Hoxc8 at successive stages of mouse MN development and observed motor deficits. Our in vivo findings suggest that Hoxc8 is not only required to establish, but also maintain expression of several MN terminal differentiation markers. Data from in vitro generated MNs indicate Hoxc8 acts directly and is sufficient to induce expression of terminal differentiation genes. Our findings dovetail recent observations in Caenorhabditis elegans MNs, pointing toward an evolutionarily conserved role for Hox in neuronal terminal differentiation.Spinal motor neurons (MNs) constitute cellular substrates for several movement disorders. Although their early development has received much attention, how spinal MNs become and remain terminally differentiated is poorly understood. Here, we determined the transcriptome of mouse MNs located at the brachial domain of the spinal cord at embryonic and postnatal stages. We identified novel transcription factors (TFs) and terminal differentiation genes  with continuous expression in MNs. Interestingly, genes encoding homeodomain TFs , previously implicated in early MN development, continue to be expressed postnatally, suggesting later functions. To test this idea, we inactivated  Motor neurons (MNs) represent the main output of our central nervous system. They control both voluntary and involuntary movement and are cellular substrates for several degenerative disorders . Due to The terminal differentiation features of every neuron type are determined by the expression of specific sets of proteins, such as NT biosynthesis components, NT receptors, ion channels, neuropeptides, signaling molecules, transmembrane receptors, and adhesion molecules . The genTo elucidate the molecular mechanisms that enable spinal MNs to acquire and maintain their terminal differentiation features, we took advantage of the orderly anatomical relationship between MN cell body location and muscle innervation, referred to as ‘topography’ . In the Caenorhabditis elegans identified Hox proteins as critical regulators of cholinergic MN terminal differentiation  of the nematode ntiation . Among tectivity . Here, wl stages , similarMnx1-GFP (green fluorescent protein) reporter mouse   and postnatal day 8 (p8). We chose e12 because: (i) spinal e12 MNs begin to acquire their terminal differentiation features, such as NT phenotype , and ii. We choser mouse  as it prer mouse . Due to Cre mice  with thedTomato) . At p8, dTomato) . Taking Ai9 mice . ThroughAi9 mice , suggestnal cord .Subsequent gene ontology (GO) analysis on proteins from embryonically enriched (e12) transcripts revealed an overrepresentation of molecules associated with neuronal development, such as regionalization, dendrite formation, and axon guidance . NotablySlc10a4, Nrg1, Nyap2, Sncg, Ngfr, Glra2, Cldn1, Cacna1g) and evaluated their expression at different life stages. Through RNA ISH, we found six genes  with continuous expression in putative brachial MNs at embryonic (e12) and early postnatal (p8) stages  stages . Availab) stages . The venSlc10a4, Nrg1, Nyap2, Sncg, Ngfr, Glra2) in brachial MNs. Their embryonic initiation and maintenance could be controlled by separate mechanisms involving distinct combinations of TFs solely dedicated to either initiation or maintenance. Alternatively, initiation and maintenance can be achieved through the activity of the same, continuously expressed TF (or combinations thereof). Recent invertebrate and vertebrate studies on various neuron types support the latter mechanism   known to be expressed in brachial MNs at embryonic stages  RNA-Seq dataset continue to be expressed at postnatal stages . We init, Hoxc8) . Through, Hoxc8) , Table 2c stages , confirmc stages . Moreoveand Lhx3 .Irx1, Irx2, Irx3, Irx5, Irx6, Creb5, Esrrg, Neurod6, Arid5b, Pou3f3, MafB, Zfhx4, Elk3, Epas1, Heyl] and 7 TFs from the p8 dataset ). We detected persistent expression for 14 of these TFs in the embryonic (e12) and early postnatal (p8) brachial spinal cord. Expression was evident at the ventrolateral region, which is populated by MNs , is persistent at early postnatal stages (p8). Moreover, we identified 14 novel TFs from different families with expression in embryonic and postnatal (p8) brachial MNs , Table 2Hoxc4, Hoxa5, Hoxc5, Hoxa6, Hoxc6, Hoxa7, Hoxc8) are continuously expressed - from embryonic to postnatal stages - in brachial MNs  in nerve cord MNs   . Hoxc8 ihial MNs . HoweverHoxc8 gene activity in brachial MNs. Because Hoxc8 is also expressed in other spinal neurons  is unaffected in these animals at e12   summarized in Neuregulin 1 (Nrg1), a molecule required for neuromuscular synapse maintenance and neurotransmission  biosynthesis proteins 7[ChT1]) . Consist dataset . Howeverrly mice . Next, wsmission , is redurly mice , likely  animals , promptiHoxc8 MNΔ early::Mnx1-GFP and control mice at e12 . We found dozens of significantly (p<0.05) upregulated (55) and downregulated (84) transcripts in MNs lacking Hoxc8   and evaluated their expression with RNA ISH in brachial MNs at different stages. Five of these genes  are continuously expressed in brachial MNs at embryonic and postnatal stages  but not postnatal brachial MNs, and this embryonic expression depends on Hoxc8   and Hoxc8 itself . GO anal8 itself . We focu8 itself . We selel stages . Importaerfamily , and Papelopment , is redurly mice . SimilarH method . In addion Hoxc8 . Becauseguidance , its deprly mice .Slc10a4, Nrg1, Nyap2, Sncg, Ngfr, Glra2, Slc44a5, Mcam, Pappa, Pex14, Tagln2), 3 of which  constitute Hoxc8 targets  in brachial MNs. However, the persistent expression of Hoxc8 both in embryonic and early postnatal MNs raises the intriguing possibility of a continuous requirement  we found affected in Hoxc8 MNΔ early mice is also reduced in brachial MNs of Hoxc8 MNΔ late mice at p8   at embryonic stages (Ret and Sema5a at e12 and Glra2 at p8), we also found that Hoxc8 is continuously required (both at e12 and p8) for expression of several terminal differentiation genes  (In the context of function . That is (Glra2)  is affecce at p8 . No effee at e12 , indicatguidance , is seles at e12 , but remls at p8 , suggestc stages , Table 1, Pappa)  and 4C.Altogether, these findings suggest that, in brachial MNs, Hoxc8 modifies the suite of its target genes at different developmental stages . In DiscHoxc8 expression was induced with doxycycline  and new  Hoxc8 target genes  datasets on MNs derived from mouse embryonic stem cells (ESC), in which ycycline . Our RNAet genes . Moreovese genes , suggestate mice  suggest Nrg1, Mcam) we identified in vivo are upregulated in iHoxc8 ESC-derived MNs  in brachial MNs  and new  Hoxc8 target genes  ani defects , and (c)hial MNs . Supporthial MNs , respectet genes .Hoxc8 MNΔ early and Hoxc8 MNΔ late animals by evaluating their motor coordination   . Although we cannot exclude the possibility that the terminal differentiation defects of Hoxc8 MNΔearly mice are a consequence of their early MN specification defects, this is unlikely as Hoxc8 binds directly to the cis-regulatory region of terminal differentiation genes   .Hoxc4, Hoxa5, Hoxc5, Hoxa6, Hoxc6, Hoxa7, Hoxc8) is maintained from embryonic to early postnatal stages  (Hoxd9) and lumbar  MNs  . Our anaa11) MNs . We furta11) MNs . While ta11) MNs , their cHoxc8, identified its target genes, and uncovered a continuous requirement for Hoxc8 in the establishment and maintenance of select MN terminal differentiation features. Our findings dovetail recent Hox studies in the C. elegans nervous system    and 4F, Hoxc8 knock-out or Hoxc8 MNΔ early mice reported aberrant connectivity of forelimb muscles  within the LMC . We found that all these molecules are expressed continuously in embryonic and postnatal (p8) brachial MNs. By removing Hoxc8 gene activity either at an early (Hoxc8 MNΔ early mice) or late (Hoxc8 MNΔ late mice) developmental stage, we uncovered a continuous Hoxc8 requirement for the initial expression and maintenance of Mcam, Pappa, and Nrg1. Intriguingly, we also found evidence for temporal modularity in Hoxc8 function, that is, the suite of Hoxc8 targets in brachial MNs is partially modified at different developmental stages. Two lines of evidence support this notion: (a) expression of the terminal differentiation gene Glra2 is only affected in Hoxc8 MNΔ late mice, indicating a selective Hoxc8 requirement for Glra2 maintenance in MNs, and (b) expression of two axon guidance molecule  is only affected in MNs of Hoxc8 MNΔ early mice.In this study, we propose that Hoxc8 controls select features of brachial MN terminal differentiation, such as the expression of the glycine receptor subunit , as well as axon guidance molecules, such as Ret (Sema5a (this study) in order to ensure proper MN-muscle connectivity. Consistent with this idea, similar axon guidance defects occur in Hoxc8 and Ret mutant mice   . Althougtiation) . We surmDrosophila ventral nerve cord  is required to maintain neuromuscular synapses  and posterior (egl-5) Hox genes control the expression levels of the LIM homeodomain protein MEC-3, which in turn controls touch receptor terminal differentiation  and posterior (egl-5) Hox genes control distinct terminal differentiation features of midbody and posterior MNs, respectively  and is required for their maintained expression in MNs during postembryonic stages  to be continuously expressed in brachial MNs, potentially acting as Hoxc8 collaborators. We made similar observations in thoracic (Hoxd9) and lumbar  MNs  is evolutionarily conserved from invertebrates to mammals. Similar to a11) MNs . Moreovea11) MNs . To dateC. elegans support the idea that continuously expressed TFs  establish during development and maintain throughout postembryonic life the identity and function of individual neuron types by activating the expression of terminal differentiation genes  (Numerous genetic studies in the nematode lecules) . Multipllecules) . Howeverlecules) .Hoxc8 in the control of MN terminal differentiation. First, Hoxc8 is expressed continuously, from embryonic to early postnatal stages, in brachial MNs. Second, our in vivo data and in vitro analysis suggest Hoxc8 is both necessary and sufficient for the expression of several of its target genes in MNs - such mode of action is reminiscent of terminal selectors , which is required for early induction of genes necessary for ACh biosynthesis in mouse spinal MNs and the in vitro generation of MNs from human pluripotent stem cells  with continuous expression in brachial MNs  of the University of Chicago (Protocol No. 72463). The generation of d/floxed , Olig2CrOlig2Cre , Mnx1-GFMnx1-GFP , ChAT-IRIRES-Cre , and Ai9Cre . All samples had high quality scores between 9 and 10 RIN. After cDNA library preparation, RNA-Seq was performed using an Illumina HiSeq 4000 sequencer .For the analysis shown in http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Unique reads were aligned into the mouse genome (GRCm38/mm10) using the HISET2 alignment program https://usegalaxy.org). The heatmaps were generated using the Morpheus program developed by the Broad Institute (https://software.broadinstitute.org/morpheus). Gene hierarchical clustering was performed using a Pearson’s correlation calculation.Raw sequence data were subjected to quality control using the FastQC algorithm  and embedded in optimal cutting temperature compound. Cryosections were generated and processed for ISH or immunohistochemistry as previously described .2O2 solution and permeabilized in PBS/0.1% Triton-X100. Upon hybridization with DIG-labeled RNA probe overnight at 72°C and washes in SSC, the anti-DIG antibody conjugated with peroxidase (Roche) and primary antibody against Foxp1  were applied overnight (4 °C) to the sections. The next day, the sections were incubated with the secondary antibody , and detection of RNA was performed using a Cy3 Tyramide Amplification system (Perkin Elmer). Images were obtained with a high-power fluorescent microscope (Zeiss Imager V2) and analyzed with Fiji software . Embryonic (1381 out of 2904) and postnatal (1348 out of 2699) MN genes were categorized into protein classes using the algorithms built into Panther  and seven (Cre::Hoxc8fl/flOlig2) mice were used at the age of 4–5 months. For the Hoxc8 MNΔ late analysis, 8 control (fl/flHoxc8) and 10 (IRESCre::Hoxc8fl/flChat) mice were used at the age of 2–5 months. A computer-controlled rotarod apparatus  with a rat rod (7-cm diameter) was set to accelerate from 4 to 40 revolutions per minute (rpm) over 300 s, and recorded time to fall. Mice received five consecutive trials per session, one session per day .Female mice were trained on an accelerating rotarod for 5 days. The experimenter was blind to the genotypes. For the Hoxc8 MNΔ early analysis, seven control (fl/flHoxc8) and seven (Cre::Hoxc8fl/flOlig2) mice were used at the age of 4–5 months. For the Hoxc8 MNΔ late analysis, 8 control (fl/flHoxc8) and 10 (IRESCre::Hoxc8fl/flChat) mice were used at the age of 2–5 months. We followed the manufacturer’s protocol. In brief, the meter was positioned horizontally on a heavy metal shelf (provided by the manufacturer), assembled with a grip grid. Mice were held by the tail and lowered toward the apparatus. The mice were allowed to grasp the metal grid only with their forelimbs and were then pulled backward in the horizontal plane. The maximum force of grip was measured, and we used the average of six measurements for analysis. Force was measured in Newton and Grams. The experimenter was blind to the genotypes.The forelimb strength of female mice was measured using a grip strength meter from Bioseb (model BIO-GS3). For the Hoxc8 MNΔ early analysis, seven control (fl/flHoxc8) and seven (Cre::Hoxc8fl/fl flOlig2) mice at the age of 4–5 months were placed onto the walking compartment. The treadmill was turned on at a speed of 15 cm/s. For the Hoxc8 MNΔ late analysis, 8 control (fl/flHoxc8) and 10 (IRESCre::Hoxc8fl/flChat) mice at the age of 2–5 months were placed onto the walking compartment. The treadmill test was conducted at two different speeds (15 cm/s and 25 cm/s). The 30-s long videos were obtained for each mouse. Videos were analyzed and data were binned into four categories based on the duration of each mouse’s stay on the treadmill .The treadmill test was conducted on female mice by using the DigiGait system , which is equipped with a motorized transparent treadmill belt and a high-speed digital camera that provides images of the ventral side of the mouse . For thet-test (two-tailed). Differences with p<0.05 were considered significant. For the rotarod performance test, two-way ANOVA was performed (Prism Software). For the treadmill experiment, we used Fisher’s exact test.For data quantification, graphs show values expressed as mean ± SEM. With the exception of the rotarod and treadmill experiments, all other statistical analyses were performed using the unpaired  C. elegans, and thus pointing towards this type of gene regulation as important in building diverse nervous systems.This manuscript will be of interest to developmental geneticists interested in neuroscience, and how spinal motor neurons maintain their unique identities in adulthood after fate decisions are made in the embryo. The work here demonstrates that a Hox transcription factor acts as a terminal selector to control motor neuron identity, thus mirroring recent studies in  public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Piali Sengupta as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Aaron D Gitler (Reviewer #2).Thank you for submitting your article "Control of spinal motor neuron terminal differentiation through sustained Hoxc8 gene activity" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers are in agreement that this is a very interesting study, addressing a fundamental topic in developmental genetics, with important implications for the field. Nonetheless, the reviewers have coalesced around several items that need to be addressed before we can consider this paper for publication.1. The behavioral data is potentially important to this study, but not robustly performed. The N numbers need to be increased and additional behavioral tests should be added. Moreover, deleting Hoxc8 expression earlier in these studies, as was done for other experiments, would be useful.2. The quality of many of the RNA in situ experiments was not good, making it hard for reviewers to easily assess the data. The authors should repeat the in situs where necessary or even consider newer, more quantitative RNA in situ approaches.3. Authors should clarify their conclusions about the role of Hoxc8 in brachial motor neuron differentiation because at least from the data presented it does not seem to have a very significant role, and the authors should consider the potential involvement of other collaborating factors.4. Authors should clarify the potential caveats by using two different methods for labeling MNs at the two time points and possibly coming up with a better method or way to normalize the data.Reviewer #1 (Recommendations for the authors):1. My major concern with this paper is the lack of specificity for the findings and ease in building a cohesive story. The paper is largely centered around RNAseq and RNA in situ . I appreciate the large amount of effort put into this study, but it would be nice to know how many of these gene regulatory networks are direct (ie. TF binding to consensus motifs in promoter regions). I was unable to follow a clear logic as to why certain genes were chosen for follow up, or to focus large parts of the story on.2. In regards to Figure 1, how specific are HB9::GFP and Chat::Ai9 to motor neurons, or neurons in general. Authors should co-stain for other neuronal and glial markers. Also, why not use a reporter from one of the identified terminal differentiation factors which is on early and late, so that a single reporter is used?3. The RNA in situs in Figures3A, 4E, 5B and some of the supplemental images are not of the highest quality and one has to read the manuscript to know what to conclude. Perhaps newer approaches like RNAscope, or similar probes, would be more quantitative and easier to interpret. Also some images have white dotted circles around them cells of interest and other images do not – please be consistent.4. Although sympathetic to the need to use the Hoxc8 late mice for behavioral experiments due to perinatal lethality with the Hoxc8 early mice, these experiments demonstrate there is no phenotype and the author's assertion that mutant mice "tend" to perform worse is an unfair stretch. Moreover, with an N number of 3 and 5 mice on some of the experiments, its hard to make conclusions. The authors should consider consulting with or collaborating with a more behavior focused lab to do these experiments in a more robust manner, adding more tests and mice, or consider leaving the behavior experiments out of this paper.Reviewer #2 (Recommendations for the authors):We have the following comments and suggestions for the authors to consider:1) Hox factors are known to control motor neuron subtype/pool specification . It would be helpful to know if Hoxc8 early or late deletions change motor neuron subtype/pool identity rather than their maturation, which could be the reason for the differential gene expression This could be confirmed with co-labeling of reporter and subtype/pool markers, as in the Irx experiments.2) Based on Figure 4F, the authors seem to distinguish terminal differentiation genes  from axon guidance molecules and transcription factors. However, there are times when these seem to be treated interchangeably (e.g. discussion of Sema5a and Ret on page 12). The authors should more clearly and consistently state what they consider terminal differentiation markers in the text.3) It would be worthwhile to have co-labeling with reporter (Hb9-GFP vs ChAT-tdTom) for at least some of the selected genes  to see if there is specific reduction in the cells of interest rather than just through correlation based on spatial pattern.4) The Irx2 knockout experiments suggest that this downstream target of Hoxc8 may be involved in specification of limb-generating motor neurons, but not in the expression of terminal differentiation markers regulated by Hoxc8. This result seems tangential to the paper as 1) the knockouts are performed very early (prior to cell specification) and therefore does not provide any additional information about how Hoxc8 regulates motor neuron development past specification stage and 2) do not a clear functional link between Hoxc8 and terminal differentiation regulation.C. elegans Irx2 (fosmid) experiments add to this paper. The loss of MNs entirely seems to detract from the overall point of this paper, which is that Hoxc8 helps to establish/maintain brachial motor neuron identity/maturation.5) It is unclear what the 6) It is unclear what the behavioral experiments add to the paper since would be very difficult to attribute any phenotype to a specific cause. Also, unclear why the experiments were performed only in late Hoxc8 deletion and not the early deletion as well.7) The quality of some of the in situs should be improved 8) It might be of interest to overexpress Hoxc8 in other regions of the spinal cord  using chick electroporation for example to see if it can lead to overexpression of some of these terminal differentiation genes. This gain-of-function experiment may lend additional support to the proposal that Hoxc8 is required to maintain expression of these genes, although I am not sure that this is a requirement for distinguishing terminal selectors.Reviewer #3 (Recommendations for the authors):1. The authors used two different genetic systems to label brachial MNs at embryonic day 12 (e12) and postnatal day 8 (p8) as it was not possible for them to label MNs with just a single genetic system. Using these reagents they conclude that Hoxc8 regulates some of the same and some different targets. But can they rule out that some of these results is a consequence of using two different labeling systems? Are they certain that the cells labeled at both time points are the same cells? Perhaps using a lineage tracing tool and/or normalizing with some of the genes they discover in their RNA-seq experiments may be a way add confidence in the similarities and differences that they find in the RNAseq datasets are accurate.2. Along the same lines, the RNA ISH of Hoxc8 at e12 looks dense, suggesting that all MNs in LMC and MMC regions express Hoxc8. On the other hand, expression of Hoxc8 seems sparse at p8, suggesting few Hoxc8+ MNs at p8 compared to e12 stage. Performing dual RNA ISH with Hoxc8 and endogenous genes of interest would provide greater confidence that the same number of Hoxc8+ MNs are present at two different time points.3. The authors state that Hoxc8 MN late mutants perform worse in rotarod performance test and forelimb grip strength in 3-month old mice. However, the statistical analysis suggests there is no significant change. It was unclear why the authors chose such a late time point for these assays, when the mice potentially have time to compensate for a compromised motor system. Redoing this experiment at earlier time points, such as with 1 month old mice (there is precedence for this in the literature), might reveal significant differences. Essential revisions:The reviewers are in agreement that this is a very interesting study, addressing a fundamental topic in developmental genetics, with important implications for the field. Nonetheless, the reviewers have coalesced around several items that need to be addressed before we can consider this paper for publication.1. The behavioral data is potentially important to this study, but not robustly performed. The N numbers need to be increased and additional behavioral tests should be added. Moreover, deleting Hoxc8 expression earlier in these studies, as was done for other experiments, would be useful.earlyHoxc8 MNΔ mice in which Olig2-Cre is active in motor neuron (MN) progenitors, and 2) lateHoxc8 MNΔ mice in which ChAT-IRES-Cre is active in post-mitotic MNs.We completely agree. In the revised manuscript, we have included new data on three behavioral tests . We increased the number of animals (N) and also included in the analysis two sets of mice with conditional Hoxc8 manipulations: 1) We have added the following paragraph in Results that summarizes our findings:“Hoxc8 gene activity is necessary for brachial motor neuron functionearly and Hoxc8 MNΔ late animals by evaluating their motor coordination , forelimb grip strength  and treadmill performance. No defects were observed in Hoxc8 MNΔ early and Hoxc8 MNΔ late mice during the rotarod performance test , suggesting balance and motor coordination are normal in these animals. Next, we evaluated forelimb grip strength because brachial MNs innervate forelimb muscles. We found a statistically significant defect in Hoxc8 MNΔearly mice, but not in Hoxc8 MNΔlate mice . Lastly, we tested these animals for their ability to run on a treadmill for a period of 30 seconds (sec). At a low speed (15 cm/sec), we observed statistically significant defects in Hoxc8 MNΔearly mice. That is, 64.28% of Hoxc8 MNΔearly mice fell off the treadmill in the first 5 sec of the trial compared to 28.57% of control mice (p = 0.0108) . Moreover, 0% of Hoxc8 MNΔearly mice were able to stay longer than 20 sec on the treadmill compared to 42.85% of control mice . On the other hand, statistically significant defects were observed in Hoxc8 MNΔlate mice only when the treadmill speed was increased to 25 cm/sec . That is, 43.33% of Hoxc8 MNΔ late mice fell off the treadmill in the first 5 sec of the trial compared to 17.39% of control mice (p = 0.0461) . Together, these data show that Hoxc8 MNΔlate mice display a milder behavioral phenotype compared to Hoxc8 MNΔearly mice. This is likely due to the fact that Hoxc8 MNΔearly mice display a composite phenotype i.e., defects in early MN specification and axon guidance  combined with terminal differentiation defects [this study], whereas the Hoxc8 MNΔlate mice only display terminal differentiation defects (this study). Although we cannot exclude the possibility that the terminal differentiation defects of MNΔearly mice are a consequence of their early MN specification defects, this is unlikely as Hoxc8 binds directly to the cis-regulatory region of terminal differentiation genes  .”We next sought to assess any potential behavioral defects in adult Hoxc8 MNΔ 2. The quality of many of the RNA in situ experiments was not good, making it hard for reviewers to easily assess the data. The authors should repeat the in situs where necessary or even consider newer, more quantitative RNA in situ approaches.Pappa, Glra2, Sema5a) we identified in vivo .We conducted new RNA fluorescent ISH (FISH) experiments coupled with a MN-specific marker (Foxp1). These new data corroborate our original conclusion: Hoxc8 controls the expression of several target genes in brachial MNs . To complement these in vivo findings, we now provide evidence in in vitro generated MNs that Hoxc8 is sufficient to induce the expression of the same target genes , likely due to the existence of additional factors that partially compensate for loss of Hoxc8 gene activity.”“We found that expression of Neuregulin 1 (Nrg1), a molecule required for neuromuscular synapse maintenance and neurotransmission, is reduced (but not eliminated) in e12 brachial MNs of Hoxc8 MNΔ “Importantly, not all Hoxc8 target genes  we identified in vivo are upregulated in iHoxc8 ESC-derived MNs . This is likely due to the lack of Hoxc8 collaborating factors in these in vitro generated MNs. A putative collaborator is Hoxc6 because (a) Hoxc6 and Hoxc8 are co-expressed in embryonic brachial MNs , (b) animals lacking either Hoxc6 or Hoxc8 in brachial MNs display similar axon guidance defects , and (c) Hoxc6 and Hoxc8 control the expression of the same axon guidance molecule (Ret) in brachial MNs . Supporting the notion of collaboration, our analysis of available ChIP-seq data for Hoxc6 and Hoxc8 from iHoxc6 and iHoxc8 ESC-derived MNs, respectively, showed that these Hox proteins bind directly on the cis-regulatory region of previously known  and new  Hoxc8 target genes .”In Discussion:“In the spinal cord, we found several other Hox genes  to be continuously expressed in brachial MNs, potentially acting as Hoxc8 collaborators.”“This residual expression indicates that additional TFs are necessary to control brachial MN terminal differentiation. As mentioned in Results, one such factor is Hoxc6, which is co-expressed with Hoxc8 in brachial MNs during embryonic and postnatal stages  . Importantly, Hoxc6 and Hoxc8 bind directly on the cis-regulatory regions of the same terminal differentiation genes in the context of mouse ESC-derived MNs . Another putative Hoxc8 collaborator is the LIM homeodomain protein Islet1 (Isl1), which is required for early induction of genes necessary for ACh biosynthesis in mouse spinal MNs and the in vitro generation of MNs from human pluripotent stem cells.”4. Authors should clarify the potential caveats by using two different methods for labeling MNs at the two time points and possibly coming up with a better method or way to normalize the data.We completely agree. The potential caveat is that our FACS-sorted cells also include a minority of cells that are not motor neurons. We acknowledge this possibility in Results:“Two factors that could contribute to these transcriptional differences between the e12 and p8 datasets are: (1) different levels of gene expression (see next Section), and (2) a small fraction of the FACS-sorted cells are not MNs. Indeed, Hb9 (Mnx1) and ChAT, in addition to MNs, are also expressed in small, non-overlapping neuronal populations in the spinal cord  .”However, we believe this caveat is not of a major concern for the following reasons:(a) The end goal of using these two different methods for labeling brachial MNs was to identify genes  that are expressed continuously in these cells. Our extensive validation of RNA-Seq results with RNA ISH suggests we met this goal .(b) As we mention in our reply to Reviewer 1 (please see R1 – Response 2), we conducted additional experiments to test the specificity of our genetic labeling approach .(c) As we mention in Results, the RNA-Seq of genetically labeled brachial MNs was: (a) sensitive because it identified genes expressed in subtypes of brachial MNs , and (b) region-specific because it identified specific Hox genes, known to be expressed only in brachial MNsReviewer #1 (Recommendations for the authors):1. My major concern with this paper is the lack of specificity for the findings and ease in building a cohesive story. The paper is largely centered around RNAseq and RNA in situ . I appreciate the large amount of effort put into this study, but it would be nice to know how many of these gene regulatory networks are direct (ie. TF binding to consensus motifs in promoter regions). I was unable to follow a clear logic as to why certain genes were chosen for follow up, or to focus large parts of the story on.We regret that in some parts the original version of the manuscript lacked cohesion. To address this, we have now made significant changes in Results and also removed all data on Irx transcription factors, as suggested by Reviewers 1 and 2. We hope the revised manuscript is now more focused and communicates effectively its main conclusion, i.e., sustained Hoxc8 activity is necessary for brachial motor neuron terminal differentiation.To address the comment on lack of specificity, we have now performed RNA fluorescent in situ hybridization (FISH) coupled with a motor neuron-specific marker (FoxP1). These new findings  corroborate the initial results reported in this paper.Pappa, Glra2) we found downregulated in brachial motor neurons of earlyHoxc8 MNΔ  and lateHoxc8 MNΔ  mice are upregulated in motor neurons derived from ES cells (ESC-MNs), in which Hoxc8 expression is induced  . Lastly, we found that Hoxc8 binds directly at the cis-regulatory region of the terminal differentiation genes  we identified in vivo, and is sufficient to induce their expression in the context of ESC-MNs .Motivated by the reviewer’s comment to identify direct Hoxc8 target genes, we analyzed recently published RNA-Seq and ChIP-seq datasets from motor neurons derived from ES cells (ESC-MNs) in which Hoxc8 expression can be induced upon doxycycline treatment . Our analysis showed that the same Hoxc8 target genes . However, we cannot exclude the possibility that a very small population of the HB9::GFP+ cells we isolated at e12.5 are spinal interneurons. The original paper  describing the generation of HB9::GFP mice notes: “GFP expression was found in motor neuron cell bodies, dendrites, and axons in HB9::GFP embryos. In this line a very low level of expression (10- to 20-fold lower than in motor neurons) was detected in DRG neurons and a subset of ventral interneurons at e10.5, but not later”.To our knowledge, the HB9::GFP line – mature oligodendrocytes are not present at e12.5 in the spinal cord, as the peak of myelination in mice occurs at postnatal day 21 (p21). Moreover, the aforementioned studies  have not reported any HB9::GFP expression in glia.It is also unlikely that we isolated any glia at e12.5 with the ChAT::IRES::Cre; Ai9 [Rosa26-CAGpromoter-loxP-STOP-loxP-tdTomato] mouse line. Double immunofluorescence staining using antibodies against ChAT (cholinergic motor neuron marker) and tdTomato (expressed in cells in which Cre is/was active) revealed robust co-localization of ChAT with tdTomato in most  spinal motor neurons .Prompted by the reviewer’s suggestion, we conducted additional experiments to evaluate the specificity of labeling spinal motor neurons at p8 with the ChAT::IRES::Cre line reported similar observations and suggested that this is likely due to earlier Cre expression in the lineage of these tdTomato positive cells (PMID: 33931636).We also observed sparse tdTomato expression, but not ChAT, in a few cells (5-10 cells per section) located more dorsally and medially in the spinal cord, which are not motor neurons . A recent study that used the same ChAT::IRES::Cre; Ai9 spinal cords at p8 . We also attempted to stain for a microglia marker CD11b , but we did not detect any staining likely due to technical reasons. Altogether, these new data suggest that the tdTomato-expressing cells we isolated at p8 by FACS are mostly spinal motor neurons. Please, see also ER- Response 4 (page 3 of this document).At p8, there are many astrocytes present in the spinal cord but few mature oligodendrocytes (myelination peaks at p21). We therefore stained against the astrocyte marker GFAP  and found no colocalization with tdTomato-expressing cells in Also, why not use a reporter from one of the identified terminal differentiation factors which is on early and late, so that a single reporter is used?This is a great suggestion but it would require the generation and characterization of new mouse lines, as well as redoing the RNA-Seq experiments at e12 and p8. Such endeavor will take 4-5 years to complete judging from the amount of time it took to complete the current study. However, we are planning on generating in the future new reporter/Cre lines based on the terminal differentiation markers identified from this study.3. The RNA in situs in Figures3A, 4E, 5B and some of the supplemental images are not of the highest quality and one has to read the manuscript to know what to conclude. Perhaps newer approaches like RNAscope, or similar probes, would be more quantitative and easier to interpret. Also some images have white dotted circles around them cells of interest and other images do not – please be consistent.Pappa, Glra2, Sema5a) we identified in vivo . Lastly, we are now consistent with the white dotted circles. We only use them when we show Hoxc8 antibody staining or Hoxc8 RNA ISH data because Hoxc8 is also expressed in other cells of the spinal cord. We decided against using white dotted circles in all figures in order to be minimally invasive and avoid excessive drawing on top of our microscopy images. There is, however, a spinal cord schematic next to each image to orient the reader on what part of the spinal cord is actually shown.We conducted new RNA fluorescent ISH (FISH) experiments coupled with a MN-specific marker (Foxp1). These new data corroborate our original conclusion: Hoxc8 controls the expression of several target genes in brachial MNs . To complement these in vivo findings, we now provide evidence in in vitro generated MNs that Hoxc8 is sufficient to induce the expression of the same target genes . Prompted by Reviewers 1 and 3, we have added a new test  on both earlyHoxc8 MNΔ and lateHoxc8 MNΔ mice. Please see our detailed answer in ER – Response 1 (page 1 of this document).Thank you for this excellent suggestion. By expanding our mouse colony, we have now conducted behavioral experiments both on Reviewer #2 (Recommendations for the authors):We have the following comments and suggestions for the authors to consider:1) Hox factors are known to control motor neuron subtype/pool specification . It would be helpful to know if Hoxc8 early or late deletions change motor neuron subtype/pool identity rather than their maturation, which could be the reason for the differential gene expression This could be confirmed with co-labeling of reporter and subtype/pool markers, as in the Irx experiments.This is an excellent point that we now clarified by text changes and new experiments.earlyHoxc8 MNΔ mice. However, the same study found that the expression of two motor neuron pool markers  at e12.5 is partially affected, suggesting defects in motor neuron pool specification when Hoxc8 is deleted early. In the current manuscript, we report that brachial motor neurons are generated in normal numbers in earlyHoxc8 MNΔ mice , but the expression of terminal differentiation genes  is affected in these mice . Moreover, Hoxc8 binds directly on the cis-regulatory region of these genes .A previous study  found that the overall organization of brachial MNs into columns is normal in lateHoxc8 MNΔ mice, we attempted to stain brachial motor neurons with the motor neuron pool markers  at e14.5 because at 14.5 we detect robust Hoxc8 depletion in these mice . However, these markers are transiently expressed and become downregulated in wildtype samples after e12.5. This prevented us from evaluating their expression at e14.5 in lateHoxc8 MNΔ spinal cords. Taken together, our analyses show that the expression of several terminal differentiation markers  is affected in brachial MNs of lateHoxc8 MNΔ mice, which could explain their milder behavioral phenotype when compared to the earlyHoxc8 MNΔ mice .For the late mice display a milder behavioral phenotype compared to Hoxc8 MNΔearly mice. This is likely due to the fact that Hoxc8 MNΔearly mice display a composite phenotype  combined with terminal differentiation defects [this study], whereas the Hoxc8 MNΔlate mice only display terminal differentiation defects (this study). Although we cannot exclude the possibility that the terminal differentiation defects of MNΔearly mice are a consequence of their early MN specification defects, this is unlikely as Hoxc8 binds directly to the cis-regulatory region of terminal differentiation genes  .”In Results, we mention: “Together, these data show that Hoxc8 MNΔ2) Based on Figure 4F, the authors seem to distinguish terminal differentiation genes  from axon guidance molecules and transcription factors. However, there are times when these seem to be treated interchangeably (e.g. discussion of Sema5a and Ret on page 12). The authors should more clearly and consistently state what they consider terminal differentiation markers in the text.Thanks, we recognize the confusion and have modified the text accordingly in several occasions:“Two simple, but not mutually exclusive mechanisms can be envisioned for the continuous expression of terminal differentiation genes  in brachial MNs.”“Lastly, Hoxc8 can only activate expression of Sema5a (member of Semaphorin family) at embryonic stages .”“In this study, we uncovered several Hoxc8 target genes encoding different classes of proteins  , suggesting Hoxc8 controls different aspects of brachial MN development through the regulation of these genes.”3) It would be worthwhile to have co-labeling with reporter (Hb9-GFP vs ChAT-tdTom) for at least some of the selected genes  to see if there is specific reduction in the cells of interest rather than just through correlation based on spatial pattern.earlyHoxc8 MNΔ mice. Consistent with what we originally reported, these experiments showed that the expression of several Hoxc8 target genes  is affected in brachial motor neurons . Moreover, most of the target genes  that are downregulated – in vivo – in motor neurons of earlyHoxc8 MNΔ and Hoxc8 MNΔlate mice are upregulated upon inducible Hoxc8 expression in vitro generated motor neurons . Hence, Hoxc8 appears to be both necessary and sufficient for the expression of its target genes.To address this important point, we coupled RNA fluorescence in situ (FISH) with antibody staining for FoxP1  on spinal cords of 4) The Irx2 knockout experiments suggest that this downstream target of Hoxc8 may be involved in specification of limb-generating motor neurons, but not in the expression of terminal differentiation markers regulated by Hoxc8. This result seems tangential to the paper as 1) the knockouts are performed very early (prior to cell specification) and therefore does not provide any additional information about how Hoxc8 regulates motor neuron development past specification stage and 2) do not a clear functional link between Hoxc8 and terminal differentiation regulation.We completely agree and have decided to remove the mouse Irx data from this manuscript. The manuscript is now more focused on Hoxc8 and its conclusions are strengthened by the addition of new molecular and behavioral data, two new main figures [Figure 5 – 6] and 5 new supplementary figures [Figure 1—figure supplement 1; Figure 3—figure supplement 1; Figure 4—figure supplement 1; Figure 5 —figure supplement 1; Figure 6 —figure supplement 1).C. elegans Irx2 (fosmid) experiments add to this paper. The loss of MNs entirely seems to detract from the overall point of this paper, which is that Hoxc8 helps to establish/maintain brachial motor neuron identity/maturation.5) It is unclear what the C. elegans Irx data from the manuscript.Again, we agree and have removed the 6) It is unclear what the behavioral experiments add to the paper since would be very difficult to attribute any phenotype to a specific cause. Also, unclear why the experiments were performed only in late Hoxc8 deletion and not the early deletion as well.earlyHoxc8 MNΔ and Hoxc8 MNΔlate mice. Our new behavioral data  nicely complement the molecular analysis of brachial motor neurons in earlyHoxc8 MNΔ and Hoxc8 MNΔlate mice.Please see ER – Response 1 (page 1 of this document). We conducted new behavioral tests  on 7) The quality of some of the in situs should be improved Hoxc8 MNΔlate mice .We have conducted new RNA FISH experiments to improve the quality of our findings . Moreover, we performed double immunofluorescence (instead of RNA ISH) to demonstrate Hoxc8 protein depletion in brachial MNs of 8) It might be of interest to overexpress Hoxc8 in other regions of the spinal cord  using chick electroporation for example to see if it can lead to overexpression of some of these terminal differentiation genes. This gain-of-function experiment may lend additional support to the proposal that Hoxc8 is required to maintain expression of these genes, although I am not sure that this is a requirement for distinguishing terminal selectors.This is an excellent idea. In the absence of an established chick electroporation system in our lab, we analyzed recently published RNA-Seq and ChIP-seq datasets from motor neurons derived from mouse ES cells (ESC-MNs), in which Hoxc8 expression is induced with doxycycline (Dox) treatment . We included this analysis in Results as shown below:“Hoxc8 is sufficient to induce its target genes and acts directlyearly and Hoxc8 MNΔ late mice  suggest that Hoxc8 is necessary and sufficient for the expression of several of its target genes in spinal MNs.To gain mechanistic insights, we analyzed recently published RNA-Seq and chromatin immunoprecipitation-sequencing (ChIP-seq) datasets on MNs derived from mouse embryonic stem cells (ESC), in which Hoxc8 expression was induced with doxycycline . Our RNA-Seq analysis showed that induction of Hoxc8 (iHox8) resulted in upregulation of previously known  and new  Hoxc8 target genes . Moreover, ChIP-seq for Hoxc8 in the context of these iHoxc8 ESC-derived MNs revealed binding in the cis-regulatory region of all these genes , suggesting Hoxc8 acts directly to activate their expression. This in vitro data together with the in vivo findings in Hoxc8 MNΔ Importantly, not all Hoxc8 target genes  we identified in vivo are upregulated in iHoxc8 ESC-derived MNs . This is likely due to the lack of Hoxc8 collaborating factors in these in vitro generated MNs. A putative collaborator is Hoxc6 because (a) Hoxc6 and Hoxc8 are co-expressed in embryonic brachial MNs , (b) animals lacking either Hoxc6 or Hoxc8 in brachial MNs display similar axon guidance defects , and (c) Hoxc6 and Hoxc8 control the expression of the same axon guidance molecule (Ret) in brachial MNs . Supporting the notion of collaboration, our analysis of available ChIP-seq data for Hoxc6 and Hoxc8 from iHoxc6 and iHoxc8 ESC-derived MNs, respectively, showed that these Hox proteins bind directly on the cis-regulatory region of previously known  and new  Hoxc8 target genes .”C. elegans are both necessary and sufficient. Our findings on mouse Hoxc8 support the idea that key features of terminal selector function are conserved across species (please see last paragraph in Discussion).Altogether, our in vivo data and in vitro analysis suggest that Hoxc8 is necessary and sufficient for the expression of at least some its target genes. Indeed, most terminal selector-type TFs in Reviewer #3 (Recommendations for the authors):1. The authors used two different genetic systems to label brachial MNs at embryonic day 12 (e12) and postnatal day 8 (p8) as it was not possible for them to label MNs with just a single genetic system. Using these reagents they conclude that Hoxc8 regulates some of the same and some different targets. But can they rule out that some of these results is a consequence of using two different labeling systems? Are they certain that the cells labeled at both time points are the same cells? Perhaps using a lineage tracing tool and/or normalizing with some of the genes they discover in their RNA-seq experiments may be a way add confidence in the similarities and differences that they find in the RNAseq datasets are accurate.Indeed, this is a very important point which we now address with text changes and new experiments. Please see our detailed responses in ER – Response 4 (page 3 of this document) and R1 – Response 2 (page 6). In brief, Hb9-GFP expression is faint at post-natal stages, which necessitated the use of an additional reporter strategy  for the discovery of genes expressed in brachial MNs via RNA-Sequencing. In Results, we acknowledge the limitations of using two different genetic labeling strategies. We also conducted additional experiments to evaluate the specificity of motor neuron labeling by staining for glia and cholinergic motor neuron markers .We note that the two different labeling systems were used in wild-type animals to identify enriched transcripts in embryonic and postnatal MNs . Hence, they do not affect our key conclusions on the function of Hoxc8 in motor neurons for the following reasons:earlyHoxc8 MNΔ mice .(a) The new Hoxc8 targets were discovered through RNA-Seq by using a single reporter (Hb9::GFP), not two different labeling systems, in (b) We used additional methods  to independently validate our RNA-Seq results and identify new Hoxc8 target genes .(c) We also provide new data to show that Hoxc8 controls terminal identity gene expression specifically in brachial MNs (with FoxP1) .Pappa, Glra2, Sema5a) we found downregulated in brachial motor neurons of earlyHoxc8 MNΔ  and lateHoxc8 MNΔ  mice are upregulated in motor neurons derived from ES cells (ESC-MNs), in which Hoxc8 expression is induced upon doxycycline (Dox) treatment  .(d) Our new analysis revealed that the same Hoxc8 target genes  of the spinal cord , which necessitated the need of Cre lines  to specifically inactivate it in motor neurons at early and late stages of development.Yes, the reviewer is correct. Consistent with our findings , a previous study showed that, at e12, Hoxc8 is expressed both in MMC and LMC motor neurons . Like most Hox genes, In wild type animals, spinal motor neurons are normally generated in excess and a large fraction of them (~50%) undergoes physiological/developmental cell death after e12, resulting in fewer motor neurons at later developmental stages (PMID: 10928282). Hence, the number of brachial motor neurons expressing Hoxc8 is high at e12 and ~50% lower at p8. To address the reviewer’s point, we conducted double immunofluorescence and RNA ISH. We found that Hoxc8 continues to be expressed in spinal motor neurons at late embryonic (e18.5) and early postnatal (p8) stages .3. The authors state that Hoxc8 MN late mutants perform worse in rotarod performance test and forelimb grip strength in 3-month old mice. However, the statistical analysis suggests there is no significant change. It was unclear why the authors chose such a late time point for these assays, when the mice potentially have time to compensate for a compromised motor system. Redoing this experiment at earlier time points, such as with 1 month old mice (there is precedence for this in the literature), might reveal significant differences.earlyHoxc8 MNΔ and Hoxc8 MNΔlate mice and significantly increased the number of animals. Please see ER – Response 1 (page 1 of this document) for a detailed discussion of the behavioral data, which nicely complement the molecular analysis of brachial motor neurons in earlyHoxc8 MNΔ and Hoxc8 MNΔlate mice.We have now conducted new behavioral tests  both on


Hydroxypyridinones (HOPOs) have been used in the chelation therapy of iron and actinide metals. Their application in metal-based radiopharmaceuticals has also been increasing in recent years. This review article focuses on how multidentate HOPOs can be used in targeted radiometal-based diagnostic and therapeutic radiopharmaceuticals. The general structure of radiometal-based targeted radiopharmaceuticals, a brief description of siderophores, the basic structure and properties of bidentate HOPO, some representative HOPO multidentate chelating agents, radiopharmaceuticals based on HOPO multidentate bifunctional chelators for gallium-68, thorium-227 and zirconium-89, as well as the future prospects of HOPO multidentate bifunctional chelators in other metal-based radiopharmaceuticals are described and discussed in turn. The HOPO metal-based radiopharmaceuticals that have shown good prospects in clinical and preclinical studies are gallium-68, thorium-227 and zirconium-89 radiopharmaceuticals. We expect HOPO multidentate bifunctional chelators to be a very promising platform for building novel targeted radiometal-based diagnostic and therapeutic radiopharmaceuticals. More than 80% of all nuclear medicine procedures use 99mTc-based radiopharmaceuticals [68Ga]Ga-DOTATATE (in 2016), [177Lu]Lu-DOTATATE (in 2018), [68Ga]Ga-DOTATOC (in 2019), [64Cu]Cu-DOTATATE (in 2020) and [68Ga]Ga-PSMA-11 (in 2020). [68Ga]Ga-DOTATATE, [68Ga]Ga-DOTATOC and [64Cu]Cu-DOTATATE were used for the PET imaging diagnosis of somatostatin receptor positive neuroendocrine tumors (NETs). [177Lu]Lu-DOTATATE was indicated for the therapy of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). [68Ga]Ga-PSMA-11 is a PET diagnostic imaging agent for prostate cancer. [177Lu]Lu-PSMA-617, another therapeutic radiopharmaceutical of prostate cancer, showed promising outcomes in a multicenter phase III randomized clinical trial (VISION) [68Ga/177Lu is a good-fit theranostic radionuclide pair. The successes of 68Ga/177Lu labeled radiopharmaceuticals in diagnoses and treatments of NETs and prostate cancer has inspired more research on other metal-based radiopharmaceuticals [89Sr]SrCl2, [223Ra]RaCl2, etc.). In targeted metal-based radiopharmaceuticals, in addition to ligands that bind firmly to metallic nuclides, a biological targeting vector/molecule is usually contained. Therefore, when designing and synthesizing targeted metal-based radiopharmaceuticals, it is necessary to consider not only the selection of suitable metallic radionuclides, but also a ligand that matches the metallic nuclide. In addition, consideration must be taken not to affect the function of the biological targeting vector/molecule. The design and synthesis of targeted radiometal-based diagnostic and therapeutic radiopharmaceuticals have always been challenging tasks [H]/[LHi] and βi as [FeLi]/[Fe3+][L]i, where L denotes ligand. pFe3+ value was normally defined as –log[free Fe(III) in solution], when [ligand]total = 10−5 M, [Fe3+]total = 10−6 M and pH = 7.4. pFe3+ is introduced for comparison of chelating ability with Fe3+. The higher the pFe3+ value, the stronger the chelator’s Fe3+ binding affinity 3N  as the tripod and 3,2-HOPO as the coordinating group  complex. They found that the stability constants of Fe(III) complexes with these two ligands are similar to that of DFO, and one of the hexadentate ligands can significantly enhance iron(III) excretion from hepatocytes and iron-overloaded mice -3,4,3-LI in healthy male and female mice and rats. This investigation showed that [14C]-3,4,3-LI could quickly reach the systemic circulation, which exhibited that 3,4,3-LI should be a promising decorporation agent for actinides such as Pu, Am, U, and Np 3doxidized .According to the hard-soft acid-base (HSAB) theory of Pearson, due to its high charge and small ion radius, Ga(III) belongs to the hard acids. Therefore, Ga(III) can interact with ionic, non-polar hard bases such as oxygen and nitrogen donor atoms, i.e., carboxylate, catecholate, hydroxamate, phosphonate, amine, etc., thus forming thermodynamically stable complexes.3. When pH value is larger than 7, Ga(OH)3 would re-dissolve into Ga(OH)4−. When Ga(OH)3 or Ga(OH)4− is formed, the kinetics of substitution reactions between multidentate ligands and these hydroxides are very slow. Therefore, the formation of Ga(OH)3 and Ga(OH)4− must be avoided. Usually the radiolabeling of 68Ga should be carried out in the presence of weak ligands such as citrate, acetate and oxalate to avoid the formation of Ga(OH)3 -heptane-dioate as another bifunctional tripodal scaffold  to synth111In- and 90Y-ibritumomab tiuxetan) is a monoclonal antibody drug that has been approved by the FDA for the diagnosis and treatment of non-Hodgkin’s lymphoma (NHL) 4d0. Similar to gallium(III) ion, zirconium(IV) ion could be considered as hard Lewis acid due to its high charge and small ion radius. As a result, zirconium(IV) ion possesses a strong tendency to interact with hard Lewis bases such as oxygen donor atoms [The element of zirconium is in group IVB and the fifth period in the periodic table. The electronic configuration of zirconium is [Kr]4dor atoms .The most ideal coordination number of zirconium is 8, even though numbers 4–12 are possible.89Zr bifunctional chelating agent has been introduced in several recent comprehensive review articles ..116].89Zr radiolabeling of 3,4,3-. 3,4,3- (10 mM) could form a stable complex with 89Zr4+ at room temperature and pH 7. The radiolabel of 89Zr-3,4,3- was stable under EDTA challenge. As a comparison, 89Zr-DFO suffered a transchelation process. In vivo experiments showed that in animal model 89Zr-3,4,3- will go through renal excretion at early time points followed by hepatobiliary clearance at later time points +. However, dissociation of 89Zr4+ from DFO and coordination to CP256 could be measured by reverse phase radiochromatography when CP256 was added to [89Zr(DFO)]+ solution. In vivo experiments in mice indicate that the 89Zr-YM103-trastuzumab showed inferior stability compared with 89Zr-DFO-trastuzumab as it will release certain amounts of 89Zr4+, leading to bone uptake. This is largely due to hexadentate coordination geometry provided by three tris(hydroxypyridinone) groups Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb. In in vitro experiments, [89Zr]Zr-3,2-HOPO-MSLN-mAb and [89Zr]Zr-DFO-MSLN-mAb both showed a high binding affinity for mesothelin (MSLN). In in vivo experiments, [89Zr]Zr-DFO-MSLN-mAb exhibited a higher tumor and lower femur uptake than [89Zr]Zr-3,2-HOPO-MSLN-mAb 6d4+. At pH = 7.4 , thorium salt is hydrolyzed to form Th(OH)4 colloidal particles. In a 9-coordination environment, the ionic radius of Th(IV) is 1.09 Å, which is the largest and stable tetravalent metal ion. Since Th(IV) has the smallest ratio of charge to ion radius, it has poor hydrolysis compared with most metal ions, and can be studied in the pH up to about 4. Th(IV) in the aqueous solution undergoes multinuclear reaction, colloid formation and carbonate complexes formation. In addition, water-containing oxides or hydroxides of Th(IV) are difficult to dissolve [In aqueous solution, the only oxidation state of thorium is Thdissolve . Th(IV) dissolve ,131.225Ac/213Bi, 212Pb/212Bi and 227Th [227Th has a long half-life, it is often used in monoclonal antibody-based radiopharmaceuticals. The current research focus of 227Th antibody-conjugated radiopharmaceuticals is in radiopharmaceuticals for breast cancer, lymphoma, kidney cancer, ovarian cancer, acute myeloid leukemia and prostate cancer [In the past ten years, the most commonly investigated radiometals in TAT are nd 227Th ,133. Bece cancer ,135.In TAT, DOTA can be used as a bifunctional chelator for thorium, but the coordination kinetics are slow, requiring a higher labeling reaction temperature, which will lead to denaturation of the targeted antibody . TherefoThe HOPO chelating agents have a strong chelating ability not only towards Fe(III) and Ga(III) but also towards actinide ions. In fact they form complexes with high stability and low toxicity with actinides and they have been used as decorporation agents for this metal ions, as the case of thorium(IV) with octa-dentate HOPO compounds ,136.4+ = 41.0(5)). This ligand was found to form strong and stable complexes with Th(IV) in aqueous solution (log β110 > 38.5) [β120 = 39.1(2)).As is shown in  > 38.5) ,138. Excβ110 = 41.7(0.3)). In animal experiments, a hepatobiliary excretion route was indicated and bone uptake was low enough to prove the stability of the thorium complex in vivo [These works boost the development of 3-hydroxy-N-methyl-2-pyridinone -based unit as the bifunctional chelator for thorium. Cuthbertson et al. developed a chelator  containi in vivo ,140.The carboxylic acid group in this chelator could be used to conjugate with antibodies. For example, after conjugating with CD33 antibody (Lintuzumab), this Me-3,2-HOPO based chelator was found to possess the ability to radiolabel with Th-227 at neutral pH and ambient temperatures. This enabled the successful delivery of Th-227 to CD33 positive cells. In a biodistribution study, while an increase of radio activity over time in the tumor region could be observed, almost no accumulation of radio activity was present in other organs. In the anti-tumor activity experiment towards CD33-TTC, almost 83% of the animals were free of any palpable tumors at the end of the study. Furthermore, the tumor inhibition was influenced by the amount of Th-227. On November 26 2019, this drug (BAY1862864) completed its phase I clinical trial .227Th by the tumor even seven days post injection [Cuthbertson et al. studied a CD70 targeted thorium-227 conjugate named as CD70-TTC, which is comprised of a CD70 targeting antibody, a 3,2-HOPO-based bifunctional chelator and alpha-emitting radionuclide thorium-227. This is till now the first report which investigated molecular targeting of CD70 expressing tumors using TAT. In vitro experiment showed that compared with the isotype control-TTC, the CD70-TTC possessed binding affinity and specific cytotoxicity to its target. A biodistribution study showed that compared with control-TTC, CD70-TTC retained significant uptake of njection .227Th(IV) and two chelators with four coordinating groups of Me-3,2-HOPO. They found that these chelators could retain high chelation yields with thorium-227 within 2.5 h or less. The thorium complexes possess high stability in phosphate-buffered saline at ambient condition over a 6-day-period [Wilbur et al. investigated the chelation reactions between y-period .Till now, radium-223 is the only approved alpha-emitting radionuclide for the treatment of metastatic castration-resistant prostate cancer. As the progenitor nuclide of radium-223 , thoriumIn metal-based radiopharmaceuticals, the concentrations of the metallic radionuclides and that of the chelators are very low. The metal-labeled radiopharmaceuticals are supposed to possess high thermodynamic and kinetic stability. Hydroxypyridinone based chelators possess low toxicity. Besides, they showed good selectivity and high stability towards some trivalent or tetravalent metal ions, such as gallium(III) ion, zirconium(IV) ion, thorium(IV) ion, etc.68Ga-THP-PSMA, which is used for the diagnosis of prostate cancer by PET/CT imaging, has been undergoing a clinical phase II study (NCT03617588). The PSMA-targeted 227Th conjugate PSMA-TTC (BAY2315497) is a human anti-PSMA antibody linked to a chelator moiety . This antibody-based therapeutic radiopharmaceutical is in a clinical phase I study (NCT03724747). The clinical phase I study of 227Th-Mesothelin-TTCs (BAY2287411) is also being carried out by Bayer (NCT03507452). This antibody-based radiopharmaceutical also uses 3,2-HOPO containing bifunctional octa-dentate chelator. Based on the promising preclinical results, 89Zr-HOPO-trastuzumab will be conducted in a clinical phase I-II study (NCT04757090). In short, HOPO-based multidentate ligands have shown clinical application prospects in the radiopharmaceuticals of gallium-68, thorium-227 and zirconium-89.As mentioned above, although some HOPO-based radiopharmaceuticals of gallium-68, thorium-227 and zirconium-89 are in different phases of clinical trials, whether these radiopharmaceuticals could finally be approved for marketing still needs further investigation.68Ga, 227Th and 89Zr radiopharmaceuticals, could HOPO multidentate chelating agents be used in other metallic radiopharmaceuticals? This is an area worthy of further in-depth study.In addition to 227Th, 225Ac, 213Bi, 212Bi and 149Tb have become promising therapeutic radionuclides [227Th, the use of HOPO-based bifunctional chelating agents in radiopharmaceuticals of 225Ac, 213Bi, 212Bi and 149Tb has not been reported.In the current TAT, nuclides . HoweverRegarding investigations of HOPO-based radiopharmaceuticals, we look forward to their deepening and expansion in the following directions:68Ga, 227Th and 89Zr should be promoted to market as soon as possible for the early diagnosis or treatment of various diseases, especially for cancers.(1) Through more extensive research, HOPO-based radiopharmaceuticals of 225Ac, 213Bi, 212Bi and 149Tb should be carried out. The design and synthesis of HOPO-base bifunctional chelating agents for these metal radionuclides should be the first step.(2) TAT is expected to become one of the most effective approaches for cancer treatment. Various studies on HOPO-based radiopharmaceuticals of 3-tren-Me-3,2-HOPO and gadolinium has a significantly high pGd value of 20.3 .Raymond et al. usually study lanthanide ions as suitable model analogues of actinide ions  when theAmong actinide decorporation agents and Gd-based MRI contrast agents, HOPO-based chelators showed strong chelating ability, which provides us with a basis in coordination chemistry for the further design of HOPO-based bifunctional chelating agents used in radiopharmaceuticals.When designing HOPO-based bifunctional chelating agents for these metallic radionuclides, we must consider the different coordination chemistry properties of each metal nuclide. For example, although both Th and Ac are actinide elements, the common oxidation state of Th in solution is 4, while that of Ac is 3, and their coordination chemistry properties are quite different. In addition, for these metal ion bifunctional chelating agents, we not only hope that they have high chelating ability , but also that they have rapid radiolabeling kinetics, in vivo stability, and a single configuration of the radiolabel. Therefore, the design and synthesis of bifunctional chelating agents containing multiple HOPO groups and a coupling group with biological targeting molecules is still challenging.(3) The synthesis of HOPO-based bifunctional chelating agents often requires multiple steps. It is very important to simplify the synthesis steps and improve the overall synthesis yield. More efficient synthetic methods should be developed and employed. The improvement in synthetic methods could help increase the overall yield, which will be good for the development of radiopharmaceuticals from bench to bedside. Probably the molecular design and synthesis of bifunctional chelating agents, as well as the interactions between radiopharmaceuticals and biological targets, may be improved by virtue of artificial intelligence development ,146.With further expansion and in-depth study, we believe HOPO-based multidentate bifunctional chelators will become a very promising platform for novel targeted diagnostic and therapeutic radiopharmaceuticals.


Clinical work and research on diabetic wound repair remain challenging globally. Although various conventional wound dressings have been continuously developed, the efficacy is unsatisfactory. The effect of drug delivery is limited by the depth of penetration. The sustained release of biomolecules from biological wound dressings is a promising treatment approach to wound healing. An assortment of cell-derived exosomes (exos) have been proved to be instrumental in tissue regeneration, and researchers are dedicated to developing biomolecules carriers with unique properties. Herein, we reported a methacrylate gelatin (GelMA) microneedles (MNs) patch to achieve transdermal and controlled release of exos and tazarotene. Our MNs patch comprising GelMA/PEGDA hydrogel has distinctive biological features that maintain the biological activity of exos and drugs in vitro. Additionally, its unique physical structure prevents it from being tightly attached to the skin of the wound, it promotes cell migration, angiogenesis by slowly releasing exos and tazarotene in the deep layer of the skin. The full-thickness cutaneous wound on a diabetic mouse model was carried out to demonstrate the therapeutic effects of GelMA/PEGDA@T + exos MNs patch. As a result, our GelMA/PEGDA@T + exos MNs patch presents a potentially valuable method for repairing diabetic wound in clinical applications.The online version contains supplementary material available at 10.1186/s12951-022-01354-4. The global prevalence of adult diabetes is projected to increase to 7.7% by 2030 , complicThe traditional wound dressing procedure is lengthy and a source of a considerable psychological burden on patients. At present, the diabetic wound healing treatment methods primarily include local drug administration and cellular therapy . BiomateAs a minimally invasive tool, microneedles (MNs) have attracted progressively more attention in recent years. They have many outstanding merit including painlessness, self-management and easy handling. Numerous studies have shown that MNs can successfully deliver small molecule drugs and macromolecular drugs  through the skin. MNs also have the advantage over traditional bandages and hydrogels of transporting drugs through deep skin and improving drug delivery efficiency. MNs are divided into soluble MNs and insoluble MNs. Insoluble MNs are more conducive to maintaining stability and sustaining the release of drugs than soluble MNs. Materials commonly used in MNs preparation include gelatin, PLGA, PVA and chitosan –17. PrevExosomes (exos) are cell-released small vesicles with a diameter of 50–150 nm, they regulate communication between cells by transferring contained bioactive cargos including miRNAs, mRNAs and proteins –20. Rece2 to carry tazarotene. MNs promote the directional release of drugs and exos in a mouse model, and accelerate cell proliferation, migration and angiogenesis both in vitro and in vivo. We also hypothesized that endothelial cells-derived exos contribute to accelerate the diabetic wound repair.Based on this premise, we sought to encapsulate the exos and tazarotene in MNs to avoid the loss of the biomolecules and injecting them into the wound site to increase the use effect. We used GelMA and PEGDA to prepare MNs, compounded exos in MNs, and grafted β-CD-AOIGelMA was synthesized according to a previously described protocol . Briefly2 was synthesized according to a previously described protocol [2 atmosphere. The reaction product was recrystallized with acetone and dried under vacuum to obtain the final product for subsequent experiments.β-CD-AOIprotocol . A simpl2 were collected from 0.7 ml sample (10 mg ml−1) dissolved in D2O by NMR system . In addition, in order to characterize the structural characteristics of PEGDA  and GelMA, Fourier transform infrared spectroscopy (FT-IR)  was used. FT-IR spectra were collected in transmission mode from 4000 cm−1 to 400 cm−1.The 1H NMR spectra of the GelMA and β-CD-AOIThe mechanical properties of hydrogels with different PEGDA contents  were analyzed by a low-force mechanical testing system. Hydrogels with a diameter of 1 cm and a height of 4 mm were prepared for testing mechanical properties. The maximum loading force was set to 50.0 N, and the downward speed of the force sensor was set to 1 mm/min. The variation curve of compression force with deformation is recorded. The microstructure of hydrogels was analyzed by scanning electron microscopy. Firstly, the normal hydrogel was brittlely broken after freeze-drying, and its cross-section was observed and analyzed by scanning electron microscopy . At the same time, we concentrated the precursor solution in the vacuum environment, and then obtained the hydrogel by photo crosslinking, and finally carried out a similar SEM analysis.The MNs were fabricated according to a vacuum method. Briefly, 1 g of GelMA was dissolved in 10 ml of PBS solution at 50 °C until fully dissolved. 25 mg Photoinitiator lithium acylphosphinate salt  were added to the GelMA solution for MNs fabrication. 100 μl of the above solution was poured over a polydimethylsiloxane (PDMS) mold containing an array of MNs cavities. The MN was placed in a vacuum environment at 50 °C remove air bubbles at the bottom of the MN mold while maintaining the dissolved state of GelMA. After centrifugation, the solution was exposed to UV light for gel. Finally, the MN arrays were detached from the PDMS mold after keeping the mold in the dark for 24 h to dry the GelMA/PEGDA MNs at room temperature.2 and GelMA. Then, after the dissolution of tazarotene was complete, the exos (100ug/ml) were added and mixed evenly, and 500 ul of matrix solution was filled into PDMS mold for MN preparation.To prepare a GelMA/PEGDA MN loaded with tazarotene  and exos, an appropriate amount of tazarotene (1 mg/10 ml) was dissolved in a precursor solution containing PEGDA, β-CD-AOIThe prepared MNs were put into 50 ml PBS solution for degradation experiment. The MNs were taken out at different time points, wiped with filter paper, and dried after freezing. The MNs were characterized by scanning electron microscope .The drug release test was further carried out. Each MN sample was immersed in 10 ml PBS (pH = 7.4) at 37 °C. At each determined time point , 2 ml of release medium was added into 2 ml of methanol, and then ultrafiltrated for detection. The release medium extracted from the original release system was replaced with fresh PBS of the same volume. The amount of tazarotene released from the mixture was analyzed by microplate reader at 351 nm. In vitro, a GelMA/PEGDA@Tazarotene + exosomes (GelMA/PEGDA@T + exos) MNs patch was immersed with PBS at 37 °C. The number of exos proteins were detected using Micro BCA Protein Assay Kit  following the manufacturer’s instructions. In vivo, MNs patch loaded with PKH26 labeled HUVECs-exos and tazarotene was covered to the shaved back skin of the diabetic C57BL mice, the skin covered with MNs patch was harvested and fluorescence detection was then performed on day 2, the original MNs patch was continued to be covered to the other area of the skin, the fluorescence detection was then performed on day 4, and so on, the same treatment was carried out on day 6 and 8.2 at 37 °C. Human foreskin fibroblasts were isolated using previously described protocols [Human immortalized keratinocytes (HaCAT)  and human umbilical vein endothelial cells (HUVECs)  were obtained from the China Center for Type Culture Collection  and cultured following the manufacturer’s instructions. HaCAT and HUVECs were cultured in Dulbecco's modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were cultured in a humidified atmosphere containing 5% COrotocols .Exosomes were isolated using a previous protocol . When HUThe qualification of HUVECs-exos was performed by transmission electron microscope . The size distribution of HUVECs‐exos was measured by Nanoparticle tracking analysis . The total protein level was quantified Pierce BCA Protein Assay Kit .To determine uptake of the HUVECs-exos by HaCAT, fibroblasts and HUVECs, HUVECs-exos were incubated with red fluorescent dye  for 5 min and the neutralize redundant dye was removed by 0.5% BSA/PBS. Then, the residual dye was removed by centrifugation again to obtain the labeled HUVECs-exos. Cells were seeded in the confocal dish, and then treated with different concentrations  of labeled HUVECs-exos. The cells were washed with PBS after incubation for 24 h, and then fixed with paraformaldehyde (4%) for 10 min. FITC phalloidin  and DAPI  were used to stain the cytoskeleton and nucleic respectively.HaCAT, HUVECs, and Human foreskin fibroblasts were used to investigate the toxicity of the GelMA/PEGDA hydrogel in vitro. GelMA/PEGDA hydrogel was immersed in a complete medium containing DMEM (high glucose) with 10% FBS for different periods of time . Then the extract liquid was collected for the biocompatibility testing. The LIVE/DEAD assay  was used to test the cell activity.4 cells were seeded in 96-well plates with 100 μl extract liquid per well for 24 h, with three parallel controls for each group. Next, 10 μl of the working reagent was added to each well, and incubated at 37 °C for 2 h.Cell viability was estimated by the CCK-8 assay . Approximately 1.2 × 10The scratch assay was performed in a 12-well plate. When the cells reached approximately 95% confluency with the extract liquid (48 h) per well, the scratch was made by using a 200 μl pipette tip. A phase contrast microscope was used to visualize the images. The images were analyzed with Image J version 5.0.4 cells per well) were seeded with the extract liquid (48 h) per well in 96-well culture plates that had been coated with 70 μl Matrigel Basement Membrane Matrix . Tube formation was detected by using the microscope at 0 h, 2 h, and 6 h incubation. Results are represented as mean ± SEM in three in- dependent experiments.HUVECs (2.5 × 10−1 at least 1 month were defined as diabetic mice model. Then the mice randomized into four groups randomly (n = 7 per group): the control group, GelMA/PEGDA MNs patch group, GelMA/PEGDA@Tazarotene (GelMA/PEGDA@T) MNs patch group, GelMA/PEGDA@Tazarotene + exosomes (GelMA/PEGDA@T + exos) MNs patch group. The C57BL mice were anesthetized by 0.3% phenobarbital sodium (0.1 ml/10 g) through intraperitoneal injection. A 10-mm full-thickness cutaneous wound was excised on the midline line of the mouse back. Silicone ring was sutured to the wound edge to prevent wound contraction. The wounds were covered with PBS, GelMA/PEGDA MNs patch, GelMA/PEGDA@T MNs patch and GelMA/PEGDA@T + exos MNs patch. Digital photographs were taken at days 0, 5, 10 and 15. The wound area was measured using the Image J software.All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. Eighteen healthy eight-week-old male C57BL mice, weighing 20–23 g, were purchased from the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology. Streptozotocin (STZ) was used to induce healthy C57BL mice to develop diabetes by intraperitoneal injection. The blood glucose levels higher than 16.7 mmollAfter 15 days, the skin was cut into 10 mm*12 mm strips. Under the mechanical testing machine, the 50 N sensor stretched the skin at a speed of 1 mm/min. The wound area skin was removed and fixed with 4% paraformaldehyde for 48 h. The samples were embedded in paraffin after dehydration. A microtome was used to obtain 5 μm-thick serial sections, which were stained with hematoxylin–eosin (H&E), Masson trichrome, and immunofluorescence analysis. The sections of wound areas were respectively incubated with primary antibodies α-smooth muscle actin  and CD31  overnight at 4 °C. Then they were incubated with the second antibody (Servicebio) for 1 h at room temperature. Images were taken by using a fluorescence microscope and then analyzed by Image J software.Statistical analysis of data was performed by the unpaired Student's t-test and one-way ANOVA among multiple groups. P < 0.05 was considered significant. Statistical analysis was performed with the GraphPad Prism v 5.0 software. Data represent the mean ± SD of three independent experiments.Our MNs patch design aims to release targeted drugs and exos for diabetic wound repair. To obtain stable MNs, we used light-curable gelatin as the primary material, and simultaneously introducing cyclodextrin, which reacts with double bonds to realize drug loading. The addition of short chain PEGDA was used to enhance the cross-linked network. The mechanism of hydrogel formation is shown in Fig. 2 indicated that approximately two-CH=CH2 bonds were grafted at each β-CD-AOI2 molecule. The infrared results showed that the vibration peak of the amide in gelatin was at 1539 cm−1, and the stretching vibration peak of the C = O in amide was at 1649 cm−1, which existed in the infrared spectra of gelatin and the infrared spectra of the reactants  was defined as: residual wound area at day ‘X’) / Wound area at day ‘0’ × 100%. As shown in Fig. Reduced angiogenesis in diabetic wound bed are considered critical to the slow healing of the skin. Endothelial cells migrate to the damaged area to form new blood vessels, carrying oxygen and nutrients to the wound site to support angiogenesis. Reportedly, tazarotene promotes vascular regeneration. To further scrutinize the angiogenesis in these MNs patch-treated mice, immunofluorescence staining analysis of CD31 and α-SMA was conducted to assess the angiogenesis near the wound areas. Positively stained vessels of CD31 and α-SMA in the GelMA/PEGDA@T MNs patch group were considerably higher than in the GelMA/PEGDA MNs patch group at day 15 Fig. a–c, signSubstantial differences were noted in collagen deposition, re-epithelialization and enhanced CD31 and α-SMA expression between control group and GelMA/PEGDA MNs patch group on 15 day Fig. a–c. The The incidence of diabetes is increasing, and the survival rate of diabetes-related amputations is remains nonideal . DFU patMNs have more prominent advantages over traditional drug delivery systems . While t2 for tazarotene delivery. The incorporated exos are released into the tissue through MNs, and the transcutaneous delivery efficiency of tazarotene was improved. We first analyzed the material’s properties, using infrared and nuclear magnetic methods to establish the successful preparation of GelMA and β-CD-AOI2 [This study used GelMA and PEGDA as matrix materials and add β-CD-AOI-CD-AOI2 . Next, w-CD-AOI2 , 43. ThrThree steps include inflammation, new tissue formation, and remodeling were involved in the process of wound repair. The proliferation, migration and angiogenesis of keratinocytes, fibroblasts, and endothelial cells are characteristics of the second step , 45. TheWe have developed a controllable biomolecules released MNs patch comprising GelMA/PEGDA hydrogel for promoting wound healing. These GelMA/PEGDA MNs can encapsulate tazarotene and HUVECs-exos to release them around the wound site, drastically accelerating collagen deposition, epithelial regeneration and angiogenesis, indicating that tazarotene-HUVECs-exos loaded GelMA/PEGDA MNs patch is a promising clinical treatment approach for DFU.Additional file 1: Figure S1. Compressive behavior of GelMA/PEGDA hydrogel. Figure S2. SEM images of hydrogel after concentrated. Scale bar = 200 μm , scale bar = 100 μm . Figure S3. Biocompatibility of the degradation products of GelMA/PEGDA hydrogel. a Survival ratio of fibroblasts, HUVECs, HaCAT treated with the degradation products of GelMA/PEGDA hydrogel after 7, 14 days, scale bar: 100 μm. b Cell viability was determined by CCK8 assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure S4. HUVECs-Exos were loaded into MNs. a, b Planar imaging of the Dil-labeled GelMA/PEGDA@T+exo MNs. Scar bar in a, 1000 μm. Scar bar in b, 100 μm. c 3D reconstruction of confocal layer by layer scanning. Figure S5. Tensile stress of the back skin of mice at 15 post-wounding. a Tensile force detection device. b Tensile force curve of the four groups of the mice.


The recent identification of diverse stromal cell populations that reside underneath intestinal crypts has enabled the characterization of key growth factor cues supplied by these cells. The nature of these signals and how they are delivered to drive intestinal epithelial development, daily homeostasis and tissue regeneration following injury are being investigated. It is clear that aside from EGF, other ligands of the family, including Neuregulin 1 (NRG1), have distinct roles in supporting the function of intestinal stem cells through the ErbB pathway.Epidermal Growth Factor (EGF) has long been known for its role in promoting proliferation of intestinal epithelial cells. EGF is produced by epithelial niche cells at the base of crypts  A balance between cell proliferation and differentiation is exquisitely controlled in the intestinal epithelium throughout life . This isThe EGF family of ligands includes eleven structurally related proteins, namely EGF, transforming growth factor α (TGF-α), amphiregulin (AREG), epigen (EPGN), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EREG), betacellulin (BTC), and the neuregulins (NRG1-4) . These min vivo, other cellular sources can compensate for this loss, resulting in the maintenance of an intact epithelium (low cells residing at the base of crypts, where they express Grem1 and antagonize BMP signaling. These cells described as trophocytes are capable of fully supporting the growth of organoids ex vivo (via a paracrine mechanism while epithelial-produced ligands such as EGF, BTC, HB-EGF, and TGFα regulate cellular function in an autocrine manner. It is important to note that the processes and signaling pathways regulating ligand production during intestinal homeostasis in these particular sub-types is not clearly defined. In addition, a comparative analysis of ligand-mediated downstream signaling pathways in normal intestinal cells is not available.In mice and humans, Paneth cells are localized within the epithelium adjacent to intestinal stem cells in the base of crypts. These specialized cells secrete WNT3, EGF and NRG1 during intestinal homeostasis . Althougithelium . The strithelium  and PDGFithelium  that alsithelium . Single  ex vivo . A simil ex vivo . MAP3K2  ex vivo . Macroph ex vivo . A unify ex vivo . Studies ex vivo . Both EG ex vivo . Other m ex vivo . EREG is ex vivo . In cont ex vivo . EGFR is ex vivo . Taken tNrg1, which results in aberrant heart and neural development leading to embryonic lethality (Numerous mouse knockout studies have investigated the functional roles of individual ligands (summarized in ethality . Althougethality ; Table 1ethality . Geneticethality . Individethality . HoweverThe intestinal epithelium is a selective permeable barrier that permits uptake of nutrients from the luminal contents while forming a barrier against the toxic by-products of digestion and pathogenic bacteria . As the in vivo (in vivo niche signals (in vivo, including WNT and EGF enrichment at the bottom of intestinal crypts, are required. Indeed, recent technological advances have allowed preservation of such complex tissue systems and gradients in culture using intestine- and organoid-on-a-chip models (It is clear that coordination of stem cell maintenance, progenitor proliferation and differentiation of mature cell types in the intestinal epithelium is orchestrated by gradients of active growth factors, agonists and antagonists in vivo . The beh signals , 2011 wi signals . Althoug signals , in whic signals . Howeverp models . These sThe relative proportion of different epithelial cell types can be manipulated in organoids by altering the culture conditions. For example, addition of CHIR99021 to elevate WNT signaling can enrich for Lgr5+ stem cells and inhibition of Notch induces secretory cells . TherefoIt is important to keep in mind that the composition of the extracellular matrix surrounding epithelial cells in adult tissues, which is not fully replicated in organoid culture, might also affect the downstream molecular response to EGFR/ErbB activation . Other lA striking feature of the intestinal epithelium is the high turnover of cells which occurs on a daily basis, the dramatic expansion of tissue during embryonic development and the rapid remodeling observed in response to injury. Members of the EGF family of ligands and their receptors contribute substantially to these processes where the current evidence suggests there are distinct functions for particular ligands but also some functional redundancy . The dowHA and TJ wrote the manuscript. WC prepared the figures. HA, TJ, and WC edited and approved the final manuscript. All authors contributed to the article and approved the submitted version.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.