Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a  CCDC28B was originally identified as a modifier of BBS, whereby a reduction in CCDC28B levels was associated with a more severe presentation of the syndrome. Different mechanisms, all relying on functional redundancy, have been proposed to explain these genetic interactions. The characterization of BBS proteins supported this functional redundancy hypothesis: BBS proteins play a role in cilia maintenance/function and subsets of BBS proteins can even interact directly in multiprotein complexes.BBS is caused by mutations in any one of 22 genes known to date. In some families, BBS can be inherited as an oligogenic trait whereby mutations in more than one BBS gene collaborate in the presentation of the syndrome. In addition, CCDC28B in cells results in cilia shortening and targeting ccdc28b in zebrafish also results in early embryonic phenotypes characteristic of other cilia mutants. In this work, we generated a Ccdc28b mutant mouse to determine whether abrogating Ccdc28b function would be sufficient to cause a ciliopathy phenotype in mammals, and to generate a tool to continue dissecting its modifying role in the context of BBS. Overall, Ccdc28b mutant mice presented a mild phenotype, a finding fully compatible with its role as a modifier, rather than a causal BBS gene. In addition, we found that Ccdc28b mutants showed behavioral phenotypes, similar to the deficits observed in rodent autism spectrum disorder (ASD) models. Thus, our results underscore a novel causal link between CCDC28B and behavioral phenotypes in mice.We have previously shown that CCDC28B also participates in cilia biology regulating the length of the organelle: knockdown of  BBS1-BBS22; [Bardet-Biedl syndrome  is a rare disorder characterized by retinal degeneration, polydactyly, intellectual disability, gonadal/renal malformations and obesity among other features . BBS is 1-BBS22;  and refe1-BBS22; –14, comp1-BBS22; ,16). The1-BBS22; ,18. Whil1-BBS22; –28.The functional characterization of BBS proteins has provided a cellular/molecular explanation to the oligogenicity observed in BBS, a phenomenon that typically relies on the presence of complementary pathways, complexes and/or some degree of functional redundancy . In thisCCDC28B mutations in a BBS patient cohort identified a synonymous single base change at the penultimate base of exon 3 (the gene is composed of 6 exons). A minigene analysis demonstrated that this change affected exon 3 definition on the 5’ end favoring the production of an aberrant mRNA lacking the first 5 bases of exon 3, which results in a frameshift and the introduction of a premature stop codon (PTC). Thus, the net result of this mutation was shown to be a decrease (but not elimination) in CCDC28B mRNA levels. Importantly, while this mutation was not sufficient to cause BBS, it was shown that the resulting reduction in CCDC28B levels, in a genetic background with mutations in bona fide BBS genes, correlated with a more severe presentation of the syndrome [As mentioned, oligogenicity is typically caused by some degree of functional redundancy. Thus, the identification of proteins that could physically/functionally interact with the BBS proteins was used both to gain insight on the cellular basis of BBS and to identify novel candidates that could contribute to the pathogenesis of the syndrome. In that context, CCDC28B (coiled-coil domain-containing protein 28B), a protein of unknown function at the time, was identified as an interactor of both BBS4 and other known BBS proteins  . Importasyndrome . Overallsyndrome ,37–39.CCDC28B in hTERT-RPE cells results in shortened cilia and a reduction in the percentage of ciliated cells. Targeting ccdc28b in zebrafish results in a distinct external phenotype characterized by a shortened body axis, increased body curvature, craniofacial and pigmentation defects and smaller eyes, phenotypes that have been described in other cilia zebrafish mutants, including BBS mutants  allow us to determine whether loss of function of this gene is sufficient to cause cilia dysfunction and associated phenotypes in mammals, and ii) serve as a tool to continue studying its modifier effect in the context of BBS. We therefore targeted Ccdc28b in mice using CRISPR/Cas9 and performed an in-depth phenotypic characterization of mutant animals (Ccdc28b mut). We focused on phenotypes that have been described for BBS mouse mutant lines, which include the development of obesity driven by hyperphagia, and retinal degeneration. We show that depletion of CCDC28B is not sufficient to cause overt ciliary defects in cells or tissues, although a trend towards presenting a reduction in the percentage of cilia and mild cilia length defects were seen in a subset of the analyzed tissues. In agreement with this observation, Ccdc28b mut animals are viable, reproduce at mendelian rates and do not develop obesity or show signs of photoreceptor loss. However, Ccdc28b mut mice show social behavior defects, phenotypes that are being documented in both BBS patients and animal models (see for example [In this work we aimed to generate a  example –49). OurCcdc28b gene (Gene ID 66264) is located on chromosome 4 and is composed of six exons with an open reading frame spanning from exon 2 to exon 6. Although several splicing isoforms have been reported (Ensembl ENSMUSG00000028795), as in humans, only two transcripts are predicted to encode full length proteins in mice. These are proteins of 200 and 205 amino acids respectively, differing in their C-terminal regions . We crossed two of those founder mice with C57BL/6J females (Jackson Lab stock # 000664) to segregate the mutations, and finally, we chose to continue our work with a mutation consisting of two one base pair deletions at the 5’ end of exon 3, thus predicted to result in a frameshift and a PTC  due to the PTC  with primers located at the far most 5’ and 3’ ends of the reported isoforms and sequenced the PCR products using Oxford Nanopore technology taking advantage of its long-read sequencing. We detected the expected mutations and did not find any evidence of novel alternative transcripts in Ccdc28b mut samples .As a first step we aimed to confirm that the expression of pressed; ). As expA levels , althoug samples . Next, wCcdc28b mut and C57BL/6J (wt) E14 embryos. We quantified the proportion of ciliated cells by counting nuclei and cilia per field, using anti γ-tubulin and anti-acetylated tubulin antibodies to visualize the basal body and ciliary axoneme respectively. We measured cilia length by analyzing at least eight randomly selected fields from each of Ccdc28b mut and wt MEFs. Our data showed that depletion of CCDC28B did not result in shortened cilia nor affected ciliation in these cells: the median cilia length of wt MEFs was 2.33 ± 0.70 μm compared to 2.31 ± 0.78 μm in Ccdc28b mut cells  compared to C57BL/6J females (approximately 7 weeks). We then evaluated cilia in the kidney, a tissue where cilia are readily observed. Kidneys from both wt control and Ccdc28b mut animals of 36 weeks of age were processed for immunofluorescence as described in the methods section. No major anatomical or histological differences were observed between Ccdc28b mut and wt animals and cilia were readily observed projecting into the lumen of tubules in both genotypes. We quantified the number of cilia per field (assessing areas of comparable cell density) and cilia length. Although we could not find statistically significant differences neither in cilia number or length, we did observe a trend towards a reduction in cilia density in Ccdc28b mut preparations, both in the kidney cortex and medulla, compared to wt mice  retinal degeneration has been shown to be progressive, first evident as a thinning of the outer nuclear layer (photoreceptors) by as early as 6 weeks of age and characterized by complete loss of the outer segment in older animals Bbs2, Bbs4, Bbs6, and Bbs12 KOs, as well as a Bbs1M390R/M390R knock-in animals, develop obesity driven by hyperphagia [Ccdc28b mut animals presented similar phenotypes we measured i) weight gain on normal diet, ii) weight gain on high fat diet (HFD), iii) food consumption and iv) systemic glucose handling  was quantified as described in methods. Both Ccdc28b mut and wt controls showed a significantly higher number of interactions with the novel object , two conditions with a high degree of genetic overlap [CCDC28B mRNA splicing thus resulting in reduced, but not abolished, CCDC28B levels [families . Also, wd organs –42. Conscephaly  and for BBS models. Overall, our data indicate that targeting Ccdc28b in the mouse is not sufficient to cause a strong ciliary defect and accordingly, does not cause phenotypes that are highly penetrant in different mouse BBS models, such as retinal degeneration or obesity. Thus, we did not obtain evidence to support a causal role for CCDC28B in BBS. We cannot rule out at this point the possibility of Ccdc28b mut animals presenting a predisposition to develop BBS like phenotypes. Older animals (we assessed mice up to 9 month of age) will have to be evaluated to test this possibility.We therefore decided to target CCDC28B. Ccdc28b mut mice did not show signs of retinopathy and did not develop obesity at least up to nine months of age. Moreover, using metabolic cages we were able to show that Ccdc28b muts are not hyperphagic. Interestingly however, when animals were fed on HFD, Ccdc28b mut mice show both a trend towards presenting a worst performance than wt controls in the GTTs and presented significantly higher glucose basal levels after 7 weeks on HFD. Thus, our results suggest a mild phenotype related to glucose handling. Interestingly, the consequences of BBS gene mutations on systemic glucose management are still not entirely clear. For example, while Bbs4 KO animals have been shown to present impaired glucose handling, mainly due to defective insulin secretion [Bbs12 KO animals have been shown to present an improved glucose metabolism [CCDC28B could contribute to modulating the presentation of obesity in BBS. Crossing this new Ccdc28b mut mouse line with available BBS mutants will allow us to start tackling this issue.Our results however are fully compatible with a second site modifier role for ecretion , Bbs12 Ktabolism . Likewistabolism ,59. Thusvs zebrafish vs mouse), one possibility is that our Ccdc28b mut animals are not complete knockouts. We believe this to be unlikely considering our qRT-PCR, sequencing, and western blot results. Also, the mutant Ccdc28b mRNA could encode an 88 amino acid polypeptide which would only include the first 56 amino acids of CCDC28B. We favor a second scenario which relies on genetic compensation, a mechanism that has been shown to be particularly important to understand differences between targeting genes at the mRNA level versus at the genomic level [egfl7  genomic mutants, but not morphants, upregulate genes that are functionally related to egfl7 [CEP290 (NPHP6), a ciliopathy gene linked to BBS, in zebrafish at the mRNA level resulted in severe cilia-related phenotypes, only a mild defect restricted to photoreceptors was observed in genomic mutants. Interestingly, the authors found that the mild presentation of the later was associated with the upregulation of several genes associated to ciliary function [Ccdc28b will be required to determine if this is the case in our model. However, rather than a weakness of this Ccdc28b mut mouse model, its mild phenotype could be seen as an opportunity to perform transcriptomic/proteomic studies attempting to dissect this compensation mechanism. Such data will likely shed important insight to understand the biological role of CCDC28B and the cellular/molecular pathways involved in cilia regulation.This work also underscores differences with our previous data working on cells and zebrafish where we observed clear ciliary phenotypes. Besides differences in models . In eriments  will be Ccdc28b mut animals did not show differences with wt controls in the open field, the EPM, or the NOR tests, thus ruling out significant locomotor, anxiety, and memory dysfunctions. Ccdc28b mut animals however did show social interaction defects as well as stereotypical phenotypes often observed in mouse ASD models. Interestingly, ASD-like behaviors have been described in BBS patients . Mor example . Thus, im cohort ,72. WhilCcdc28b mut mouse model could provide an opportunity to study cellular/molecular aspects of ASD-like phenotypes. While our results in mice demonstrate a direct link between Ccdc28b and the development of behavioral phenotypes, whether these phenotypes are caused by a CCDC28B-dependent cilia defect and/or CCDC28B extra-ciliary functions is still not known. For example, it was shown recently that CCDC28B plays a role in immune synapse assembly by regulating T-cell antigen receptor, importantly, by affecting actin polymerization and endosomal trafficking, cellular processes that might prove relevant in the context of neuronal homeostasis [Our eostasis . In our eostasis . Observieostasis , 76, sugeostasis –79, thusAll experimental protocols were opportunely approved by the Institutional Animal Ethics Committee (protocol number 007–18), in accordance with national law 18.611 and international animal care guidelines  regardiad libitum. A C57BL/6J mouse colony was bred and maintained in our institutional facility and was used to both obtain zygotes for genome editing and to generate wt control animals. Both wt controls and Ccdc28b mut animals were housed in the same conditions and in the same room of the facility, thus minimizing environmental differences.All animal procedures to generate the mutant line were performed at the SPF animal facility of the Laboratory Animal Biotechnology Unit of Institut Pasteur de Montevideo. Mice were housed on individually ventilated cages  containing chip bedding , in a controlled environment at 20 ± 1°C with a relative humidity of 40–60%, in a 14/10 h light-dark cycle. Autoclaved food  and autoclaved filtered water were administered Ccdc28b mut animals are fertile and thus the colony was maintained by crossing homozygous mutant animals.Cytoplasmic microinjection was performed in C57BL/6J zygotes using a mix of 30 ng/μl Cas9 mRNA , and 15 ng/μl of each sgRNA (2 guides were used) (Synthego), diluted in standard microinjection buffer. Viable embryos were transferred into the oviduct of B6D2F1 0.5 days post coitum (dpc) pseudo-pregnant females , following surgical procedures established in our animal facility . For surGapdh as a reference gene.Tissues were homogenized in TRIzol (Invitrogen) for RNA extraction according to the manufacturer’s protocol. Reverse transcription was done using SuperScript II RT (Invitrogen) and qRT-PCR was performed using SYBR FAST Universal 2X qPCR Master Mix (Kapa) on a QuantStudio 3 RT-PCR System (Thermo Fisher Scientific). All samples were run in triplicate and the CT value was normalized to calculate relative expression of each gene. The fold expression was calculated using the ΔΔCt method with Tissues were lysed using RIPA buffer  supplemented with a protease inhibitor cocktail (Sigma). Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) and 100μg of total protein were loaded into SDS-PAGE gels, transferred to PVDF membranes and probed with anti-CCDC28B  overnight at 4°C. HRP-conjugated secondary antibody was used.2. For immunofluorescence studies cells were cultured on glass coverslips and at 80% confluency, MM was replaced with medium containing 0.5% FBS for 24 h to stimulate ciliation. Cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer saline (PBS), permeabilized with 0.1% Triton-X100, blocked with 5.5% FBS and stained with anti-gamma and anti-acetylated tubulin primary antibodies (Sigma) followed by the corresponding secondary antibodies conjugated to AF488 or TMRM (Invitrogen). Nuclei were stained with DAPI (Invitrogen). Images were taken in a Zeiss LSM 880 confocal microscopy. Eight randomly selected confocal fields from cultured MEFs from at least two Ccdc28b mut and two wt embryos were analyzed. Cilia length was measured using the freehand ROI selection tool of the FIJI image processing package [Mouse embryonic fibroblasts (MEFs) were obtained from E13.5-E14.5 following standard procedures and maintained sub-confluent in DMEM Glutamax with 10% FBS, Hepes 10mM, penicillin 10000 U/mL and streptomycin 10000 μg/mL  under controlled conditions at 37° C with 5% CO package . The proThe perfusion of the mice was performed during the light (resting) phase of the sleep-wake cycle . The animals were anaesthetized with ketamine/xylacine  and perfused with PBS followed by 4% PFA. Brains, kidneys and eyes were immediately dissected out and fixed by immersion in 4% PFA overnight (ON). Thereafter, the brains were cryoprotected in 30% sucrose solution in 0.1 M PBS for 48 h and frozen. Coronal sections (30 μm) were obtained by a cryostat . Sections containing, hippocampus, amygdala and striatum  were collected and stored in an anti-freeze solution at −20°C until immunostaining procedures were performed. Kidneys and eyes were embedded in paraffin and cut into 6 micron slices. Eye sections were dewaxed, rehydrated and stained with Hematoxilin and Eosin following standard procedures.Cilia were detected in brain and kidney sections by immunofluorescence. Renal cilia were stained as previously described , using bad libitum, first with a normal control diet  and then with a high-fat diet . Body weight was recorded weekly. For the weight gain control in ND a group of 4 wt , and 7 Ccdc28b mut  mice were followed up from week 4 to week 21. For weight gain in HFD a group of 7 wt and 7 Ccdc28b mut  from 15–17 weeks of age were used to start the experiment (week -2), then changed to HFD (week 0) and maintained in that diet for 11 weeks when all mice were euthanized. For glucose tolerance testing, mice were starved for 16 h before receiving a single intraperitoneal glucose injection . Glycemia was measured from tail vein blood using a hand-held glucometer . For food intake measurements, mice were transferred to metabolic cages, and after 24 h of adaptation, food was weighed every 24 h for three consecutive days.The animals used in this study were raised and maintained according to standard protocols that were approved by the ethical committee at the Institut Pasteur de Montevideo (protocol number 003–19). Mice were fed wt males, 8 Ccdc28b mut males, 8 wt females and 8 Ccdc28b mut females. Cohort 1 consisted of all 16 males and 3 females of each group, while cohort 2 consisted of 5 females of each group. All assays were recorded using Any-maze software, except the marble maze test, and for all assays where automatization was not possible, videos were scored by investigators blinded to genotype.Behavioral assays were conducted in a total of 32 mice, all between 11 and 13 weeks old, divided as follows: 8 Locomotor activity was assayed in an open field opaque white plexiglass chamber (39 x60x50 cm), where animals were left for 6 min sessions to explore freely and afterwards were returned to their home cages. Any-maze software was used to measure total distance, time freezing and time in central area.Marble maze test can be used to assess repetitive, compulsive-like behaviors . BrieflyGrooming can be used as a repetitive behavior assay . Cages wThe reciprocal interaction test was used to assess social behaviors and interactions towards an age- and sex matched partner. To this end, mice were placed in standard mouse cages (15x27x13 cm), and interactions were recorded for 10 min, which is the period during which most social interactions happen . The tesThe Elevated Plus-Maze test is used as a model of anxiety ,91. The This test is constructed to assess the mouse’s ability to recognize a novel object in the environment, and is divided in three phases: habituation, familiarization, and test phase . BrieflyP values were smaller than 0.05.All data were analyzed using GraphPad Prism 9. Unpaired t-test was used to compare the fold expression in the qRT-PCR analysis. For GTT analysis, the area under the curve was calculated in GraphPad Prism 9, and then compared between both groups using the unpaired t-test. Basal glucose between both groups at different times was compared with the 2-way ANOVA with multiple comparisons. The proportion of cilia positive cells was calculated by counting the number of cilia with clear acetylated-tubulin signal over the total number of nuclei and the comparison between the different samples was performed using a test of Hypothesis specific for comparison of two proportions (hypothesis test for proportions). For the analysis of cilia length, and number of cilia per field, we first tested the data sets for normal distribution, using the Shapiro-Wilk test. If normal distribution was proved, unpaired t-test was used for comparison, and if data did not have a normal distribution, comparisons were performed using the Mann-Whitney test. In all behavioral analysis  we first tested the datasets for normal distribution, using the Shapiro-Wilk test and identified outliers. After normal distribution was probed and outliers excluded, unpaired t-test was used to compare the two groups. In all cases, differences were considered significant when S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigConfocal images showing A) amygdala (bar = 50 μm), B) hippocampal CA1 (bar = 50 μm), C) dentate gyrus (bar = 50 μm). DAPI (magenta) and anti-ACIII antibody (green) were used for nucleus and cilia visualization respectively.(PDF)Click here for additional data file.S4 FigThree animals per genotype and two photos per animal were analyzed. The number of cilia per field was quantified.(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.


While inflammation induced by Toll-like receptor (TLR) signaling is required to combat infection, persistent inflammation can damage host tissues and contribute to a myriad of acute and chronic inflammatory disorders. Thus, it is essential not only that TLR signaling be activated in the presence of pathogens but that TLR signaling is ultimately terminated. One mechanism that limits persistent TLR signaling is alternative pre-mRNA splicing. In addition to encoding the canonical mRNAs that produce proteins that promote inflammation, many genes in the TLR signaling pathway also encode alternative mRNAs that produce proteins that are dominant negative inhibitors of signaling. Many of these negative regulators are induced by immune challenge, so production of these alternative isoforms represents a negative feedback loop that limits persistent inflammation. While these alternative splicing events have been investigated on a gene by gene basis, there has been limited systemic analysis of this mechanism that terminates TLR signaling. Here we review what is known about the production of negatively acting alternative isoforms in the TLR signaling pathway including how these inhibitors function, how they are produced, and what role they may play in inflammatory disease. The inflammatory response is critical for fighting infection. However, inflammation that is overly robust in an acute setting or inflammation that becomes persistent in a chronic setting can damage tissues and cause disease. Thus, it is not only critical that inflammation be turned on when needed upon infection, but it is also vital that the inflammatory response ultimately is turned off. Numerous epidemiological studies demonstrate the opposing roles of inflammation on infectious and inflammatory disease. For example, patients with rheumatoid arthritis who received anti-TNF therapy for >12 weeks were at a 2-fold increased risk of serious infection  signaling pathway. TLRs are a family of single-pass transmembrane receptors that recognize components of pathogens known as Pathogen-Associated Molecular Patterns (PAMPs) , 4. For Tnfaip3, the gene that encodes A20, in mice leads to an increased inflammatory response, hyper-responsiveness to LPS stimulation, and premature death  that interacts with the DD in the downstream IRAK1 kinase. Exon 2 encodes an intermediate domain (ID), and exons 3-5 encode the Toll-Interleukin 1 Receptor (TIR) domain that interacts with Toll-like receptors and the IL-1 receptor. Dimerization of TLRs leads to recruitment and multimerization of MyD88, recruitment of the IRAKs, and thus formation of a large signaling complex known as the Myddosome , alternative splicing of the MyD88 pre-mRNA also produces a shorter isoform (MyD88-S) in which exon 2 is skipped. This short isoform contains an in frame 135 bp deletion that still produces a functional protein. This short isoform has been observed in mice, humans, and other mammals, and in multiple tissues and cell types including monocytes, macrophages, dentritic cells, T cells, B cells, epithelial cells, and glial cells. Thus, like the canonical TLR signaling pathway components, MyD88-S is likely present in most tissues and cell types in the body.MyD88-L serves as a signaling adaptor that bridges signaling between the TLRs and the downstream IRAK kinases, ultimately leading to the activation of the NF-κB and AP1 transcription factors. In contrast, MyD88-S serves a very different function. MyD88-S, which was first described around 20 years ago, is a dominant negative inhibitor that prevents NF-κB activation . In thatIntriguingly, while MyD88-S inhibits IL-1 and TLR-induced NF-κB activation, it does not inhibit AP1 activation . In factThese various studies investigated the effect of MyD88-S overexpression. Vickers et al.  used a dThe inhibitory effect of MyD88-S is not unique to immune cells. MyD88-S also acts as a negative regulator in epithelial cells , 84 and As observed for many negative regulators in the TLR signaling pathway, prolonged PAMP stimulation leads to increased production of MyD88-S Figure 5in vivo, as intratracheal instillation of LPS increases MyD88-S production in alveolar macrophages in mice  that terminates persistent pro-inflammatory signaling. Analogous to the study of how MyD88-S regulates TLR signaling, far more has been learned about the mechanisms controlling production of MyD88-S than production of the other negative regulators that have been identified Figure 5LPS stimulation is sensed by TLR4 and the downstream TLR4 signaling pathway, so it is logical that some component(s) in this pathway will mediate the effects of LPS on the induction of MyD88-S levels. Indeed, studies that activated and inhibited different components in the TLR4 signaling pathway demonstrated that the entire signaling pathway including NF-κB itself is necessary and sufficient for LPS-induced MyD88-S production in murine macrophages . ConsistThus, perhaps not too surprisingly, components of the TLR4 signaling pathway and a few other signaling and epigenetic regulators have been identified that regulate MyD88 alternative splicing. Ultimately, these signaling regulators must impact the splicing machinery to modulate MyD88 splicing. As described in the Introduction, removal of introns from pre-mRNA is mediated by the spliceosome, which is composed of five small nuclear RNAs (snRNAs) called U1, U2, U4, U5, and U6 and associated proteins that interact with each snRNA . Alternative splicing, in turn, can be regulated by RNA binding proteins that modulate spliceosome activity. For example, members of the SRSF (Serine and Arginine Rich Splice Factor) bind mRNAs, and often favor inclusion of the bound exon in the mature mRNA; in contrast, members of the HNRNP (Heterogeneous Nuclear Ribonucleoprotein) often, but not always, will bind to pre-mRNA and will inhibit exon inclusion –124.The core spliceosome regulates MyD88 alternative splicing. siRNA-mediated inhibition of core spliceosome components associated with the U2 snRNP  or U5 snRNP (Eftud2) in mouse and/or human macrophages weakens LPS-induced inflammatory cytokine production, in part, by upregulating production of MyD88-S , 126. PhInterestingly, inhibition of core splicing components also upregulates other negatively acting splice forms including smTLR4 and IKKβb, and these negatively acting splice forms also regulate LPS-induced cytokine production when the spliceosome is inhibited . Our intThe hypothesis that TLR pathway genes that produce negatively acting splice forms are poised to undergo alternative splicing is supported by the analysis of the intron preceding the skipped exon 2 in MyD88-S. Key sites that regulate splicing include the AG at the 3’ end of the intron and the polypyrimidine (pY) tract that lies just upstream of this dinucleotide; the U2AF spliceosome component binds to these sites . UpstreaOne interpretation of these data is that these splicing regulatory sequences in TLR pathway genes act as the “thermostat” that makes it relatively easy for signaling to induce splice switching and the termination of inflammation. The observation that even moderate inhibition of any of multiple spliceosome components leads to splice switching and the production of negative regulators is consistent with this possibility. A second, not mutually exclusive possibility is that the expression or activity of one or more of these core spliceosome components is regulated by LPS. Which spliceosome component this could be and how its activity might be modulated is unknown.It is also possible, perhaps even more likely, that other RNA binding proteins such as members of the SRSF or HNRNP families are regulated by LPS and that these family members in turn regulate alternative splicing of these negative regulators. RNAi-mediated Inhibition of SRSF2 increases MyD88-S production , althougAs described above, much less is known about mechanisms regulating production of other negatively acting isoforms in the TLR signaling pathway. The inclusion of the additional exon between exons 2 and 3 in IRF3a has been examined in one mechanistic study. SR protein family members that are likely to be SRp40 (SRSF5) and SRp55 (SRSF6) bind to an IRF3 pre-mRNA fragment encompassing part of exon 2 and the entire alternate exon 3a . OverexpBeyond these few studies examining the mechanisms controlling the alternative splicing of MyD88 and IRF3, little is known about how these negatively acting isoforms are generated. Moreover, a related open question is how much of the mechanism controlling alternative splicing of these negative regulators is shared: is there one unifying mechanism or are there 13 different mechanisms? The answer probably lies somewhere in between, but for now remains a mystery.TNFAIP3, which encodes the A20 negative regulator. Mis-splicing of this gene leads to loss of production of this negative regulator, which leads to enhanced NF-κB activation and contributes to inflammatory diseases including Still’s Disease and rheumatoid arthritis . The underlying cause of ARDS is some type of lung trauma, often infection –155; ARDA similar shift in MyD88 splicing was observed in a cohort of patients with a second inflammatory lung disease. MyD88 splicing in PBMCs was also shifted in a pro-inflammatory direction in patients with Interstitial Lung Disease (ILD) who were undergoing an acute exacerbation . This coB. pseudomallei infection)  . Moreove-S ratio . So the + T cells from patients with Chronic Obstructive Pulmonary Disease (COPD) (MyD88 splicing changes have been observed in other disease cohorts as well. MyD88-L but not MyD88-S levels were increased in B cells from patients with B cell lymphomas . The MyDe (COPD) . Changese (COPD) , 161, ane (COPD) –144.While MyD88-S has been the negatively acting isoform most examined in these association studies, there is a little data available about other negative regulators. TBK1s levels but not wild type TBK1 levels were increased in PBMCs from patients infected with Hepatitis C Virus (HCV) . A putatin vivo to assess the effect of this alternative splicing in a system where causality can be demonstrated. As yet, few such in vivo studies have taken place.Numerous cell line studies using overexpression or RNAi indicate that production of these negatively acting isoforms can regulate inflammation. These association studies further suggest the potential importance of this regulatory mechanism in the context of infectious and inflammatory disease. Ultimately, it will be necessary to manipulate splicing of these gene via intraperitoneal injection with oligonucleotides that increased MyD88-S production. Following this treatment, they challenged the mice with an intravenous injection of IL-1β. While an intriguing therapeutic modality, they only analyzed one in vivo marker of inflammation, and reported that SAA-1 mRNA was decreased in liver of these mice. It would be interesting to examine the extent of the anti-inflammatory effects of modulating MyD88 splicing in vivo in this fashion using their described reagents.Vickers et al.  used antin vivo  sensitization model; MD-2s expression before HDM treatment decreased subsequent markers of allergic airway inflammation. These lung MD-2s studies are proof of principal that manipulating production of these negative regulators could have clinical benefit.A more thorough analysis has been performed assessing the anti-inflammatory effect of MD-2s expression in vivo . This stin vivo . Expressin vivo in mouse experiments designed to test causality. Thus, while these studies are very suggestive, many questions remain about the effect of these negatively acting splice forms in disease pathogenesis.In summary, there are numerous association studies that suggest that production of these negatively acting splice forms could impact infectious or inflammatory diseases. In contrast, very few studies have tested the effect of these negative regulators In many ways, there are more questions than answers about the negatively acting splice forms in the TLR signaling pathway. To start, it is unclear if there are other negatively acting isoforms remaining to be discovered in this pathway. The TLR signaling pathway has been extensively studied because of its functional significance. The reported divergent ratios of some of these isoforms may make it difficult for them to be found in unbiased RNAseq studies depending on the read coverage and the nature of the analysis. Thus, it is perhaps not surprising that most of these inhibitory isoforms have been identified on a gene-by gene basis. Thus, we expect that there very well could be more negatively acting isoforms in this pathway that have not yet been identified. Moreover, while we have focused on the TLR signaling pathway in this review, there are examples of negatively acting splice forms in other innate immune signaling pathways. For example, alternative splice forms of NOD2 and MAVS that encode negative regulators have been identified , 165. SoThe precise mechanisms used by most of these negative regulators to inhibit signaling are still unclear. In many cases, there are only a few or in some cases even one publication describing these negatively acting splice forms, so there is still lots of mechanistic investigation to be done. Similarly, the mechanisms mediating production of these negative regulators in the first place are largely uninvestigated. How do PAMPs induce production of these negative regulators? Is there a common mechanisms that mediates their production or are multiple mechanisms involved. There is much still to uncover.From a disease standpoint, the published association studies suggest that defects in production of these negative regulators could be playing a role in human disease. While suggestive, it will be necessary to test these possibilities further by using mouse models that allow for manipulation of these splice forms. While manipulating individual splice forms is more complicated than knocking out a whole gene, there are a variety of mouse genetic tools including CRISPR/Cas9 available for these purposes. If evidence continues to accumulate that these splice forms do play an important role in human disease, then the ability to use splice-switching oligonucleotides to artificially manipulate splicing in this pathway could become an intriguing approach to facilitate mouse disease model studies and potentially a useful therapeutic approach to explore.Finally, there is an over-arching question of why are there so many negative regulators. This is a question that covers not just these negatively acting splice forms but the myriad of other negatively acting proteins induced following PAMP challenge. Often, ablation of individual negative regulators in mice leads to autoimmune or autoinflammatory phenotpypes. Will the same be true of these negatively acting splice forms or will there be redundancy among them? This becomes particularly relevant as the functional studies on many of these negative regulators were performed using isoform overexpression . How all these negative regulators function singly and in concert in a physiological setting is a fascinating yet complicated open question.There is hope that these answers will be forthcoming, as a growing number of studies have been examining alternative pre-mRNA splicing in different contexts including the regulation of innate immunity.FL and SA both conceived of, wrote, and edited the manuscript. Both authors contributed to the article and approved the submitted version.This manuscript was supported by NIH grants R01HL148335 and R01AI155749.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The signaling pathways associated with lipid metabolism contribute to the pathophysiology of autism spectrum disorder (ASD) and provide insights for devising new therapeutic strategies. Prostaglandin E2 is a membrane-derived lipid molecule that contributes to developing ASD associated with canonical Wnt signaling. Cyclooxygenase-2 plays a key role in neuroinflammation and is implicated in the pathogenesis of neurodevelopmental diseases, such as ASD. The endocannabinoid system maintains a balance between inflammatory and redox status and synaptic plasticity and is a potential target for ASD pathophysiology. Redox signaling refers to specific and usually reversible oxidation–reduction reactions, some of which are also involved in pathways accounting for the abnormal behavior observed in ASD. Redox signaling and redox status-sensitive transcription factors contribute to the pathophysiology of ASD. Cannabinoids regulate the redox balance by altering the levels and activity of antioxidant molecules via ROS-producing NADPH oxidase (NOX) and ROS-scavenging superoxide dismutase enzymes. These signaling cascades integrate a broad range of neurodevelopmental processes that may be involved in the pathophysiology of ASD. Based on these pathways, we highlight putative targets that may be used for devising novel therapeutic interventions for ASD. Autism spectrum disorder (ASD) is a set of heterogeneous neurodevelopmental conditions  associatThe pathophysiology of ASD includes alterations in neuronal circuits induced by defects in various signaling pathways. Most studies on ASD pathogenesis have indicated a combination of genetic mutations and various altered signaling pathways, such as those involving mTOR , Wnt, PrThe combination of various core mutations and consequent dysfunction of cell signaling pathways has already been reported in the pathogenesis of ASD in preclinical animal models and patient samples. In this review, we highlight putative therapeutic targets based on these pathways. These putative targets may be used for novel therapeutic interventions for ASD in the future. Timely, specific, and accurate modulation of these pathways may contribute to our understanding of ASD pathogenesis. This review provides new perspectives on the various cellular signaling pathways involved in the regulation of lipid metabolism in ASD pathogenesis.Lipids act as regulatory molecules that contribute to the growth and maintenance of brain functions. Altered fatty acid metabolic pathways may be involved in ASD pathogenesis. A natural lipid-derived molecule, prostaglandin E2 (PGE2), is involved in the physiological processes implicated in ASD ,7. EsterAdditionally, PGE2 receptor EP4 signaling may be part of the neuroprotective and anti-inflammatory pathways in models of neurodegenerative disease . VariousApart from these, the endocannabinoid system (ECS) may be involved in ASD pathophysiology . The ECSProstaglandin E2 (PGE2) is a well-known membrane-derived lipid signaling molecule that plays an important role in neuronal development. Abnormal PGE2 levels are due to environmental insults during prenatal development ,15. Vari−1-/- and COX−2-/-) exhibit altered COX/PGE2 signaling and, hence, are a potential model of ASD [Cyclooxygenase-2 (COX-2) is the main regulator of PGE2 synthesis . Abnormal of ASD .Collectively, PGE2 upregulates the canonical Wnt signaling pathway, which is associated with the development of ASD. The association between PGE2 and Wnt signaling during prenatal development of the nervous system, where PKA and PI-3K might act as mediators, suggests that PGE2 alters Wnt-dependent migration and proliferation of neuroectodermal stem cells, which has implications for ASD .Neurodevelopmental disorders, such as ASD, are associated with increased activity of the brain catalase of phospholipase A2 (PLA2) isoforms. As PGE2 biosynthesis has been associated with PLA2 expression , new synCyclooxygenase-2 (COX-2) plays a key role in neuroinflammation and the pathogenesis of neurodegenerative diseases such as ASD . EndogenCOX-2 knockout mice exhibited dysregulated expression of several ASD-related genes . COX-2 dRegulation of PGE2 synthesis via the modulation of COX-2 activity has been implicated in the pathogenesis of neurodevelopmental diseases such as ASD . CyclooxIrregular COX-2/PGE2 signaling leads to ASD-related behaviors and social abnormalities. Thus, COX-2 may be related to ASD-like behavioral symptoms in association with PGE2. The associations between COX-2 and PGE2 are presented in As PGE2 plays an important role in brain development, abnormalities in the COX-2/PGE2 signaling pathway due to genetic or environmental causes are linked to ASD  and may −1-/- and COX−2-/-) exhibit altered COX/PGE2 signaling and, hence, are a potential model of ASD [Cyclooxygenase-2 (COX-2) is the main regulator of PGE2 synthesis . Abnormal of ASD .−1-/- and COX−2-/-) exhibit altered COX-2/PGE2 signaling and, hence, are a potential model of ASD [With respect to pathophysiology of ASD, abnormal COX-2/PGE2 signaling is associated with behavioral symptoms of ASD . Male mil of ASD .Significantly higher levels of both COX-2 and PGE2 have been reported in the plasma samples of autistic patients. Increased plasma PGE2 levels were concomitant with significantly lower levels of α-synuclein , which pIn summary, combining α-synuclein, COX-2, and the PGE2/PEA receptor could play an important role in brain development, with abnormalities in the COX-2/PGE2 signaling pathway due to genetic or environmental causes linked to ASD .The endocannabinoid system (ECS) consists of endogenous cannabinoids and metabolic enzymes that play a critical homeostatic role in modulating polyunsaturated omega fatty acid (PUFA) signaling to maintain a balanced inflammatory and redox state .Cannabis sativa L., may regulate signaling and exert therapeutic effects via cannabinoid receptors [PUFAs may be linked to the ECS and involved in the maintenance of brain plasticity, memory and learning, blood flow, and the genesis of neural cells . The ECSeceptors . ECS’s aeceptors . ECS-indeceptors .ECS signaling regulates proliferation, differentiation, and the death of brain cells and has important consequences for neural development and brain repair via cannabinoid (CB) receptors . FurtherThe two cannabinoid receptors, CB1 and CB2, are derivatives of PUFAω-6 AA and ω-3 fatty acids Wu . The ECS–/–Shank3B mice showing substantial social interaction impairment) showed that BLA-NAc glutamatergic circuit activation reduces sociability and social reward seeking without increased anxiety or rewarding effects [As described above, the main endocannabinoid in the brain, AEA, has been reported to alter social behaviors in genetic models of ASD . Such ef effects .2-arachidonoylglycerol (2-AG)-mediated cannabinoid signaling regulates the BLA–NAc glutamatergic circuit , which wStudies based on animal models have revealed important roles for ECS combined with G protein-coupled cannabinoid receptors. Furthermore, ECS’s endogenous lipid-derived agonists may contribute to social anxiety and social reward as two key aspects of social behavior . FunctioCollectively, the role of eCB signaling may be related to social impairment in neuropsychiatric diseases such as ASD. With respect to the therapeutic potential of eCBs, the ECS plays a key role in neurodevelopment via inflammatory responses and, thus, the renowned anti-inflammatory properties of cannabinoids might be useful in treating ASD pathology .Cannabigerol (CBG) is the precursor molecule for the most abundant phytocannabinoids . CBG is The microglia harbor a completely functional ECS signaling system . In addiWith respect to the role of CB1 and CB2, CBG binds CB1 and CB2 but functions as a competitive antagonist for CB1. CB1 is important for neuronal differentiation, normal axon migration, and the establishment of neuronal connectivity . Early eAnandamide-mediated signaling may act selectively in reducing social anxiety and enhancing social rewards in relation to ASD . ImportaAlthough limited effects of CBD on ASD symptoms, a previous study reported clinical effects of CBD on ASD symptoms . MoreoveThe phytocannabinoid cannabidivarin (CBDV) has the ability to ameliorate behavioral abnormalities resembling core and associated symptoms of ASD . RestoraCollectively, BLA-NAc circuit activation decreases sociability and is regulated by eCB signaling. NAc-specific 2-AG augmentation and BLA-NAc circuit inhibition normalized social deficits in the Shank3B–/– model of ASDs . TherefoCannabis sativa, one of the world’s oldest propagated plants, and its extracts are available for medical use [n = 36) [Cannabis is derived from ical use . Accordiical use . A previ[n = 36) . Accordi[n = 36) . ASD sym[n = 36) . TherefoInterestingly, cannabinoids (CBs) reduced the frequency of seizures in autistic patients via the excitatory/inhibitory imbalance in the excitatory/inhibitory system, such as a CB-related increase in glutamate (the excitatory system) in subcortical regions  and a decrease in cortical regions . CBD increased GABA transmission (the inhibitory system) in critical and subcortical regions of normal development subjects but decreased it in the same areas of the ASD group .Collectively, CB may have important pharmacological potential for the behavioral symptoms associated with ASD and ASD-related seizures. Although they have been shown to reduce seizure frequency via the excitatory/inhibitory system , furtherA previous study reported reduced endocannabinoid (eCB) signaling in children with autism and in an animal model of VAP-induced ASD. 2-AG deficiency induced behavioral defects caused by the absence of its primary synthetic enzyme, diacylglycerol lipase α (DGLα), in dendritic mesoporous silica nanoparticles (dMSNs) . These cRedox signaling refers to the specific and usually reversible oxidation/reduction reactions involved in cellular signaling pathways . Redox sReactive oxygen species (ROS) act as secondary messengers regulating redox-sensitive signaling pathways, which elicit specific cellular responses . As redoThe redox state is predominantly reliant on an iron redox couple and is maintained within strict physiological limits. Iron acts as a redox-active cofactor in many biological processes and plays an important role in cell growth . Iron hoA recent study reported that synucleinopathy (the pathologic accumulation of α-synuclein) induces the cellular iron sequestration response, leading to neurobehavioral deficits . As S-niHuman copper transporters are subject of a complex multilayer regulation by various metabolic signals . CerulopThe antioxidant capacity of Cp is implicated in various neurodevelopmental disorders, such as ASD. Cp expression in myelinating glial cells is crucial to prevent oxidative stress and neurodegeneration in the central and peripheral nervous systems . Lower pA former clinical study indicated that 93 children with ASD had lower ferritin and higher mean corpuscular volume  compared to 74 children with no neurodevelopmental disorders other than ASD . The ASDThe evidence indicates an important interplay between the cannabinoid receptors CB1 and CB2, their synthetic and metabolizing enzymes, and various key inflammatory and redox-dependent processes. The ECS may also control oxidative stress by directly or indirectly modulating CB1 and CB2 signaling or CB receptor-independent processes via COX-2-dependent eicosanoid pathways . ModulatAccording to primary findings, cannabinoids (CBs) regulate the redox balance by altering the level and activity of antioxidant molecules . CBs tarCollectively, as described above, changes in neuromodulatory eCB signaling were found in patients with ASD and in VPA-induced rodent models of ASD. The presynaptic release of glutamate induces postsynaptic mGluR-mediated upregulation of eCB and the main endogenous agonists of cannabinoid (CB) receptors, such as anandamide and 2-arachidonoylglycerol (2-AG) , indicatWith respect to genetic factors of eCB signaling, a recent study reported that the fatty acid amide hydrolase (FAAH) C385A polymorphism was associated with this signaling in the brain . EpigeneNeuroinflammation contributes to the pathogenesis of various neurodevelopmental disorders, such as ASD. Microglia and astrocytes are the main pathological components in this process . The endThe endocannabinoid system (ECS) has been representatively examined in the fields of neuroscience and neurodevelopment. With respect to the pathophysiology of ASD, several studies indicate that eicosanoids are lipid-based signaling molecules that play a role in the pathophysiology of ASD due to their interaction with cyclooxygenase (COX)-derived prostaglandins (PGs) and PGE2 . AlthougFMR1 gene and loss of its protein product FMRP. Although it has this simple genetic origin, FXS is a phenotypically complex disorder including a range of physical and neurocognitive disruptions. Circuit hyperexcitability may be a common convergence point accountable for many wide-ranging phenotypes in FXS [Fragile X syndrome (FXS) is the most common heritable form of mental retardation and monogenic cause of ASD. FXS is characterized by motor, cognitive, and social alterations, mostly overlapping with ASD behavioral phenotypes. The severity of these symptoms and their timing may be exacerbated or advanced by environmental adversity interacting with the genetic mutation . Symptoms in FXS . The mecs in FXS .FMR1 gene, leading to the loss of fragile X mental retardation protein (FMRP), a synaptically expressed RNA-binding protein regulating translation. The ECS is a key modulator of synaptic plasticity, cognitive performance, and seizure susceptibility [The new drug ZYN002 is a transdermal cannabidiol gel formulation and targets the dysregulation of the endocannabinoid system . Treatmetibility . The cantibility . As fatttibility . CollectThis review has several limitations: (1) the discussion of neuroinflammation in general is rudimentary; (2) the clinical use of non-steroid anti-inflammatory drugs and cannabinoids does not support the conclusion.(1) With respect to the role of neuroinflammation in ASD, neuroinflammation is emerging as a key underlying mechanism of ASD . The var(2) With respect to the clinical use of non-steroid anti-inflammatory drugs and cannabinoids, the current evidence is suggested to be insufficient to support the prescription of cannabinoids for the treatment of psychiatric disorders involving ASD Cannabis has been used to alleviate symptoms associated with ASD  . MoreoveAdditinaly, a recent clinical study conducted in 53 children (median age: 11 years) indicated that cannabidiol may improve symptoms of comorbidities of ASD; however, the long-term effects should be evaluated in a large-scale study .With respect to the risks of endocannabinoid therapy, cannabinoid signaling may be involved in the social impairment and repetitive behaviors observed in individuals with ASD due to a possible role of the EC system in excitatory–inhibitory (E-I) imbalance and immune dysregulation in ASD . MoreoveThe signaling pathways associated with lipid metabolism help to identify abnormalities and new strategies for treating the pathophysiology of ASD. The available experimental evidence links to several common signaling pathways, such as prostaglandin-E2 (PGE2) and cyclooxygenase-2 (COX-2), endocannabinoids (eCBs), redox signaling, iron signaling, and copper signaling.The alteration of PGE2 function by environmental insults has been linked to brain pathology, including ASD. PGE2 is a membrane-derived lipid signaling molecule and plays an important role in neuronal development, contributing to the risk of developing ASD associated with canonical Wnt signaling. COX-2 is an enzyme for converting arachidonic acid to prostaglandins and plays a key role in neuroinflammation, being implicated in the pathogenesis of neurodegenerative diseases, including ASD. The endocannabinoid system (ECS) consists of signaling for the maintenance of a balance between inflammatory and redox states, synaptic plasticity, and neuroendocrine functions. The ECS plays a role in epigenetic modifications, such as DNA methylation and neurodevelopment, via inflammatory responses, contributing to the pathophysiology of ASD. Abnormal COX-2/PGE2 signaling is related to the development of ASD. New synthetic inhibitors of lipolytic enzyme phospholipase A2 (PLA2), which is involved in the inflammatory atherosclerotic process, may be used for the treatment of neuroinflammation-associated neurodevelopmental disorders in cells. As described in the limitation section, the clinical use of non-steroidal anti-inflammatory drugs and cannabinoids are limited.Redox imbalance induces cellular changes, leading to abnormal behaviors in ASD. Redox signaling refers to the specific and usually reversible oxidation/reduction modification of molecules involved in cellular signaling pathways. Environmental factors related to mild maternal inflammation and subsequent alterations in brain development may induce cellular changes, leading to the abnormal behaviors in ASD. The final common pathway of enhanced reactive oxygen species (ROS) signaling may act via resultant PI3K/AKT pathway stimulation. The ECS may also control oxidative stress by directly or indirectly modulating CB1 and CB2 signaling or CB receptor-independent processes via COX-2-dependent eicosanoid pathways. The role of ROS in signaling pathways and redox-sensitive transcription factors contributes to the pathophysiology of ASD.Cannabinoids regulate the redox balance by altering the level and activity of antioxidant molecules via ROS-producing NADPH oxidase (Nox) and ROS-scavenging superoxide dismutase enzymes.Neuroinflammation contributes to the pathogenesis of various neurodegenerative diseases, such as ASD. Microglia and astrocytes are main pathological components in this process in relation to the endocannabinoid systems.FMR1 gene and loss of its protein product FMRP.We reviewed the current knowledge on the main signaling pathways that are commonly disturbed in ASD. Additionally, we highlighted some putative druggable targets along these pathways in relation to novel therapeutic interventions for ASD. Fragile X syndrome (FXS) is the most common heritable form of mental retardation and monogenic cause of ASD. FXS is a leading inherited cause of ASD and intellectual disability, resulting from a mutation in the Accurate modulation of these prominent pathways may contribute to the neurodevelopmental regulation of homeostatic patterns and could be used to treat some ASDs.


This research topic is kindly dedicated to the memory of our beloved and inspiring friend Professor Carlos Mauricio Castro Costa. It was interesting to see that, during the emergence of a new virus which has plagued humanity for 3 years now, Human T lymphotropic virus 1 (HTLV-1) received a lot of attention from health authorities, including the World Health Organization (WHO). This was a direct response to an instigative letter calling for action to eliminate the virus . HTLV, wBandeira et al. designed an investigation that showed a low (0.4%) seroprevalence of HTLV-1 infection among prisoners from 12 closed prisons in Mato Grosso do Sul state, Central Brazil, with the predominance of the Cosmopolitan subtype Transcontinental subgroup. In contrast, Abreu et al. identified a high prevalence (3.0%) of HTLV infection among the Warao Indigenous Refugees in the Brazilian Amazon, with the presence of HTLV-1 subtype 1A (Cosmopolitan) and the Transcontinental subgroup and HTLV-2b, highlighting the need to create public health policies for the population of immigrants running from their own country to the Brazilian territory. Important information came from quilombos (slave remnant communities) in the state of Pará, Brazil where Brito et al. described a prevalence of 0.5% of HTLV (HTLV-1 and HTLV-2), reinforcing the need for infection control policies among these communities. Oliveira-Filho et al. investigated intra-family transmission among injecting drug users in a large geographical area of the state of Para and both HTLV-1 and HTLV-2 were found, again demonstrating the urgent intervention for infection control and prevention to reduce the spread of HTLV. A complex epidemiological model was set by Miranda C et al. to investigate a 10-year (2007–2016) analysis of HTLV-1/2 infection in first-time blood donors from four blood banks in Brazil. This revealed that HTLV-1/2 prevalence was not decreasing with a trend observed toward an increase in HTLV-1/2 infection among younger people, males, white skin color and higher education.The Research Topic received interesting papers in regard to the prevalence of HTLV-1 and subtypes found among vulnerable communities. Pereira et al. reported that in one of the most important epidemiological settings in Brazil, the state of Bahia, the prevalence of HTLV infection in people living with HIV-1 (2.4%) was much higher than among people with negative HIV-1 serology (0.5%), and that the highest frequency was among women. Three interesting and comprehensive reviews were presented. A systematic review by Ye et al. showing the importance and higher frequency of Strongyloides stercoralis infection among people living with HTLV-1 than in HTLV-1 seronegative and a strong association between severe strongyloidiasis and HTLV-1 infection. This was also reported by Montaño-Castellón et al. who also reported that HIV-1 and HTLV-1 coinfection and HIV-1 and HTLV-1/2 triple coinfection were associated with shorter survival, higher mortality rate, and faster progression to death, while coinfection by HIV-1/HTLV-2 seems to have neutral association with longer survival, slower AIDS progression, and lower mortality rate. The review by Rosadas and Taylor highlighted several factors related to the impact of HTLV-1 on co-infections and vice versa. Among other aspects, they emphasize that large scale prospective controlled studies on the prevalence and impact of HTLV-1 in co-infections are needed.Franco et al. reported a multi-epitope protein, expressed in a prokaryotic system, including epitopes from Gag, Tax and Env proteins, aiming to develop a serological screening test for both HTLV-1 and HTLV-2, that showed great potential to be used to detect mono or co-infected individuals. Rocha Júnior et al. developed a promising, rapid, and sensitive duoplex-RT-PCR aiming to confirm and discriminate both HTLV-1 and HTLV-2. The new approach was validated by a multicenter study across Brazil. The assay presented adequate efficiency for HTLV-1/2 differentiation showing high sensitivity and specificity. Similarly, Gonçalves et al. developed and validated an in-house multiplex quantitative real-time PCR assay targeting the pol and tax genes. The laboratory tests demonstrated high sensitivity and specificity, leading the authors to conclude that their method is efficient and reliable for diagnosis. As a novel approach Machado et al. used the phage display to select HTLV-1 epitopes for diagnosis and described four clones with HTLV-1 mimetic peptides which aligned with gp46, protease and Tax, showing the potential of the technique for bioprospecting HTLV-1-related peptides with good potential either for diagnostic tests or as possible vaccine components for future studies.Although there are multiple commercial assays suitable for screening, a repeated concern has been the need for cheaper tests to type and confirm HTLV infections. Lopes et al. describe the first-year results of implementing an HTLV-1 serodiagnostic service. Notably infection was confirmed and typed, all patients found to have HTLV-1/2 infection were referred to a counseling service and into follow up whilst two patients with suspected myelopathy were directed into neurological care, a model of care that should be provided wherever screening is introduced. Kimura et al. report the impact of HAM/TSP on health-related quality of life (HRQoL) in Japan with the interplay between the various elements of HAM contributing to impaired role functioning. They argue the case for comprehensive care to address the poor QoL. Meanwhile Aben-Athar et al. noted the absence of HTLV related care plans and therefore set out to develop and test a nursing care plan. The resulting detailed plan for care in the community aims not only to improve quality of life by tackling each of the many facets of HTLV-1-associated myelopathy but also to prevent avoidable deterioration such as falls leading to fractures which further impact pain, mobility and thus social participation and day to day living activities. From the same group, Sampaio et al. look at the problem from the occupational therapist's perspective identifying the key areas both in the home and the work environment that the team can help to address to reduce social exclusion, increase independence and thus improve quality of life. Galvao-Castro et al. continue this theme reporting on the importance of integrated care for people living with HTLV-1 and the difficulties experienced despite 20 years of an HTLV service. The importance of dedicated multi-disciplinary services is also picked up by Federico et al. not just in regions were HTLVs are common but also in non-endemic areas where knowledge is less. The impression of HTLV being invisible in their region of Argentina will resonate with persons living with HTLV around the world. Turning full circle this brings us back to the advice that is given to patients with HTLV. Often the evidence base for recommendations is limited, such as for HTLV management during pregnancy where there are no guidelines. Barr et al. take a pragmatic approach to this suggesting a management algorithm which can be tested and refined but for now provides a basis for prevention of mother to child transmission multi-disciplinary team (MDT) discussions. Miura et al. published an insightful review on the complexities of the molecular mechanism behind HTLV-1 persistence, gene expression and its clinical implications.The care and management of persons with HTLV-1 infection is often complex and always multidisciplinary, starting with diagnosis. With the application of any diagnostic test comes the responsibility of the result, especially when a disease or the potential to develop disease is the diagnosed. Too often patients with HTLV-1/2 seroreactive results are sent away with anxiety and uncertainty. via milk was reviewed by Millen and Thoma-Kress who highlighted the need to develop innovative strategies that prevent vertical transmission but allow safe breastfeeding. In this regard, promising results of an in vitro study was published by Schneiderman et al. The group elegantly showed that cabotegravir, the long-acting integrase strand transfer inhibitor, potently inhibits HTLV-1 transmission and suggested that it could be used to prevent HTLV-1 transmission. Bradshaw and Taylor reviewed available information on the effects of antiretrovirals on HTLV-1. They focused their work in the context of pre-exposure prophylaxis for HIV and its potential impact on HTLV-1 transmission. The authors reinforced the importance of conducting more studies in this promising area, being in accordance with current recommendations from WHO.The mechanism of HTLV mother-to-child transmission Tu et al. Regarding HTLV treatment, Gutowska et al. analyzed the potential of pomalidomide (Pom) treatment to reduce HTLV-1 viral burden using a rhesus macaque model. The hypothesis was that by increasing cellular surface expression, Pom would increase the susceptibility of HTLV-1-infected cells to NK and CTL killing. Indeed, Pom treatment resulted in immune activation, increase in specific humoral response and in the frequency of detection of HTLV proviral DNA. However, authors highlighted that Pom may not be effective as a single-agent therapeutic to control HTLV-1 infection.The state of the art and perspectives for the development of both preventive and therapeutic vaccines for HTLV-1 infection was addressed in the work by Rosadas et al. highlighting the most relevant points from each presentation. Another interesting paper by Fowler and Einsiedel investigated the perception of patients and health personnel toward HTLV-1 in Australia, the geographical area with the highest prevalence of HTLV-1 in the world, affecting mainly the vulnerable aborigine population group and observed striking differences between community and healthcare workers. In a similar way, Martel and Gotuzzo, discussed and emphasized the importance of the sexual transmission of HTLV-1 and the relevance to the prevention and control of the virus. Miranda A. E. et al. prepared a novel approach to evaluate the public health policies in Brazil, using a methodology for strategic planning commonly used in business and economics. The SWOT  analysis, allows policy makers to shed light into the advances and the setbacks that need further attention. During the preparation of the present Research Topic, it became clear to the guest editors that we were facing a problem in regard to the name of the virus. Vallinoto et al. prepared a formal technical opinion to the HTLV academic community stressing the importance of using the formal nomenclature established by the International Committee on the Taxonomy of Viruses, ICTV, and the negative implications to refer to the virus other than Human T lymphotropic virus 1, in regard to its prevention and control, considering the present search systems commonly used in academic settings.As a consequence of the recent initiatives from the WHO to coordinate efforts to control and eliminate HTLV-1, there was a second webinar sponsored by HTLV Channel/PAHO/WHO/Brazilian Ministry of Health, to celebrate the 2021 HTLV World Day and discuss public health policies worldwide. The webinar was covered by a report from Prevention and control are key points to achieve the ultimate goal to eliminate the circulation of an infectious agent and its associated diseases. So far, few viruses were successfully eliminated. HTLV-1 is a candidate for elimination despite the nature of the persistent infection that results in some chronic diseases as strategies that could achieve this are known. The manuscripts published in the present issue are highly significant as they provide important information that can be easily applied to many geographical areas in the world. Prevalence studies are still needed to support a comprehensive international discussion that will determine adequate methodological approaches and define targets for the prevention and control of HTLV-1/2. The contributions fulfilled the majority of the suggested subjects of the Research Topic, but there is still a need to develop economic analysis focusing on strategies to prevent and control HTLV infection.We hope the data presented here will contribute to improve the quality of life of those living with HTLV and are applied halt further infections by HTLV-1/2.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.GT is supported by the Imperial NIHR Biomedical Research Centre. RI (#312979/2018-5), AV (#302935/2021-5), and LM (#314209/2021-2) are Research Grantees of the Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques. It has been shown that hMSCs have the potential to differentiate into different lineages, including osteoblasts (bone), neuroblasts , adipoblasts (fat), myoblasts (muscle), and chondroblasts (cartilage)4. In particular, osteogenic induction of hMSCs differentiation in conjunction with tissue engineering approaches has been exploited to provide an alternative method besides autologous bone graft to replace or restore damaged bone tissues5. This evidence demonstrates the application of hMSCs as a promising cell source in bone tissue engineering, given their regenerative properties. Although the differentiation capacity of hMSCs’ has been studied extensively, the mechanisms that control their plasticity, especially how hMSCs can be differentiated into osteoblasts and make bones, are hindered by the availability of technologies for detecting behaviors of cells with single-cell resolution. Despite conceptual advancements in stem cell differentiation, the cellular and molecular mechanisms that govern hMSCs during osteogenic differentiation remain poorly understood.Human mesenchymal stem cells (hMSCs) are adult stem cells isolated from adult tissues such as bone marrow and adipose tissue. hMSCs have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their multipotency and easy accessibility10. Many studies have demonstrated the critical role of Notch signaling in cell fate determination, cell proliferation, differentiation, and apoptosis in various cell types14. Notch signaling pathway is initiated when notch ligand binds to the notch receptor, leading to a cascade of events that eventually cleavage of Notch intracellular domain (NICD). Subsequently, the released NICD then translocates to the nucleus, which regulates transcription of target genes15. In mammalian cells, there are multiple ligands that are involved in Notch signaling, for example, Delta-like  and Jagged (Jag1 and Jag2). Among these five Notch ligands, Dll4 is one of the most notable ligands that are involved in development. In recent years, numerous studies have shown that notch signaling is required for stem cell differentiation, including myogenic differentiation16, neural stem cell17, intestinal stem cells18, hematopoietic stem cells19, and human embryonic stem cells20. For example, previous studies have shown that Notch signaling pathway promote murine mesenchymal stem cells (C3H10T12 cells) differentiation by enhancing BMP9 signaling21; Bagheri et al. showed that Notch signaling is active during osteogenic differentiation induced by pulsed electromagnetic fields; Wagley et al. have showed that canonical Notch signaling is required for BMP-mediated human osteoblast differentiation22. However, the fundamental mechanisms of how Notch signaling regulates osteogenic differentiation are unclear due to a lack of effective tools to detect gene expression dynamics at the single cell level. Current approaches for characterizing the regulatory role of Notch signaling in osteogenic differentiation are often limited because they are based on the fixation or physical isolation of cells to identify specific phenotypes. The tissue microenvironment plays an essential role in the regulation of cell functions, including migration, proliferation, and differentiation. Current available biosensing techniques, such RNA in situ hybridization23, single cell transcriptomics25, are limited to fixed cells and cannot track the dynamic profiling of gene expression at the single cell level. The dynamic behavior of cells is inherently lost when studied in isolation or fixed. Fluorescent protein tagging, such as MS2 (MS2 bacteriophage coat protein) and SunTag, coupled with single molecule imaging techniques are powerful strategies for monitoring dynamic gene expression in live cells28. Nevertheless, protein tagging techniques are limited by transfection efficiency, and the requirement of genetic modification to express engineered transcripts containing multiple tandem repeats of the binding sequence. Thus, dynamic monitoring of gene expression in individual cells in native and engineered tissue environments will uncover unrecognized features and regulatory processes of the cells, which will open opportunities to develop novel therapeutic approaches for tissue engineering and regenerative medicine. Previous studies have shown that Notch signaling has dual effects of induction and inhibition on stem cell osteoblastic differentiation which depends on cell differentiation stage and the time of Notch activation29. Therefore, it is essential to monitor the dynamic gene expression in single cells during osteogenic differentiation to elucidate the unrecognized characteristics and regulatory processes of the cells. Moreover, given its importance in stem cell proliferation and differentiation, a better understanding of Notch1-Dll4 signaling in both 2D and 3D microenvironments will provide new insights for stem cell therapy and tissue regeneration.Notch signaling is an evolutionarily conserved, intercellular, and contact-dependent signaling pathway which regulates cell fate decisions and plays a significant role during development and organogenesisTo address the unmet needs, we present an approach using a double-stranded locked nucleic acid (LNA)/DNA nanobiosensor for monitoring gene expression dynamics during osteogenic differentiation in 2D and 3D microenvironments. In this study, we demonstrated the usage of this LNA/DNA nanobiosensor to investigate the role of Notch signaling in regulating hMSCs osteogenic differentiation in 2D and 3D microenvironments. By incorporating this biosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of Dll4 mRNA expression profiles during the early stage of osteogenic differentiation. We further investigated the role of Notch1-Dll4 signaling in regulating osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. hMSCs differentiation efficiency and spheroid sizes were investigated and compared under different perturbations of Notch signaling. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Moreover, we identified that Dll4 is a molecular signature of hMSCs osteogenic differentiation. Inhibition of Notch1-Dll4 signaling reduced osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid size. Taken together, the capability of this LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis. Our findings regarding the role of Notch signaling in regulating osteogenesis will provide new insights to improve cell-based therapies and organ repair techniques.30. The LNA donor sequence is designed to be complementary to the target mRNA sequence. This LNA donor probe will spontaneously bind to the quencher sequence to form a LNA donor–quencher complex. The fluorophore at the 5′ end of the LNA donor is quenched by quencher sequence (Iowa Black Dark Quencher) due to its quenching ability31. The LNA donor–quencher complex is then transfected into hMSCs following manufacturers’ instructions. This LNA probe is designed to have a higher binding affinity with the target sequence. Once transfected into cells, in the presence of a target mRNA, the LNA probe is thermodynamically displaced from the quencher, to bind to specific target sequences. This displacement is due to larger difference in binding free energy between LNA probe to target mRNA versus LNA probe to quencher sequence. This displacement reaction permits the fluorophore to fluorescence, thus detecting the gene expression at single cell level , acquire lineage , and early lineage progression . Although previous gene expression studies have identified several critical regulatory signaling pathways that are involved in hMSCs early osteogenic differentiation, it is obscure how the gene expression dynamics relate to early osteogenic different progression. To examine the early osteogenic differentiation process, we monitored the transcription factor Dll4 mRNA expression dynamics using the LNA/DNA nanobiosensor. A random probe was included as a control  enzyme activity during hMSCs osteogenic differentiation using LNA/DNA nanobiosensor and ALP staining assay (Sigma Aldrich) Fig. . We next39. Jag1 peptide can activate Notch signaling by inhibiting the function of endogenous Jag1, a Notch ligand that has weak signaling capacity but competes with Dll440. hMSCs were treated with DAPT (20 μM) and Jag1 (20 μM) during osteogenic differentiation to examine the drug effects. A control group was designed without osteogenic induction. The osteogenic differentiation and Dll4 mRNA expression under different treatments were evaluated and compared. Osteogenic differentiation after induction was evaluated by quantifying ALP activity under different treatments. Our results showed pharmacological treatments with DAPT and Jag1 effectively induced changes in osteogenic differentiation  in the expression of Dll4 mRNA. Meanwhile, with the treatment of γ‐secretase inhibitor DAPT, a significant decrease of Dll4 mRNA (32.7%) was observed compared to the osteogenic induction group Fig. B. Jag1 t45. Moreover, it has been reported that the formation of 3D aggregates, or spheroids enhanced the regenerative capacity of hMSCs by promoting the secretion of proangiogenic and chemotoxic factors, and improved cell retention, and survival in preclinical studies46. Here, we investigated the effects of Notch1-Dll4 signaling in regulating osteogenic differentiation of 3D hMSCs spheroids. We first fabricated 3D self-organized spheroids of hMSCs using the hanging drop approach47. The self-organized 3D spheroids were collected and seeded in Matrigel  techniques, this method only requires a small number of cells without lysis. The LNA/DNA nanobiosensor can be designed in a short sequence to detect gene expression activities at a single-cell level. This ability enables us to correlate the Dll4 mRNA expression to the cell behaviors and to monitor the dynamic gene expression profiles during osteogenic differentiation. It is noted that our LNA/DNA nanobiosensor is applicable to other different cell types and tissue environments. In addition, this nanobiosensor is inert to nonspecific binding and stable in the cells for the duration of the experiment.49. This pathway plays a crucial role in lineage-specific differentiation of stem cells, and multiple stages of organismal development50. There are four Notch receptors  and five Notch ligands (Delta-like 1 (Dll1), Dll3, Dll4, Jag1, and Jag2). It has been reported that in recent years Notch signaling is involved in MSCs differentiation. For instance, Semenova et al. reported that Notch signaling regulates osteogenesis is dose-dependent, low-dose activation enhances osteogenic differentiation, while high-dose activation leads to apoptosis51. Cao et al. studied the involvement of Notch receptor Notch1 and Notch ligand Dll1 in osteogenic differentiation21. They observed that Notch1 inhibition reduced ALP activity during BMP-induced osteogenic differentiation of hMSCs in vitro. In contrast, it has been reported that inhibition of Notch signaling promotes adipogenic differentiation of MSCs52, indicating that Notch involvement is lineage-dependent during MSCs differentiation. Although numerous studies have demonstrated the involvement of Notch receptors or ligands, the involvement of Notch ligand Dll4 in osteogenic differentiation has not been explored. Here, we studied the involvement of Notch ligand Dll4 during hMSCs osteogenic differentiation in 2D and 3D microenvironment. By exploiting an LNA/DNA nanobiosensor, we examined the dynamic expression of Dll4 mRNA during osteogenic differentiation. Our results showed Dll4 mRNA is highly expressed in differentiated hMSCs and has a dynamic change at the early stage of differentiation , indicating that Dll4 mRNA is a molecular signature of differentiated hMSCs. In the absence of Notch inhibitor, hMSCs, cultured in osteogenic medium, differentiated towards the osteoblast phenotype, has high Dll4 expression. In conclusion, the results of this study add new information concerning osteogenic differentiation of hMSCs and the involvement of Notch1-Dll4 signaling. Further studies may elucidate the mechanisms underlying Notch1-Dll4 signaling in regulating osteogenesis.l-glutamine, and mesenchymal cell growth supplements. The hMSCs were originally isolated from normal (non-diabetic) adult human bone marrow withdrawn from bilateral punctures of the posterior iliac crests of normal volunteers. The hMSCs used in this paper are from three different purchases. The cells were cultured in a humidified incubator at 37 °C with 5% CO2 and passaged using 0.25% Trypsin–EDTA (Invitrogen). The cell culture medium was replaced every 3 days. hMSCs from passages 3–7 were used in the experiments. DAPT was purchased from Sigma Aldrich (DAPT ≥ 98% (HPLC), solid, D5942). To induce osteogenic differentiation, hMSCs were seeded at a density of 1 × 104 cells/mL with a volume of 500 μL in 12 well-plates. The basal medium was replaced with osteogenic induction medium after 2 days of cell seeding when the cells reached 80% confluency. Osteogenic induction medium were changed every 2 days. For studying Notch1-Dll4 signaling, hMSCs were treated with 20 μM γ-secretase inhibitor DAPT or 20 μM Jag1 peptide  after osteogenic induction. It is noted that both DAPT and Jag1 were added daily. Images were taken after 3 days, 5 days, 7 days, and 10 days of induction, respectively.Human mesenchymal stem cells (hMSCs) were acquired from Lonza and maintained in mesenchymal stem cell basal medium (MSCBMTM) with GA-1000, 47. A process flow illustrating the 3D hMSCs spheroids formation is shown in Fig. g to form a cell pellet. Then trypsin were aspirated and replaced with fresh medium with 20% methylcellulose  added. The final concentration of the cell solution is 4 × 105 cells/mL. Then 20 μL drops of the mixture were placed on the underside of a petri dish lid, with 5 mL of 1 × PBS inside the dish to prevent droplet evaporation. In our experiments, 75 droplets were seeded per lid. Spheroids were then incubated as hanging drops in the 37 °C, 5% CO2 incubator for 72 h. Next, the spheroids were harvested using open-end 1 mL tips to wash off the spheroids from the lid at a small amount of basal growth medium and keep the lid inclined to ensure the spheroids slipped over. Then all the spheroids were transferred into a pre-sterilized 1.5 mL microcentrifuge tube . As the spheroids are gathered in the bottom of the centrifuge tube, aspirate the medium as much as possible, then mix spheroids in growth factor reduced Matrigel . The 3D spheroids mixed in Matrigel were then placed in 24-well plates. For Dll4 mRNA detection, hMSCs were transfected with LNA/DNA probe before forming spheroids. For evaluating ALP activity in 3D spheroids, ALP staining was performed after 10 days of osteogenic induction.We fabricated 3D self-organized spheroids of hMSCs using hanging drop approach58. Briefly, the LNA probe was designed to be complementary to the target mRNA sequence with a 100% match. The binding affinity and specificity were optimized using mFold server and NCBI Basic Local Alignment Search Tool (BLAST) database. A quencher is a 10-base pair nucleotide sequence with LNA/DNA monomers that is complementary to the 5′ end of the LNA probe. An Iowa Black RQ was labeled at the 3′ end of the quencher probe. The Dll4 probe was designed based on target mRNA sequences, Supplementary Table An LNA probe is a 20-base pair nucleotide sequence with alternating LNA/DNA monomers. A fluorophore (6-FAM) was labeled at the 5′ end for fluorescence detection. The design process of LNA probes was reported previouslyTo prepare the LNA/DNA nanobiosensor, the LNA probe and quencher were initially prepared in 1 × Tris EDTA buffer at a concentration of 100 nM. The LNA probe and quencher were mixed and incubated at 95 °C for 5 min and cooled down to room temperature over the course of 2 h. To optimize quenching efficiency, the fluorescent probe and quencher probe were prepared in a number of different ratios to obtain high signal-to-noise ratio. The quenching efficiency was evaluated by measuring fluorescence intensity using a fluorescence microplate reader . The optimized LNA probe and quencher mixer can be stored in a refrigerator for up to 7 days. The prepared double-stranded LNA/DNA probe (100 nM) was transfected into hMSCs using Lipofectamine 2000 in opti-MEM from Invitrogen to determine the target gene expression. Gene expression was evaluated by measuring the fluorescence intensity of cells transfected with LNA/DNA probes.To quantify hMSCs osteogenic differentiation, cells were stained for alkaline phosphatase (ALP) using the alkaline phosphatase kit (Sigma-Aldrich) using a modified protocol. First, the staining solution were prepared by mixing fast red violet solution, naphthol AS-BI phosphate solution, and water at a ratio of 2:1:1. Next, hMSCs were fixed using 4% cold Paraformaldehyde (PFA) for 2 min which enabled the maintenance of the ALP activation. The staining solution was then added to fixed cells for 15 min under room temperature and protected from light. For nucleus staining, Hoechst 33,342 staining solution was prepared in 1 × PBS at 1:2000 dilution and added to cells for 15 min. The cells were then washed three times with 1 × PBS, 15 min each time, before taking images.t test in Excel (Microsoft). P < 0.05 was considered statistically significant.Images were captured using ZOE Fluorescent Cell Imager with an integrated digital camera (BIO-RAD) or Nikon TE 2000 with a Retiga R1 monochrome CCD Camera. All fluorescence images were taken with the same setting for comparison. Data collection and imaging analysis were performed using NIH ImageJ software. To quantify the fluorescence intensity of Dll4 mRNA and ALP, the mean intensity of each cell were first measured. The background noise were then subtracted. All the cells were quantified in the same field of view. The ALP differentiation efficiency were calculated as: differentiation efficiency = ALP expressed hMSCs/total number of hMSCs. An auto-threshold was used to identify the ALP expressed hMSCs. Experiments were repeated at least three times, and over 100 cells were quantified for each group. For 3D spheroids, at least 20 spheroids were analyzed for each group. Results were analyzed using independent, two-tailed Student Supplementary Information.