progeny of founder MR04 were then bred to a ubiquitous germline Actin-Cre mouse line to confirm Cre-dependence of Cas9 expression 2, 3 times with agitation of the powder between runs to ensure even cross-linking. One sample was not cross-linked, as a control (No XL). Powdered brains were then resuspended in 1 mL ice-cold 1X Lysis Buffer , 10 mM Na3VO4, 10 mM NaF) and homogenized with a drill (power 14) 10 times. Samples were incubated on ice for 5 min then immediately treated with appropriate concentration of RNAse If (NEB) for 3 min at 37 °C at 1200RPM. Samples were centrifuged at 20,000 × g at 4 °C for 20 min. 2% of the supernatant was kept for Input sample, the rest of the supernatant was kept for immunoprecipitation.Dissection, lysis and RNAse treatment: Five P21 C57BL/6J mice were anesthetized by inhalation of isoflurane. Mice were decapitated and brains were quickly dissected and moved to ice-cold PBS. The forebrain was removed and dropped into liquid nitrogen in a ceramic mortar and then powdered with a ceramic pestle. Powdered brains were kept in 6 cm petri dishes on dry ice until cross-linking. UV cross-linking was performed in a Stratalinker, at 400 mJ/cm(II)Immunoprecipitation: Per IP, 6 μl of QKI-6 antibody (Millipore #AB9906) or total Rabbit IgG (Jackson Immunoresearch #711-005-152) was added to 72 μl of M280 Streptavidin Dynabeads (Invitrogen) and 10 ug of Biotinylated Protein G and incubated for 1 h at room temperature with rotation. Beads were subsequently washed 5 times with 0.1% BSA (Jackson Immunoresearch) before mixed with supernatant. Immunoprecipitation proceeded for 2 h at 4 °C with rotation, after which beads were washed with 1X High Salt Wash Buffer . In the last wash, beads were split for RNA extraction (98%) and western blot (2%).(III)g for 7 min at room temperature. The aqueous layer was kept and subsequently cleaned up on the Zymo RNA Clean and Concentrator 5. Quality and quantity of RNA were assessed on an Agilent TapeStation.RNA extraction: Beads were resuspended in 1X LDS Bolt Non-Reducing Sample Buffer, heated for 10 min at 70 °C then run out on a 4–12% Novex NuPAGE gel in 1X NuPAGE MOPS buffer with a protein ladder (Bio-Rad). The gel was transferred to a 0.2 μm PVDF membrane for 2 h at 200 mA at 4 °C. Individual lanes were cut with a razorblade between 37 and 50 kDa, to obtain, on average, ~100 base length fragments. Membrane strips for each sample were digested in 200 μl of 100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA, 1% Triton-X 100 and 32 units Proteinase K (NEB) at 37 °C for 1 h, with shaking. 200 μl of 7 M Urea was added to each sample for an additional 20 min at 37 °C, with shaking. After, 400 μl of Acid-Phenol:Chloroform (pH 6.5) was added to each sample, incubated for 5 min then spun at 10,000 × (IV)Sequencing Library Preparation: Because yields of RNA after CLIP are extremely low (picograms), all RNA harvested was used for library prep. We used direct ligation of an RNA adapter to the harvested RNA after initial dephosphorylation (NEB Antartic Phosphatase) of free ends. RNA was purified using MyONE Silane Dynabeads (Thermo Fisher). Direct ligation of the A01m adapter (Supplementary Table (V)53 v1.2.1. The mouse genome (mm10) was binned into 50 nucleotide non-overlapping windows. Peaks were called under the genome windows on a merged.bam file from all QKI IP samples. Significant peaks were determined by genome-wide FDR p value < 0.05. Count analysis was performed using Subread54 v1.5.3, for each sample file. Differential expression was performed using the edgeR package in R. Per edgeR instructions, only peaks with a minimum of five counts (CPM of 41) in two or more samples were analyzed. High-confidence QKI peaks were identified by 2-fold enrichment over both IgG IP and Input samples, with an FDR-corrected p value of <0.1 for discovery purposes. A control set of Input ‘peaks’ is defined as all detected RNAs in the Input matching the CPM cut off described above.Peak Calling and Differential Expression: Peaks were called across the genome using PiranhaData are available at GEO: GSE147121.(I)MEME Analysis: MEME-CHiP v4.12.0 was performed in discriminative mode, using Input peak sequences as background. Default settings were applied with the exception of using 4 nucleotides as the minimum motif width.(II)55. Specificity indices were determined using previously published RNA-seq data21. A Chi-square test was used to determine whether the distribution of cell-types in QKI or Input samples were significantly different.Cell-Type Specific Expression Analysis (CSEA): Analysis for unique genes from QKI or Input significant peaks was performed using the pSI package(III)Gene Ontology Analysis: GO analysis was performed in Cytoscape v3.5.1 using the BiNGO plug in. Input peak genes were used as the background list. Significant biological process categories were determined by hypergeometric test with Benjamini–Hochberg correction. For analysis of CLIP targets within astrocytes, CLIP targets were subsetted to those genes enriched in astrocytes  > 0.1 in TRAP vs Input comparison, below), and the background for BiNGO analysis was set to all genes enriched in astrocytes.(IV)Analysis of sequence features of targetsCLIP Analysis: 25000 transcripts which have 3′ UTR and 3′ UTR length ≥50nt were randomly selected from all mouse (GRCm38.97) transcriptions. The 3′ UTR sequences from a subset of transcripts which have the same length distribution as the CLIP targets, as well as measurable expression in brain RNASeq data (>10 counts), were selected as control sequences. The artificial peak location of a control sequence was randomly picked within the 3′ UTR region.The conservation scores of the CLIP targets and control sequences were retrieved from UCSC basewise conservation scores (phyloP) of 59 vertebrate genomes with mouse. The average conservation scores from CLIP targets are compared with those from control sequences.Number of polyA sites of each sequence was counted by scanning the polyA signal (A[AT]TAAA). To demonstrate whether the peaks are closer to stop condon or polyA sites, the percentage of distance was calculated as (polyA_site pos − peak pos)/(polyA_site pos − stopcodon pos) Metagene plots were generated by metagene R package 2.14.0 with given alignment files from CLIP, Input and IgG. The expression level of the 1000nt region centered at stop codon, PAS, QBM and peak of CLIP were compared with those of Input and IgG, respectively.(I)Target: QKI, exon 2gRNA: ATGTACAATGACACGTTAAAQKI gRNA design and sequences: gRNAs for QKI was designed using the Zhang lab CRISPR design tool (crispr.mit.edu).(II)tg/+) or Cas9-negative littermates (for imaging) or progeny of (Rosa26:Cas9tg/+) X (Rosa26:TRAPtg/tg) for CRISPR-TRAPseq. Mice were anesthetized on ice for 5 min before injections and allowed to recover on heating pad for 15 min. Two microliters of 1012 vector genome (vg)/mL virus was injected bilaterally in the cortex, 1.5 mm from the midline in two regions: 1-mm caudal to bregma and 2-mm rostral to lambda. Injections were performed with a 33-gauge needle (Hamilton #7803–05) with a 50-μL Hamilton syringe (#7655-01). Mice were aged until P21 before being killed with CO2 and transcardial perfusion with PBS, followed by 4% PFA in PBS.QKI knockdown in vivo: QKI gRNA was cloned into AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR, a gift from Feng Zhang (Addgene plasmid # 60229). The plasmid was modified such that pCBh promoter was replaced with GFABC1D promoter, and Cre was replaced with CFP and Cre, separated by a 2a peptide. The vector was packaged with AAV9 capsid by the Hope Center for Viral Vectors at Washington University. 1 μl of high titer virus was injected into P0 lox-stop-lox Cas9 knock-in mice  at P1-3 and were sacrificed at P21 for CRISPR/TRAP-Seq. TRAP was performed on P21 cortex homogenates as described previouslyLibrary preparation was performed with 0.1–5 ng of total RNA, integrity was determined using an Agilent bioanalyzer. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech) per manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles/burst 50, time 120 s. cDNA was blunt ended, had an A base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 14 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq using paired reads extending 150 bases targeting 30 M reads per sample.Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. Sequencing results were quality checked using FastQC version 0.11.7. Reads aligned to the mouse rRNA were removed by bowtie2 version 2.3.5. Illumina sequencing adapters were removed and the remaining RNA-seq reads were then aligned to the Ensembl release 97 primary assembly with STAR version 2.5.1a. Gene counts were derived from the number of uniquely aligned unambiguous reads by htseq-count version 0.9.1. Isoform expression of known Ensembl transcripts was estimated with Salmon version 0.8.2. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. TRAP performance was assessed by PCA and comparison to prior studies. Samples were excluded if TRAP failed (no enrichment of astrocyte genes compared to input). The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 2.6.2.For gene level analyses, all gene counts were then imported into the R/Bioconductor package EdgeR5 and TMM normalization size factors were calculated to adjust samples for differences in library size. Only genes that have sufficiently large counts in the smallest group size samples were retained for further analysis using the default settings of the filterByExpr function in EdgeR. Differential expression analysis was then performed to analyze for differences between conditions. A negative binomial generalized log-linear model (GLM) was fit to the counts for each gene. Then the likelihood ratio tests (LRT) were conducted for each comparison. Similar approaches were taken for transcript isoforms, but the differential expression analysis used the R/Bioconductor package Limma with the voomWithQualityWeights function to further account for increased levels of variance at the isoform level.p value < 0.05) on P7 and P32 gene counts from the cortex, hippocampus and striatum15. Up- and down-regulated transcripts when QKI was deleted were compared with P7 and P32 astrocyte enriched transcripts by Fisher’s exact test.P7 and P32 astrocyte transcripts were identified by differential expression analysis  was used to map the cell distribution in the brain of QKI-6 targets, up- and down-regulated transcripts when QKI was deleted, respectively, as describedMice were sacrificed by inhalation of isoflurane and then rapidly perfused with ice-cold 1X PBS, followed by 4% paraformaldehyde. Brains were post-fixed overnight and then cryoprotected in 30% sucrose. Sectioning was performed on a cryostat (Leica). Immunofluorescence (IF) was performed on 14 μm slide-mounted coronal brain sections in all cases. Slides were blocked in 5% Normal Donkey Serum (Jackson Immunoresearch) with 0.25% Triton X-100 in PBS for at least 30 min at room temperature. Primary antibodies were incubated overnight at 4 °C . Surfaces were reconstructed from a 13 × 13 × 10 (µm) region of interest in which no detectable neuronal cell bodies, blood vessels or major astrocyte branches were found. The default surface rendering parameters were used to control for bias in the analysis, with the exception that the minimum fragment sized was changed from 10 to 1 to capture the smallest branches.Quantification of the neuropil volume was performed as previously described2, 0.5 mM DTT, supplemented with 1 μl rRNAsin (Promega) and 1 μl Superasin (Ambion) per ml and 1 mini EDTA-free protease inhibitor tablet (Roche) per 10 ml) using a glass homogenizer and Teflon pestle with a drill. Lysates were cleared by centrifugation at 2000 × g for 10 min. Concentration of the supernatant was determined by BCA assay (Thermo Fisher). 50 ug of total lysate was run on 4–12% SDS-PAGE gel (Bio-Rad) and transferred to 0.2 μm PVDF membrane (Bio-Rad). Ponceau image was quantified for loading control. Membranes were blocked in 5% Milk in 1X TBS with 0.5% Tween for 3 h at room temperature. Membranes were incubated in antibody overnight at 4 °C . Appropriate HRP conjugated secondary antibodies were applied after washes at room temperature for 1 h. Membranes were incubated in Clarity (Bio-Rad) chemiluminescence reagents for 5 min and then developed for 1 min in a myECL Imager (Thermo). N is represented in figure legend.Whole mouse brains were dissected after isoflurane inhalation and rapid decapitation. Brains were homogenized  or RNA extraction. The rest of the lysate was incubated with IgG or QKI-6 coupled streptavidin Dynabeads for 2 h at 4 °C . Beads were washed in 1X High Salt Wash Buffer, 4 times for 5 min each with end-over-end rotation. 10% of the beads were resuspended in 1X Laemmli buffer and loaded onto an SDS-PAGE gel to confirm immunoprecipitation of QKI. RNA was extracted from the remaining 90% of beads. For RT-PCR confirmation of QKI-6 targets . Homogenates were spun at 20,000 × Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Many remote Australian Aboriginal communities face barriers in accessing animal health care for their pets. A community that faces these barriers implemented a community-driven program with the objective of improving animal health and population management. The program was evaluated by comparing the percentage of animals desexed, the body and hair condition of dogs, and the presentations at the health clinic for dog bites before versus after 12 months of program implementation. Results show improved animal health measures and no measurable change in human presentations for dog bites. This program, with One Health considerations, could be suitable for other communities facing similar animal health care barriers.This study evaluated a community-driven animal health and management program in the remote community of Wadeye, Northern Territory. This evaluation used a pre-post design to assess changes in animal and human health outcomes over a 12-month period of program implementation, from June 2018 to June 2019. The evaluation assessed the program by comparing animal health outcomes before versus one year after program implementation and comparing human health outcomes before versus during the first 12 months of the program. Outcome measures included the desexing status of dogs and cats, body condition and hair score of dogs, and rates of people presenting to the health clinic for a dog bite. Animal health outcomes significantly improved after program implementation. From pre to post program, there was a 77% increase in the prevalence of good body condition score among dogs and a 9% increase in the prevalence of good hair score among dogs, and the prevalence of desexed dogs and cats more than doubled. There was no significant change in the number of people presenting to the health clinic for a dog bite. Consideration on how to further incorporate human and environmental health aspects into the program could be useful for future One Health programs. The World Health Organization estimates that 61% of all human pathogens are zoonotic  and 75% of emerging diseases affecting people in the last decade have originated from animals .Animals have the potential to harbour and spread exotic diseases. The North of Australia is particularly prone to the introduction of exotic diseases due to the vast coastline, remoteness of communities, and the movement of animals and people from neighbouring countries where diseases not yet present in Australia are endemic ,4. The iThis is particularly pertinent in rural and remote Aboriginal communities. People living in rural and remote areas of Australia are more likely to experience greater health concerns and poorer access to health services compared to urban communities ,6, and tA large proportion of communicable diseases in companion animal populations are easily preventable through access to animal medicines and veterinary services, regular treatment, and increased awareness ,11. HaviThere is increasing interest in addressing animal health care barriers and public health concerns in low-resourced settings using a One Health approach. One Health is a cross-disciplinary approach that considers the relationships between human, animal, and environmental health . A One HA remote community that faces barriers in accessing animal health care is Wadeye in the Northern Territory, Australia. Wadeye has a population of approximately 2280 people, 77% of whom are Aboriginal . There iDue to the barriers in accessing animal health care, the Thamarrurr Development Corporation (TDC) Rangers, along with Animal Management in Rural and Remote Indigenous Communities (AMRRIC), implemented a companion animal health and management program, with support from the West Daly Regional Council. AMRRIC is a not-for-profit organisation that works to deliver veterinary care and animal-related education in rural and remote Aboriginal and Torres Strait Islander communities using a One Health approach.⚬The TDC Rangers and AMRRIC started initial discussions in September 2016, followed by an initial veterinary visit and companion animal census in June 2017. The animal health and management program then began in June 2018 and is ongoing.Co-developed animal health and management program⚬The TDC Rangers administered preventative medicines under veterinary direction, including ivermectin (a broad-spectrum anti-parasitic), to the dog population approximately every three months from February 2018 to June 2019. Afoxolaner (a broad-spectrum anti-parasitic) was administered to dogs that had an observed high burden of parasites. Additional preventative treatments (such as vaccination) were delivered by the visiting veterinary service when required.Preventative medicines administered to the dog population⚬AMRRIC adapted the census for use in Wadeye. The TDC Rangers, with support from AMRRIC, conducted three annual companion animal censuses from June 2017 to June 2019. The Rangers assist with translating the census questions to community members, as the majority of people do not speak English as a first language.Annual companion animal census⚬The visiting veterinary service, with assistance from the TDC Rangers, undertook weeklong visits to deliver desexing programs and undertake treatments and surgeries as needed. From June 2018 to June 2019, three veterinary visits occurred in Wadeye with 259 animals receiving treatment, 183 of which were surgically desexed.AMRRIC visiting veterinary service⚬The TDC Rangers undertake training, provided by AMRRIC staff and TDC’s Trainer, on recognizing common animal health concerns, preparing and administering preventative medicines, and collecting data.Ongoing training opportunitiesThe program consists of five main components including see :Co-develEthics approval was provided by the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (2018-3176) and the Human Research Ethics Committee at the Australian National University (2018/588).Three data sources were used. A companion animal census was undertaken in June 2017, 2018, and 2019. The Rangers collected the data on a custom-designed AMRRIC app from local community households in Wadeye, with assistance from collaborating organisations. Data were captured at each household by observing the animals and asking the owners about the number of dogs and cats that reside there and their current health and reproductive status. The majority of animals were free-roaming, with mixed-breed medium-sized dogs most commonly seen.The health outcomes of interest included desexing status (collected as desexed or can breed), body condition score (assessed using the Purina body condition score system and collected as skinny (score 1–3), normal weight (score 4–5), or overweight (score 6–9) ), and haIvermectin was administered at the same time as the census. Veterinary visit data were collected using the AMRRIC app on the number of animals seen and the type of treatments delivered. Dog bite data were collected at the health clinic in Wadeye for any patient that presented with a dog bite and aggregated quarterly from July 2016 to June 2019.This was a pre-post design analysing three years of companion animal census data to assess animal health pre program implementation (2017) and over the initial 12 months of the program (2018 to 2019). Dog bite data were analysed over four years to assess presentations of dog bite patients to the health clinic, pre program implementation (2016 to 2018), and over the initial 12 months of the program (2018 to 2019). Veterinary visit data were also analysed to assess the extent and type of veterinary treatments delivered during the program (2018 to 2019).An internationally used approach to design and measure companion animal management programs in communities is the International Companion Animal Management Coalition (ICAM) programs and indicators. The indicators focus on animal health, population numbers, and public health to assess the outcomes of the program on the community. Based on the ICAM Coalitions suggested indicators for assessing change, the following indicators and questions were developed  see Tab.Census data were supplied line-listed by animal and identified by household for each census year. The main outcome variables included desexing status, which was coded as “can breed”, “desexed”, and “don’t know”. Body condition score was coded as “underweight”, “normal weight”, and “overweight”. Hair score was coded as “good hair”, “some hair loss”, and “lots of hair loss”. Exposure variables included the year which was coded as “2017”, “2018”, and “2019”. Species was coded as “cat” and “dog”. Variables were created to group the number of animals per household, with categories including “1–5”, “6–10”, “11–15”, and “16–24”. Age group was coded as “kitten”, “adult cat”, “puppy”, “young dog”, “adult dog”, and “old dog”. Sex was coded as “male”, “female”, and “don’t know”.Dog bite data were supplied as the quarterly aggregated number of people presenting to the Wadeye health clinic for a dog bite, overall and by sex. Veterinary visit data were supplied as the aggregated number of animals that received each type of treatment at each veterinary visit.Outcome variables were categorised into binary variables for regression analysis. For all outcomes, responses of “don’t know” were coded as “missing” and not included in the analysis. The regression analysis also excluded any animals missing age or sex. Body condition score was coded as “normal weight/overweight” (good body condition) versus “underweight”. Hair score was coded as “good hair” (good hair score) versus “any hair loss” (some and lots of hair loss combined). Desexing status was coded as “desexed” versus “not desexed”. Year was coded as “2018” and “2019” with 2017 coded as missing, with the exception of hair score which analysed “2017” and “2019”, with 2018 coded as missing due to a large amount of missing data.Statistical analysis was undertaken using Stata 15 and Excel.Descriptive tables showing counts and percentages were used to summarise animal census data by year for total animal population and each animal health outcome variable. The number of animals per 100 people in Wadeye for three years was calculated using the Australian Bureau of Statistics (ABS) 2016 census population data . The avep-value of < 0.05.Regression analyses were used to quantify differences in outcomes from pre to post program implementation, comparing data from 2019 (post program implementation) to data from 2018 (pre program implementation). Prevalence ratios (PR) and corresponding 95% confidence intervals (CIs) were used to compare outcomes  in 2019 compared to 2018. In the case of the hair score, data from 2017 were used as the pre-intervention time point (instead of 2018) due to a high level of missing data on the outcome in 2018. All regression models are presented unadjusted and adjusted for age group and sex of the animals because they were potentially confounding variables. Adjusted results are presented in the text. A result was considered significant at a Dog bites were presented as quarterly dog bites per 1000 population, by sex of person and overall. ABS 2016 census population data for Wadeye were used as the denominator to calculate crude rates. A time-series analysis was performed on total dog bites comparing July 2016–June 2018 (pre program implementation) to July 2018–June 2019 (the program period).From 2017 to 2019, the companion animal population decreased from 732 to 633. Across the years, dogs constituted the majority of pets (81–84%) in the community, with cats constituting the remaining 16–19%. From 2017 to 2019, there was a decrease in the number of dogs (598 to 532) and cats (134 to 101) owned as pets. There were 32.1 animals per 100 people in 2017 and 27.8 animals per 100 people in 2019. The average number of animals per household remained relatively stable, with 3.6 in 2017, 3.9 in 2018, and 3.7 in 2019 see .Across the years, less than half of the dog and cat population was desexed, with the percentage of desexed animals improving over time. The majority of dogs were of normal weight, with very few animals overweight and the underweight population improving over time. The majority of dogs had a good hair score in 2017 and 2019  see . There wThere was a 150% increase in the prevalence of desexed dogs from 2018 to 2019 . Similarly, there was a 125% increase in the prevalence of desexed cats from 2018 to 2019 . The prevalence of good body condition in dogs was 77% higher in 2019 compared to 2018 . The prevalence of good hair score was 9% higher in 2019 compared to 2017  see .The rate of clinical presentations to the health clinic for dog bites per quarter ranged from 1.3 to 7.0 per 1000 population between July 2016 and June 2019. The average quarterly rate pre program implementation was 4.7, and the average quarterly rate during the program was 4.2 dog bites per 1000 population. There was no difference in dog bites by sex of people presenting to the health clinic. Time-series analysis showed that the rate of dog bite presentations was increasing on average by 0.19 presentations per 1000 population (9.33 + 0.19 * Time) per quarter before the program was implemented. During program implementation, the rate of dog bites was decreasing on average by 0.20 presentations per 1000 population (11.20 − 0.20 * Time) each quarter. The difference in the slope of the two trends was not significant.From 2017 to 2019, the companion animal population decreased from 732 to 633. In 2019, there was an average of 3.7 animals per household in Wadeye, almost three times the national average  . Wadeye The results show that after program implementation all animal health outcomes improved significantly. The prevalence of desexed dogs and cats more than doubled in 2019 compared to 2018. There was a 77% increase in the prevalence of good body condition in dogs in 2019 compared to 2018 and a 9% increase in the prevalence of good hair score in 2019 compared to 2017. There was also a decrease in the overall animal population size, an aging dog population, and fewer households with large numbers of animals. Due to the non-randomised, pragmatic nature of the evaluation, these improvements cannot be solely attributed to the program. However, the observed improvements were consistent with the hypothesised outcomes of the program, and the improvements occurred following program implementation. We therefore expect that at least some of the observed improvements can be attributed to the program itself.Sustained animal health and management programs are important components in remote communities to keep animal populations healthy and manageable. Desexing animals can lead to a more manageable animal population and fewer animal-associated injuries . ReachinThere was no observed significant difference in dog bites pre to post program, overall and by sex; however, the dog bite findings were consistent with a decrease in the rate of dog bites in the community over the period of program implementation, compared to pre program implementation. The average quarterly rate pre program was 4.7 dog bite presentations per 1000 population, and the average quarterly rate during the program was 4.2 dog bites per 1000 population. The dog bite data relied on a dog bite injury being of sufficient severity to require medical attention; therefore, this analysis does not capture injuries that were not severe enough to require treatment.To see programs such as this be sustainable and continue in communities, ongoing access to resources is needed. Building monitoring and evaluation capacity into program designs can produce evidence of the impact of the program, assess if the program is meeting community needs, and lead to increased support and funding ,28. EnhaThe strengths of this study include the inclusion of an annual census of the animal population before the program was developed, at the start of the program, and 12 months later, allowing a quantitative evaluation of the program. The community-driven component of this program was vital, with the involvement of community members likely to lead to a greater chance of the continuation of the program. The community-driven arrangement of the program allowed the Rangers to build on their skills and knowledge about animal health and management, leading to a higher likelihood of sustainability of the program. Additionally, this program had cooperation and support from multiple sectors including the local council, federal government, a not-for-profit animal health organisation, a Ranger group, and research scientists working in partnership and fostering a One Health approach.The limitations of this study include the lack of completeness of the census data, as there is a substantial amount of missing data (including responses of “don’t know”) over the three years. In 2018, this was mainly due to technical issues. The prevalence of missing data was lower in 2019 than in previous years for most outcomes. This may be due to holding detailed refresher training with the Rangers, and the increasing experience with using the census app by delivering it annually. The analysis of dog bites had limitations as the data were supplied aggregated and the April–June 2018 quarter could not be separated into pre and post program implementation. There were also not enough time points to run an interrupted time-series analysis on the data. Collecting data on this outcome at additional time points would increase the power in conducting the interrupted time series analysis. This analysis could also be strengthened by collecting information about how the dog bites occurred, as dog bites may be in response to human behaviour and not related to the overpopulation of animals.The inability to link animal health and human health from the same household limits the relationships we can draw between improvements in health outcomes . FurtherThis study showed the positive effects on animal health observed over the initial 12 months of a community-driven and One Health approach to animal health and management in a remote community. From June 2018 to June 2019, three veterinary visits were delivered with 259 animals receiving treatment. All animal health outcomes significantly improved and there was a decrease in the overall animal population, an aging dog population, and fewer households with large numbers of animals.A One Health approach can be appropriate in this setting as it considers the relationships between animals, humans, and the environment, with improvement in animal health having the potential to improve human health and overall community wellbeing. Many programs such as this may not be supported long-term and rely on obtaining support and external funding to continue. The leadership of the community in implementing this program is vital to its sustainability, and through increasing skills and knowledge around animal health and management, this program has a greater chance of continuing. Future programs could consider incorporating a formal evaluation component to be able to produce and publish evidence of the impact of the program and assist in gaining continuing support.


Integrating computer science (CS) into school curricula has become a worldwide preoccupation. Therefore, we present a CS and Robotics integration model and its validation through a large-scale pilot study in the administrative region of the Canton Vaud in Switzerland. Approximately 350 primary school teachers followed a mandatory CS continuing professional development program (CPD) of adapted format with a curriculum scaffolded by instruction modality. This included CS Unplugged activities that aim to teach CS concepts without the use of screens, and Robotics Unplugged activities that employed physical robots, without screens, to learn about robotics and CS concepts. Teachers evaluated positively the CPD and their representation of CS improved. Voluntary adoption rates reached 97% during the CPD and 80% the following year. These results combined with the underpinning literature support the generalisability of the model to other contexts. While no consensus has been reached yet, the dialogue around emerging skills and their role in school curricula has evolved from debating the issue of whether computer science (CS) should be introduced in the standard curriculum, to how its introduction should be done  conceived for K-4 primary school education.The corresponding CS curriculum based on the decomposition into three disciplinary fields that is in line with work by Schiper  and withPresenting the model that was employed to integrate CS and Robotics in the first four grades of primary school see Fig.  and its Reporting the results of the pilot program evaluation, in relation to the above points. Specifically, the analysis revolves around two questions: How did the teachers perceive the training sessions? To what extent did the teachers change their representation of CS  program. The CPD was decomposed into multiple sessions and that are referred to as “training sessions”.The related work is structured in two parts. Section 2.1 presents the literature that helped construct the model presented in Fig. Implementing changes in the educational system is a complex process with many barriers along the way. These are documented in 1) the literature on diffusion of innovation Rogers , in 2) tAlthough the barriers to curricular reform are documented in the literature, no comprehensive model exists that regroups them and that is evaluated in-situ. As such, we present in Fig. political backing , Educational Robotics (ER) and programming activities. CSU activities  or whether the aim is understanding core CS concepts as well. We argue that both are important and are part of a larger digital education context which must be considered as a whole. As such, core CS concepts, ICT and questions about digital citizenship should be integrated in order to convey a full picture on the topic. As the curriculum is particularly dense in the proposed model, the decision was taken to design a two-year CPD program. The first year was dedicated to core CS concepts, and the second year to ICT and digital citizenship. Only the results on core CS concepts are considered in the present study.In many cases, CS CPD was not offered by the ministry of education or a central service but “can best be described as a mix of central support coupled with stakeholder-driven initiatives” . To assess their effectiveness and success, over the course of the pilot a number of evaluation metrics were collected from the participants, as presented in Section 3.3.to integrate digital education into the compulsory school curriculum, with digital education being considered as a whole and including three pillars: CS, information and communication technologies (ICT) and digital citizenship. Therefore, led by the educational department, and fuelled by the knowledge that students had to acquire the desired twenty-first century skillset, the project relied on the collaboration and expertise of all relevant institutions:The state department of education Professors, computer scientists and roboticists from the local technical university Professionals from the local university of teacher education University experts in the evaluation of policies and large-scale projects Public schools from the Canton Vaud.The development and validation of the CS and Robotics integration model for primary school is the result of the research conducted within a political initiative in the administrative region of the Canton Vaud in Switzerland. The initiative, in line with others worldwide, looks The conception of the contentThe conception of the CPD and delivery of the training sessionsThe accompaniment of teachers in schools Testing the content in the classrooms and reporting to the professionals in charge of the CPD program (henceforth referred to as trainers)The overall evaluation of the project in collaboration with the researchers.With reference to the discussion in Section 2.1.2, this initiative seems to unite all the partners required to maximise the chances of success in a unique configuration for Switzerland: political backing and collaboration between the different institutions, public schools and universities.starting from the first years of primary schoolexpand to upper secondary school. However, to ensure that the endeavour was successful, a pilot initiative with a restricted number of establishments was launched. This provided the opportunity to conduct translational research to evaluate the integration of digital education and proceed to informed adjustments before the regional deployment. The pilot initiative involved 10 schools that were selected from a pool of voluntary establishments. The pilot schools were selected to represent the diversity in the region  in terms of location, technical infrastructure, and socio-economics. Teachers from the pilot schools were required to follow a CS professional development program in the first year before progressing to the other pillars of digital education  in the second year. This pool of teachers constitutes the core of the evaluation of the initiative. In particular the evaluation of the CS-CPD and the outcomes in the following year contributes both to validating the CS and robotics integration model, as well as taking informed decisions for the upcoming generalisation and deployment to the entire region.The collaborators from the different institutions envisioned integrating the three pillars of digital education into the curriculum CS Unplugged activities, to discover the basics of algorithmicsRobotics Unplugged activities .Twelve trainers  were in charge of the conception of the CPD and the delivery of the training sessions in the establishments. They had complementary backgrounds with varying experience in the domains of teaching, teacher education and CPD, CS and robotics. Each session was led by a pair of trainers, selected to ensure the presence of both pedagogical and technical competencies. This decision was taken to help the teachers feel reassured and in confidence, as dictated by the aforementioned training principles. The devised CS-CPD program was composed of four day-long face-to-face training sessions  spread out over the school year. The CPD was designed to be hands on and interactive, encouraging collaboration between teachers. The sessions included both teacher content as well as student content  that were easily transposable to classrooms following the principle of isomorphism Lebrun  p.17 andAs anticipated in the introduction, the key goal of the CPD program can be decomposed in two main factors. The first was to ensure positive perception of the CPD format and content. The second was to make sure teachers had a positive representation of the discipline (see Section 4.2.1) and transferred (at least some of) the different activities in their classrooms. This adoption metric is especially significant since the teachers were not obliged to implement the activities in their classrooms.To evaluate the success of the pilot program along the two axes outlined above, we employed a mixed methods approach. Whenever possible, multiple data sources were triangulated in order to obtain a more reliable view on the matter. Information about the type of data acquired, from which participants, at which point in time, whether it is qualitative of quantitative and how it relates to the different axes of analysis are summarised in Table Approximately 350 teachers , participated in the four days of CS CPD see Fig.  that toofocus group was conducted during the last CS CPD session. Teachers were divided into groups of 10 and were asked to reflect on 1) the content of the training session, 2) the format, 3) how it would be possible to assess the students, 4) perspectives of the impending discipline, 5) their needs 6) representation of the discipline and 7) collaboration within the project. Their responses were collected in the form of post-its . The following year teachers electronically responded to adoption surveys to provide information about long-term adoption, as the ICT CPD did not focus on CS activities, nor reprise them. Adoption was not surveyed in Day 5 (Oct. 2019) as this was too early in the school year. The last adoption survey was conducted in March 2020 prior to schools closing due to COVID-19.Following each CS CPD session, the teachers filled out a paper-based questionnaire investigating their perception of the training sessions themselves. The questionnaire items were evaluated on a 4-point Likert scale . The first day also introduced questions on the representation of CS. Following sessions integrated question on the adoption of the CS activities proposed in the previous sessions. The different items are summarised in Table to accompany and support the teachers of their establishment in implementing the CS activities in their classrooms. Before being delivered to all teachers, each training session was piloted with the PRs, so that they could provide feedback on the content and test the activities in their classrooms before the other teachers. At the end of the CS CPD program, three focus groups of approximately 8 PRs each were organised to evaluate the implementation of CS in their respective schools and understanding what their role in it was. These followed the same procedure as the teachers’ focus groups.Across the 10 pilot schools, 23 voluntary teachers were attributed the role of the purveyors in our model . These teachers followed four days of training sessions in parallel to the others in order to experience the content first hand, acquire additional knowledge and be able The ten school directors were consulted at the start of the pilot program and following each CPD session. This had two goals: firstly, to get feedback about the progression of the program and, secondly, to collect their views on what was going on in their establishments. In particular, two questionnaires were administered, once at the beginning and the end of the CS-CPD.The following sections report the results of the pilot program evaluation along the axes outlined in the introduction  using the tools described in Section 3.all days and the overall satisfaction valuing 3.6  and the isomorphism that rendered the activities easily transposable to the classrooms . Satisfaction is not consistent across days and criteria . Specifically, the CS Unplugged and Robotics Unplugged days (Days 1 and 2) were particularly well received (average 3.7) with significantly higher scores compared to other days (Days 3 and 4) for adapted difficulty, equilibrium and content . In addition, the Robotics day (Day 2) significantly differs from all others in terms of interest . As the matrix in Fig. Day 3 and Day 4. This trend is consistent across the grades that the teachers taught . Therefore, it would appear that the content . They attribute the quality of the CPD to several elements: the supervision, support during the year and the resources that were rapidly made available to the teachers (principle: Accompanied and Supported). At the same time, they put forth elements that might hinder the effectiveness of the deployment to the entire region: logistics of the material, teacher replacement logistics, overloading the teachers, lack of coherence between the different pillars of the program  and lack of a comprehensive view of the overall project. Indeed, in terms of teacher overload, the decision was taken not to deliver a differentiated CPD program within the cycle but rather to provide the same content to all. Our rationale was to promote a common CS culture and cohesion throughout the cycle so that teachers may engage in a dynamic of the teams (principle: Collaborative and in Co-Construction), confirmed by the data collected during the teachers’ focus group that took place on the last day of the CS CPD. Specifically, teachers confirmed the impact of the CPD program in establishing a community of exchange and collaboration between colleagues (36 post-its), whether it be at the level of practices, workshops or equipment (principle: Accompanied and Supported).A positive reception of the training sessions was not only expressed by the teachers, but also by the school directors. In the final questionnaire administered in June 2019, the directors reported an average satisfaction of 3.9 with the CPD program. In the Day 1, fifteen teachers provided positive comments about the dynamism and competencies of the trainers, with the only negative comment coming from a teacher worrying that trainers might not understand the constraints of students in 1-2P (4–6 years old). This comment however did not appear again in the following questionnaires, nor in the focus groups. Similarly, two out of the three PR focus groups highlighted the fact that the trainers had an open attitude and always valued their feedback in the preparation of the teachers’ CPD (principle: Collaborative and in Co-Construction). One school director also corroborated this by commending the trainers for their attentiveness (principle: Collaborative and in Co-Construction) in the open questions at the end of the year.Finally, although no question about the trainers was explicitly asked over the course of the training sessions, the spontaneous feedback we obtained was positive. In As presented in the Introduction, it is not enough to measure the teachers’ perception of the CPD to evaluate the efficacy of the pilot program. Indeed, our main metric for success is their adoption (i.e. a measure of how much of the content presented in the training ended up being used in classrooms). This metric is especially important in this pilot since the teachers had no obligation whatsoever to conduct any of the activities. However, adoption cannot be disjointed from the teachers’ representation of the discipline, since a negative view is likely to lead to low adoption.directors of the establishments in the beginning-of-year questionnaire indicated that 16% of teachers would be reticent, 40% would need convincing and only 44% were already convinced about the integration of CS in their teaching  perceived CS as complex, abstract or had no idea about the subject.Initial estimations provided by the Day 1. As can be seen in Fig. reticence, openness and confidence with respect to the topic, 13% of teachers felt reticent, 94% were open to CS and 88% confident that they could implement it in their classrooms when considering a binary split between positive and negative responses. This does not differ between teachers of different grades . In the responses provided to the open question on the evolution of their representation (263 responses), 58% of teachers stated that their representation evolved positively and 37% that they believed that the discipline was accessible compared to their initial representation of CS (253 responses) that they characterised as “nothing” (22%), “fuzzy” (15%), “complex” (11%) and abstract (6%) with only 12% of positive comments. When explicitly asked about potential problems in the overall project, main concerns revolved around the accessibility of the resources (22%), time (21%) and classroom management (13%).Changes in representation around the discipline are visible already at the end of Day 2, in the open question on how the activities went with the students and what difficulties were encountered, the 250 teachers having conducted activities at that point reported having highly appreciated the enthusiasm of their students (16%) and facility that the students experienced when conducting the activities (12%). To complement and further support these results, in the final teachers’ focus group, out of the 1200 post-its overall , and although the teachers were not explicitly asked, the teachers again mentioned having a better understanding of CS (13 post-its) and of how computers worked (12 post-its).At the end of We think that a positive representation of the discipline and confidence are crucial ingredients for the adoption of the proposed activities in classroom. As such, an analysis of the adoption of the proposed activities can not only be viewed as a concrete, direct measure of the success of the pilot program, but also as an indirect measure of the impact that the program had on the teachers’ representation of CS. The adoption analysis presented in the following Section spans Year 1, in which the teachers were involved in the CS-CPD, and Year 2, in which CS was no longer the focus of the CPD  to 92% in March 2019 (out of 272 responses) and finally 97% in April 2019 (out of 199 responses) when considering only the teacher that could implement the activities in their classrooms  and that this was even more complex due to the fact that the material was shared within the establishments, often over several sites (24 post-its). We argue that the matter of time will cease to be an issue once the discipline is officially integrated the curriculum. However, the matter of accompaniment and support is important, notably in the first years of such a program.As a first result extracted from the PRs reported in their focus group that the teachers rarely solicited them for help in the classrooms. The PRs attributed this to high teacher self-efficacy and to the practical, concrete and transferable nature of the activities presented (principle: Adapted and Adaptable). From the teachers’ perspective, in Day 3, 75% of 1-2P teachers who conducted an activity did so with half of the class, while teachers in 3-4P did so with the entire class 80% of the time. We infer that there is a need for accompaniment, notably in the first half of the cycle. However, teachers in 1-2P often teach in pairs and therefore do not necessarily require additional assistance.Even though the PRs’ role was defined as one of accompaniment and support in the schools, the Year 2 conducted a higher number of periods , still in spite of not having any officially allocated time for CS in the curriculum. This suggests that the teachers were able to appropriate the content and gained in self-efficacy. However, when considering the proportion of teachers having conducted the activities (see Table March 2019 to 80% in March 2020. We hypothesise that 1) the teachers who did not adopt in Year 1, did not do so also in Year 2 and 2) as a sign of novelty effect wearing off, the portion of teachers that had conducted just one activity (15%) by Day 4, did not do any by Day 7 , 29% said they had other priorities, 27% that they did not have time and 19% both. Nonetheless, approximately 50% of the teachers that could be followed between day 6 and 7 went on to conduct activities in their classrooms.In the open comments in Day 6, five teachers insisted that the CS content was better adapted to the second portion of the school year. This is corroborated by the fact that 46% of those that said they did not have time and 42% of those that said they had other priorities in Day 6 conducted an activity between Day 6 and Day 7.Additionally, 50% of those who mentioned not feeling confident in Day 6 still went on to conduct activities by Day 7 (12/24). These facts suggest that there are other factors at play than time, priorities and confidence when trying to understand why some teachers consistently do not adopt.The adoption results during the CS CPD carry on in the second year of ICT CPD which did not reprise any CS related content. As Fig. number of periods with respect to the activity type , Robotics Unplugged (RU) activities, Robotics activities with Visual Programming (RVP) and Visual Programming activities (VP). It is interesting to observe that the type of activities implemented by teachers in their classrooms evolved towards more robotics unplugged activities in Year 2  bridges the gap between CS unplugged and visual programming tasks. As such it gives young students the possibility of discovering a wider range of CS concepts by exploiting modalities that are developmentally appropriate. Indeed, there is a level of concreteness fading ”) that occurs as soon as the succession proposed here in the model is implemented. Although the usual way to assess a model would be to conduct a complete assessment of all its different elements, we believe that the results of this pilot program, in addition to the evidence provided by the literature, help validate the proposed model as a whole, which can be used as an example for other initiatives attempting to integrate CS and/or robotics into their curricula.Indeed, regarding the evaluation that was conducted, changes in teachers’ representation of the discipline as well as the positive reception of the CS CPD in terms of interest, adapted difficulty, adequate equilibrium of theory and practice as well as content, are evident from the start of the program. This manifested in 94% of teachers saying that they were open to integrating CS in their classrooms by the end of the first session, compared to an initial prognostic by directors which estimated that 40% of teachers would need convincing and 16% would be reticent. The change in representation reflects in the high levels of adoption evaluated over two years. Indeed 2312 periods of CS were globally conducted by 92% of the teachers in March of the first year (0.7 periods per week), building up to 97% of adoption in April of that same year. There was little decrease in momentum in the second year in spite of not having had training sessions related to CS concepts: 80% of teachers conducted a total of 2875 periods by March, reaching the targeted 1 period per week of CS in the corresponding classrooms. We attribute the success of the project to having addressed all the barriers evoked in the literature, with in particular an adapted set of training principles and corresponding curriculum content. We also believe that the results should generalise well in the deployment phase as the 10 pilot establishments were selected to be representative of the whole region.A number of limitations were also identified. Although teachers globally appreciated the training sessions, it was complex for them to see the bigger picture and understand not only how the content was interconnected, but also how it more globally linked to the overall digital education theme, that includes CS, ICT and digital citizenship. As such, the training principles may be lacking an element on clear direction and objectives. From the analysis perspective, the data acquisition method did not help match teachers throughout the training sessions in Year 1. Such information would have been useful to construct teacher profiles and see how the adoption correlated with the feedback that the teachers provided over the course of the CPD program.Based on the obtained results, notably in terms of adoption, the next step will be to cement the curriculum for the four first years of primary school. Additionally, students were not evaluated, although teachers provided feedback on their classroom experiences. Once the curriculum is set, it will be important to expand the analysis to the students to see what they are learning and make sure that this corresponds to the learning objectives set by those drafting the curriculum.Finally, for future CPD programs, it would be important to expand on the current analysis framework and gain additional insights into teachers’ representation of CS and evaluate how it evolves throughout the program in relation to their perception of the CPD format and content. There was also insufficient indication regarding teachers’ self-efficacy and how confident they felt in integrating the different content in their classrooms, as this was just asked globally in the first CS training session. It is important to have this information at the level of the different activities as the instructional modality is likely to have an effect on the outcome. Finally, it is important to understand the factors which influence adoption, considering elements such as self-efficacy, interest and perceived utility with respect to the different content presented and not just globally. Indeed, teachers must not only find the content interesting, but also see how it benefits their students, with both elements constituting an indicator of autonomous motivation. In particular, we would like to understand what the reasons for non-adoption are in order to find solutions in the long term to ensure the sustainability of the project.


MLXIPL gene, is a transcription factor that is expressed at high levels in the liver and has a prominent function during consumption of high-carbohydrate diets. ChREBP is activated by raised cellular levels of phosphate ester intermediates of glycolysis, gluconeogenesis and the pentose phosphate pathway. Its target genes include a wide range of enzymes and regulatory proteins, including G6pc, Gckr, Pklr, Prkaa1,2, and enzymes of lipogenesis. ChREBP activation cumulatively promotes increased disposal of phosphate ester intermediates to glucose, via glucose 6-phosphatase or to pyruvate via glycolysis with further metabolism by lipogenesis. Dietary fructose is metabolized in both the intestine and the liver and is more lipogenic than glucose. It also induces greater elevation in phosphate ester intermediates than glucose, and at high concentrations causes transient depletion of inorganic phosphate, compromised ATP homeostasis and degradation of adenine nucleotides to uric acid. ChREBP deficiency predisposes to fructose intolerance and compromised cellular phosphate ester and ATP homeostasis and thereby markedly aggravates the changes in metabolite levels caused by dietary fructose. The recent evidence that high fructose intake causes more severe hepatocyte damage in ChREBP-deficient models confirms the crucial protective role for ChREBP in maintaining intracellular phosphate homeostasis. The improved ATP homeostasis in hepatocytes isolated from mice after chronic activation of ChREBP with a glucokinase activator supports the role of ChREBP in the control of intracellular homeostasis. It is hypothesized that drugs that activate ChREBP confer a protective role in the liver particularly in compromised metabolic states.The Carbohydrate response element binding protein, ChREBP encoded by the  Pklr) is induced by high glucose  composed of two enhancer (E)-boxes (CANNTG) separated by five nucleotides  and Mlxipl (Mlx interacting protein like), respectively . Accordingly, the gene names of MondoA and ChREBP are ectively .ChREBP is a large ~100 kDa protein comprising a DNA binding domain in the C-terminal region and nuclear import and export signals in the N-terminal region which interact with importins and the scaffolding protein 14-3-3 during shuttling between the cytoplasm and nucleus. The N-terminal segment contains regions conserved in Mondo homologs that comprise a low glucose inhibitory domain (LID) that controls a glucose response activation conserved element (GRACE) which mediates transcriptional activation . ChREBP vs. 864 amino acids) than the full-length ChREBP which is now called ChREBP-α  raised in conditions of markedly elevated glucose 6-P as occurs in experimental models of high glucose or glucose 6-phosphatase deficiency . Howeveratocytes , 26, 27 Two elegant complementary studies on the ChREBP-β isoform in pancreatic β-cells using si-RNA to selectively target the ChREBP-β transcript, showed that the latter isoform mediates glucose-induced β-cell proliferation and induction of ChREBP target genes  but it aPklr, Txnip, Acc1) (G6pc and Gckr (The expression of ChREBP-β is driven by both ChREBP-α which is activated by high glucose and by ChREBP-β itself (which is constitutively nuclear and active) through a positive feed-back loop resulting in a sustained increase in ChREBP-β mRNA in conditions of high glucose . The elep, Acc1) . This imp, Acc1) , 28, 29,and Gckr , 27, 30,per se is ineffective because mannoheptulose and 5-thioglucose which inhibit glucose phosphorylation block the induction of ChREBP target genes by high glucose  region that has the nuclear export and import sequences and within the DNA binding domain . Phosphorylation affects the binding to importin proteins which promote translocation to the nucleus and also promotes binding to 14-3-3 which favors sequestration in the cytoplasm . Covalenvia cellular levels of acetyl-CoA and the NAD+/NADH redox state   , 54, 55 REBP-/-) , 59. WhiREBP-/-)  and in aREBP-/-) . In adipREBP-/-) . SelectiREBP-/-) .-/- models are more susceptible to fructose-induced toxicity than LiChREBP-/- as shown by the high mortality when transferred to diets high in fructose or sucrose (Khk-C), aldolase B (Ald-B), and triose kinase (Tkfc), as well as fructose 1,6-bisphosphatase (Fbp1) and glucose 6-phosphatase (G6pc) enabling conversion of triose phosphates to glucose  and inorganic phosphate  activity, a marker of hepatocyte damage, establishing a protective role for hepatic ChREBP in fructose metabolism  which generates glyceraldehyde 3-P; aldehyde dehydrogenase (Aldh) which oxidizes glyceraldehyde to glycerate; and alcohol dehydrogenase (Adh) which generates glycerol  is not limiting unlike Glut5 in relation to fructose metabolism  was supported by the aggravated oxidative stress and inflammation resulting from Tkfc knock-down  and other components of metabolic syndrome including raised blood triglycerides, uric acid and markers of inflammation is attributed to the increased consumption of fructose in the diet . Therefoet genes , 80, pretabolism  and furtget gene . Accordiand G6pc , 23, 30 fructose , 54, 55 learance  and likehenotype  and in cotective . Loss-of failure . Two hypficiency , 84. Secock-down .Gckr gene, thereby stimulating hepatic glucose disposal  encoding enzymes or regulatory proteins of the glycolytic and gluconeogenic pathway. Genome wide analysis had identified, two of the subunits of AMPK as candidate target genes of ChREBP , in hepatocytes from control mice but not from the GKA treated mice which showed improved ATP homeostasis  but not by other (G6pc) ChREBP target genes  but not other (G6pc) ChREBP target genes in models of varying resilience of ATP homeostasis. This indicates that the vulnerability or resilience of hepatocytes to maintaining ATP is a major determinant of the ChREBP target genes that are induced by a substrate challenge, and furthermore that the raised phosphate ester level is not the best marker. In this context a better understanding of ChREBP function may emerge from experimental models of mild versus severe metabolic stress, where different target genes of ChREBP may have greater or lesser roles.In the past 20 years since the discovery of ChREBP, a major focus has been on lsewhere , 55. Thelsewhere . The eviPrkaa1,2  is of paWriting—original draft preparation, LA. Writing—review and editing, LA, SC, and BF. Funding acquisition, LA. All authors contributed to the article and approved the submitted version.Work in the authors’ laboratory is funded by the Medical Research Council, UK (MR/P002854/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The participants were 202 elementary school students (average age: 11.6 ± 1.5 years). The garden-based integrated intervention program was conducted during regular school hours for a total of 12 weeks. The program, based on a mediator model for improving children’s eating behavior, included gardening, nutritional education, and cooking activities utilizing harvests. In order to examine effects of the program, the mediating factors related to children’s eating behavior were evaluated using pre-post questionnaires. As a result of the program, dietary self-efficacy, outcome expectancies, gardening knowledge, nutrition knowledge, vegetable preference, and vegetable consumption were significantly increased, and food neophobia was significantly decreased. In addition, there were positive correlations between most mediating factors. Thus, the garden-based integrated intervention developed in this study was effective in improving children’s eating behavior for vegetables.This study was conducted to develop and verify the effects of a garden-based integrated intervention for improving children’s eating behavior for vegetables. A pre-pos Nutrition in growing children is very important; dietary habits that affect the nutritional state established in infancy and childhood, are solidified in adolescence . The dieNutritional status at school age affects not only physical aspects but also emotional and intellectual aspects . AccordiAccording to the Korea National Health and Nutrition Examination Survey, the average daily vegetable consumption of children aged 6 to 11 years is about 148 g, which is about one-third (420 g) the recommended daily vegetable consumption for this age group. As a result, more than half of all children in South Korea consumed less than the recommended amount of nutrients such as calcium and vitamin C, which are derived mainly from vegetables .Accordingly, various methods such as school garden programs, nutrition education, or cooking activities have been attempted to improve children’s eating behavior worldwide ,12,13. IHowever, there are not enough studies to investigate the effect of garden-based integrated intervention and to analyze the mediating effects of factors affecting children’s eating behavior. Therefore, this study was conducted to develop and determine the effects of a garden-based integrated intervention program combining nutrition education and cooking activities, based on mediating factors, for improving the eating behavior to vegetables of elementary school students.For recruitment of participants, a letter regarding the garden-based integrated intervention program was sent to elementary schools in the Seoul area, South Korea. Based on school garden environment, available grade levels, and number of students who agreed to participate, elementary schools C and K were selected. Promotional materials about the study and the program were sent to all parents of 3rd in the school C and 6th graders in the school K. Finally, the parents of all students provided consent for them to participate in the study (95 3rd graders in the school C and 107 6th graders in the school K). The average age of the participants was 11.6 ± 1.5 years. With regard to gender distribution by grade, there were 45 (47.4%) boys and 50 (52.6%) girls in the 3rd grade and 54 (50.5%) boys and 53 (49.5%) girls in the 6th grade. Before the program, the researcher visited the schools and conducted an orientation session in which the purpose of this study and schedule were described, precautions were listed, and written informed consent was obtained from participants. This study was approved by the Institutional Review Board of Konkuk University (7001355-201805-HR-242).A 12-session integrated intervention program, incorporating gardening, nutrition education, and cooking activities using harvests, was developed . This prIn order to select mediating factors affecting children’s eating behavior for vegetables, related studies were identified by using the following keywords in databases such as Web of Science, ProQuest Dissertation and Theses, Academic Search Premier, and Korean National Assembly Digital Library: (1) gardening-related keywords , (2) eating behavior-related keywords , and (3) children-related keywords . As per the search results, 40 research papers were used as reference to verify the effectiveness of the garden-based program to improve children’s eating behavior. The five factors that mediated the association between garden-based activity and children’s eating behavior for vegetables and fruits extracted from 40 studies were “skills required to engage in gardening” ,24,25, “Moreover, sub-elements of the social cognitive theory were incorporated in each session of the program according to the relevant themes . Social Solanum tuberosum), lettuce (Lactuca sativa), tomato (Lycopersicon esculentum Mill.), eggplant (Solanum melongena), carrot (Daucus carota), and beet (Beta vulgaris var. cicla). Moreover, the gardening knowledge education was focused on the cultivation, harvesting, and management methods of each crop, referring to Unit 2-1 (Growing Plants in Everyday Life) of a 6th-grade Korean elementary school textbook [The activities in the 12-session program were focused on gardening, nutrition education, and cooking activities using the harvests. The gardening activities mainly included plant cultivation, such as making plant beds, transplanting, watering, weeding, and harvesting plants. The participants cultivated seasonal plants, such as potato  with their friends and teachers. An example of the 11th session in the garden-based integrated intervention is presented in The 12-session garden-based integrated intervention was conducted once a week and 40 min per session. It was run by researchers who are a horticultural therapist certified by the Korean Horticultural Therapy Association and two volunteers during regular class hours. Gardening activities were conducted in the schools’ outdoor gardens, with a size of about 6 mIn order to identify the mediating factors influencing children’s eating behavior, self-reported questionnaires were used before and after the program. A pilot test was conducted by 10 elementary students to determine the level of difficulty and suitable questions for elementary school students before starting the study.Self-efficacy was assessed with a dietary self-efficacy questionnaire on fruit and vegetable consumption for elementary students . A 24-itTo assess outcome expectancies for vegetable consumption, an outcome expectation questionnaire for children’s fruit and vegetable consumption, developed by Domel et al. , was modIn order to assess aversion to new foods, the 10-item food neophobia scale developed by Pliner and Hobden  and tranTo investigate vegetable preferences, a list of representative vegetables from the Dietary Reference Intakes for Koreans 2015 was used . Some ofTo assess nutrition knowledge, the textbook for Korean elementary students published by the Ministry of Food and Drug Safety and the Ministry of Education was referred to in order to select items that matched the educational contents of this program. The items were revised and supplemented after consultation with elementary school teachers. In order to classify the degree of difficulty by grade, 15- and 21-item were graded by referring to textbooks for 3rd graders and 6th graders, respectively . Each nutritional knowledge item had three response options: “Yes,” “No,” or “I don’t know.” Participants received 1 point for a correct answer and 0 points for an incorrect answer. The perfect score of the 3rd students’ questionnaire is 15 points, and that of the 6th students’ questionnaire is 21 points. Higher scores were indicative of greater nutrition knowledge.For the assessment of gardening knowledge, the 6th grade textbook of Korean elementary was referred to in order to select items that matched the educational contents of this program, and after consulting with an expert such as elementary school teachers and specialists in horticulture, the researcher revised and supplemented the scale. The final 10-item scale included questions about environmental factors necessary for plants to grow, knowledge about plant cultivation, and so on . For each question, the participant received 1 point for the correct answer and 0 points for an incorrect answer; the total score ranged from 0 to 10. Higher scores were indicative of greater gardening knowledge.To investigate daily vegetable consumption, a dietary record sheet was developed to note the amount of food consumed based on the 2010 Dietary Reference Intakes proposed by the Korean Nutrition Society. The contents of the record sheet included the type and quantity of foods eaten during the day and how to measure and record food consumption. Before and after the program, children were instructed to record two days’ meals with their parents. Based on the dietary record sheet, the type and amount of food consumed were entered into the CAN 4.0 program developed by the Korean Nutrition Society to calculate the frequency of vegetable consumption. The average vegetable consumption frequency two days before and after the program was calculated and the values analyzed.The researchers revised the questionnaires developed by Park et al.  and Lee We also developed a satisfaction questionnaire for parents and teachers. It consisted of the following items: overall satisfaction, satisfaction with education and newsletter contents, re-participation, possibility of recommending the program to others, whether the children’s eating behavior improved after the program, and so on.t-test was conducted. Cronbach’s alpha was also calculated to test the reliability of the evaluation tools. Pearson’s correlation analysis was conducted to examine the correlations between mediating factors for improving eating behavior. A correlation coefficient of less than ± 0.2 means little correlation, that of ± 0.2 to ± 0.4 means low correlation, that of ± 0.4 to ± 0.7 means somewhat high correlation, and that of more than ± 0.7 means high correlation [p < 0.05. Satisfaction with the program and demographic information were analyzed using spreadsheet software in Excel .To compare and analyze the mediating factors related to eating behavior for vegetables before and after the program, a paired relation . All stap < 0.001; p < 0.05).After the program, the self-efficacy, outcome expectancies, gardening knowledge, nutrition knowledge, and vegetable preference scores of all participating children significantly improved , but there were no significant differences in the scores of 6th graders (p > 0.05).Grade-level analysis of gardening knowledge, nutrition knowledge, outcome expectancies for vegetable consumption, self-efficacy, and vegetable preference showed significantly improved scores for both grades  were satisfied with the overall program and more than half were satisfied with the per session and sessions per week. The most preferred activity was cooking with the harvests (68.0%). Harvesting was the next most popular activity, at 8.8%. The next preferred activities were watering (8.8%), planting (7.7%), making a garden plot (2.1%), managing the garden (2.1%), weeding (2.1%), and sowing seeds (0.5%). Additionally, 81.9% (158 students) answered that they hoped for a continuation of the program and 83.9% (162 students) responded that they would recommend the program to friends.According to parents’ overall program satisfaction, 75.3% (113 people) answered that they were satisfied. Regarding the program contents, 87.3% (130 people) responded that they were satisfied with the horticultural activities, cooking activities, and nutrition education. Regarding post-program changes, 84.4% (130 people) answered that there were positive changes in their children’s eating behavior.Regarding teachers, all indicated that they were very satisfied with the overall program. In addition, all responded that they were satisfied with the professionalism and attitude of the instructor, the per session, and sessions per week of the program. Additionally, all answered that there were positive changes in their students’ eating behavior after the program.This study was to investigate the effects of a garden-based integrated intervention for children’s eating behavior to vegetables and to identify the mediating factors that affect eating behavior for vegetables in children. As the result of the program, dietary self-efficacy, outcome expectancies, gardening knowledge, nutrition knowledge, vegetable preference, and vegetable consumption were significantly increased, and food neophobia was significantly decreased . MoreoveThe garden-based integrated intervention significantly increased dietary self-efficacy of children who participated in this study . MoreoveOutcome expectancies related to vegetable consumption of children significantly increased after the garden-based integrated intervention . The outChildren’s food neophobia scores significantly decreased after participating in the garden-based integrated intervention . MoreoveChildren’s gardening and nutrition knowledge scores improved after the garden-based integrated intervention. Gardening can be an effective strategy to improve children’s knowledge of horticulture and increase their vegetable consumption by involving them in the process of planting, growing, harvesting, and preparing vegetables . PreviouChildren’s vegetable preferences and consumption significantly increased . This reIn conclusion, the purpose of the present study was to investigate the effects of the garden-based integrated intervention for improving children’s eating behavior for vegetables by measuring the mediating factors related to eating behaviors in children. The result shows the potential of garden-based integrated intervention for improving children’s eating behavior for vegetables. Future study of the topic would be informed by a larger cluster randomized controlled trial to verify the effects of the intervention. Moreover, it would be valuable to verify the lasting effects on eating behaviors through the intervention. Furthermore, it would be interesting to involve parents who have a direct impact on their eating behaviors in the intervention.