Differentiation

BackgroundDuring development, axons are organized into bundles, a process known as axonal fasciculation. The zebrafish lateral line nerve has been used as a model to study axonal fasciculation; however, the underlying mechanisms are not yet fully understood. Although Fgf3 and Fgf10a are well known to regulate the migration of the lateral line primordium along which the lateral line nerve projects, their roles in the organization of the lateral line nerve itself have not been clarified. Resultsfgf3,10a double mutants exhibited lateral line axonal defasciculation accompanied by an increased number of Schwann cells. Live imaging revealed a marked increase in Schwann cell proliferation and demonstrated that newly divided Schwann cells migrate along axons and infiltrate interaxonal spaces, thereby expanding these spaces and disrupting axonal fasciculation. Pharmacological manipulations further implicated a contribution of Nrg1-ErbB signaling to this phenotype. ConclusionsOur findings suggest that Fgf3 and Fgf10a are required to restrict Schwann cell proliferation and infiltration, thereby ensuring axonal fasciculation during lateral line development.

BackgroundCranial neural crest cells (cNCC) generate craniofacial cartilage, bone, and peripheral neurons and glia, and birth defects arise when the cartilage/neuronal/glial progenitor fail to differentiate. PRDM16 is a transcriptional regulator containing both zinc-finger and SET domains, implicated in craniofacial development and orofacial clefting, but its role in cranial sensory ganglion formation has not been defined. ResultsHere, we demonstrate that prdm16 is required for trigeminal ganglion (TG) assembly and sensory neurogenesis from cranial neural crest lineages. In zebrafish, prdm16 is expressed in TG beginning by 18 hours post fertilization (hpf) and persists through the later developmental stage at 48 hpf. In the prdm16 loss-of-function zebrafish, fewer HuC+ TG neurons are present at 24 hpf and 48 hpf, along with reduced overall ganglion size. Live imaging in Tg(sox10:mRFP; elavl3:GFP) embryos demonstrates similar numbers of sox10+ cNCCs migrating to the TG region and reduced cell numbers and overall smaller size of TG in prdm16-/-. Acetylated {beta}-tubulin immunostaining shows fewer trigeminal axon projections early and an altered projection pattern by 48 hpf. A reduction in a defined sensory neuron population, p2rx3b+ cells displayed a weaker signal and decreased cell number in prdm16-/- TG. Transcriptomic analysis of FACS-isolated sox10+ cranial neural crest cells supported reduced expression of key neurogenic and sensory lineage genes. Finally, in mouse embryos, PRDM16 is expressed in TG neurons, and Prdm16csp1/csp1 embryos exhibited reduced TG volume, area and fewer HuC+ neurons at E18.5. ConclusionTogether, these data identify Prdm16 as a conserved regulator of trigeminal ganglion growth and sensory neuron differentiation, linking PRDM-family chromatin regulators to the development of the peripheral sensory nervous system.

The development of cranial nerves, including the trigeminal nerve, and the formation of neuromuscular junctions (NMJs) are crucial processes for craniofacial motor function. Mllt11/Af1q/Tcf7c (henceforth Mllt11), a novel type of cytoskeletal-interacting protein, has been implicated in neuronal migration and neuritogenesis during central nervous system development. However, its role in peripheral nerve development and NMJ formation remains poorly understood. This study investigates the function of Mllt11 during trigeminal ganglion development and its impact on motor innervation of the masseter muscle. We report Mllt11 expression in the developing trigeminal ganglia, suggesting a potential role in cranial nerve development. Using a conditional knockout mouse model to delete Mllt11 in Wnt1-expressing neural crest cells, we assessed trigeminal ganglion development and innervation of the masseter muscle in the jaw. Surprisingly, we find that Mllt11 loss does not affect the initial formation of the trigeminal ganglion but disrupts its cellular composition, with a reduction in the ratio of neural crest-derived Sox10+ cells relative to placode-derived Isl1/2+ cells. Furthermore, our study demonstrates that conditional Mllt11 knockout leads to reduction of neurofilament density and NMJs within the masseter muscle, indicating altered trigeminal motor innervation. Our findings show that Mllt11 regulates the cellular composition of the trigeminal ganglion and is essential for proper trigeminal motor innervation in the masseter muscle.

BackgroundIn humans and other mammals, spinal cord injury (SCI) can lead to a permanent loss of sensory and motor function, due to the inability of damaged neurons and axons to regenerate. However, other vertebrate species including zebrafish exhibit complete spinal cord regeneration and functional recovery after SCI. Wnt signaling is required for neurogenesis and axon regrowth in a larval zebrafish SCI model, but the genes regulated by this pathway and the cell types that express them remain largely unknown. ResultsIn this study, we used bulk RNA-sequencing (RNAseq) to identify candidate genes regulated by Wnt signaling that are expressed after SCI. Using this unbiased screen, we identified multiple genes previously unassociated with SCI in larval zebrafish, and confirmed by in situ hybridization that their expression is injury-responsive, Wnt-dependent, and localized to fibroblast-like cells surrounding the spinal cord. ConclusionsTogether, our data reveal potential novel gene targets and cell populations that may play important roles in spinal cord regeneration.

Background & AimsEnteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. Sox10 deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How Sox10 deficiency alters ratios of enteric neuron subtypes is unclear. Sox10s prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine Sox10Dom effects ENS progenitors and early differentiating enteric neurons. MethodsENS progenitors, developing neurons, and enteric glia were isolated from Sox10+/+ and Sox10Dom/+ littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) validated expression changes detected in scRNA-seq. ResultsscRNA-seq profiles revealed three neuronal lineages emerging from cycling progenitors via two transition pathways accompanied by elevated activity of Hox gene regulatory networks (GRN) as progenitors transition to neuronal fates. Sox10Dom/+ scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. Hoxa6 was differentially expressed in the neuronal lineages impacted in Sox10Dom/+mutants and HCR identified altered Hoxa6 expression in early developing neurons of Sox10Dom/+ ENS. ConclusionsSox10Dom/+ mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in Sox10Dom/+ scRNA-seq profiles including multiple Hox genes. This is the first report that implicates Hox genes in lineage diversification of enteric neurons.

BackgroundEndocytosis constitutes a fundamental cellular process governing development through coordinated regulation of plasma membrane remodeling and ciliogenesis, processes essential for cell shape changes and embryonic development. Although Twist1 null embryos display complete cranial neural tube closure defects and conditional knockout in neuroectoderm disrupts cranial neural crest cell fate determination and delamination, the function of TWIST1 in neural tube morphogenesis remains unknown. We investigated the basis underlying neuroectodermal morphological abnormalities in TWIST1 mutant embryos, specifically the formation of ectopic lateral bending points and cellular disorganization, by examining TWIST1 function in cilia formation, adherens junction integrity, and endocytic vesicle dynamics. ResultsImmunofluorescence analysis revealed that cytosolic TWIST1 colocalizes with {beta}-catenin and endocytic regulators LRP2 and RAB11B along the apical surface of cranial neuroectoderm. Twist1 knockout resulted in reduced ciliary length and number. Quantitative PCR and Western blot analyses demonstrated upregulation of RAB11B and {beta}-catenin at mRNA and protein levels in Twist1 mutants. This molecular dysregulation coincided with increased accumulation of apical endocytic vesicles and altered expression profiles of endocytic component genes, ultimately modifying the apical neuroectodermal cell-cell junctions. ConclusionOur findings establish TWIST1 as a regulator of neuroectodermal morphology, demonstrating its ability to modulate ciliogenesis, endocytic vesicle dynamics, and cell-cell integrity.

BackgroundNeural crest cells (NCCs) are migratory embryonic stem cells that give rise to a diverse set of cell types. Here we describe the dynamic distribution of NCCs in developing embryos of the common wall lizard Podarcis muralis inferred from ten markers. Our aim is to provide insights into the NCC development of lacertid lizards and to infer evolutionary modifications by comparisons to other tetrapods. ResultsNCC migration is ongoing at oviposition, following three streams in the head and multiple in the trunk. From 21ss, we observe expression patterns indicating the beginning of differentiation towards mesenchymal and neuronal fates. By 35ss, migration is restricted to caudal levels, and fully differentiated chromaffin cells are observed. ConclusionsWe find that some markers show patterns that differ from other tetrapods. For example, the antibody HNK-1 labels three NCC streams from the hindbrain while some comparable reptile studies describe four. However, the information emerging from all markers combined shows that the overall spatiotemporal distribution of NCCs in the common wall lizard is largely conserved with that of other tetrapods. Our study highlights the dynamic nature of seemingly canonical marker genes and provides the first description of spatiotemporal NCC dynamics in a lacertid lizard.

BackgroundActivity of the receptor tyrosine kinase PDGFR and the tyrosine phosphatase SHP2 are critical for vertebrate craniofacial development. We sought to determine the effect of SHP2 binding to PDGFR via phenotypic and biochemical analyses of an allelic series of mouse embryos with combined loss of both proteins in the neural crest lineage. ResultsWe demonstrated that SHP2 preferentially binds PDGFR/ homodimers among the three PDGFR dimers. Analysis of allelic series mutant embryos revealed increased cell death in the lateral nasal and maxillary processes at E10.5, variably penetrant facial blebbing, facial hemorrhaging, midline clefting and loss of the mandibular region at E13.5, and widespread craniofacial bone and cartilage defects at birth. Further, we showed that loss of SHP2 leads to increased phosphorylation of PDGFR and the downstream effector Erk1/2 in E10.5 allelic series mutant embryo lysates. ConclusionsTogether, our findings demonstrate additive effects on craniofacial development upon conditional ablation of PDGFR and SHP2 in the mouse neural crest lineage and indicate that SHP2 may negatively and positively regulate PDGFR signaling through distinct mechanisms.

Craniofacial development is a complex and tightly regulated process and disruptions can lead to structural birth defects, the most common being nonsyndromic cleft lip and palate (NSCLP). Previously, we identified FOS as a candidate regulator of NSCLP through family-based association studies, yet its specific contributions to oral and palatal formation are poorly understood. This study investigated the role of fos during zebrafish craniofacial development through genetic disruption and knockdown approaches. Fos was expressed in the periderm, olfactory epithelium and other cell populations in the head. Genetic perturbation of fos produced an abnormal craniofacial phenotype with a hypoplastic oral cavity that showed significant changes in midface dimensions by quantitative facial morphometric analysis. Loss and knockdown of fos caused increased cell apoptosis in the head, followed by a significant reduction in cranial neural crest cells (CNCCs) populating the upper and lower jaws. These changes resulted in abnormalities of cartilage, bone and pharyngeal teeth formation. Periderm cells surrounding the oral cavity showed altered morphology and a subset of cells in the upper and lower lip showed disrupted Wnt/{beta}-catenin activation, consistent with modified inductive interactions between mesenchymal and epithelial cells. Taken together, these findings demonstrate that perturbation of fos has detrimental effects on oral epithelial and CNCC-derived tissues suggesting that it plays a critical role in zebrafish craniofacial development and a potential role in NSCLP. Summary statementPerturbation of fos, a candidate gene associated with nonsyndromic cleft lip and palate in humans, causes a distinctive orofacial phenotype in zebrafish as a result of abnormal development of craniofacial tissues. Disruption of fos in the oral epithelial, cranial neural crest and Wnt responsive cell populations around the oral cavity causes anomalies that suggest a potential role in the etiology of NSCLP.

LMX1B, a LIM-homeodomain family transcription factor, plays critical roles in the development of multiple tissues, including limbs, eyes, kidneys, brain, and spinal cord. Mutations in the human LMX1B gene cause the rare autosomal-dominant disorder, Nail-patella syndrome which affects development of limbs, eyes, brain, and kidneys. In zebrafish, lmx1b has two paralogues: lmx1ba and lmx1bb. While lmx1b morpholino data exists, stable mutants were previously lacking. Here we describe the characterisation of lmx1b stable mutant lines, with a focus on development of tissues which are affected in Nail-patella syndrome. We demonstrate that the lmx1b paralogues have divergent developmental roles in zebrafish, with lmx1ba affecting skeletal and neuronal development, and lmx1bb affecting renal development. The double mutant, representing loss of both paralogues (lmx1b dKO) showed a stronger phenotype which included additional defects to trunk muscle patterning, and a failure to fully inflate the notochord leading to a dramatic reduction in body length. Overall, these mutant lines demonstrate the utility of zebrafish for modelling Nail- patella syndrome and describe a previously undescribed role for lmx1b in notochord cell inflation. SUMMARY STATEMENTZebrafish lmx1ba and lmx1bb knockout lines exhibit paralogue-specific roles in cartilage, renal, muscle, and notochord development, establishing zebrafish as a viable model for dissecting LMX1B- related pathologies such as Nail-patella syndrome.

Robinow Syndrome (RS) is a rare disease characterized by craniofacial malformations and limb shortening linked with mutations in seven WNT pathway genes. Our objective was to investigate the functional effects of frameshift mutations the intracellular adaptor protein, Dishevelled (DVL1; c.1519{Delta}T, p.Trp507Glyfs*142) on chicken craniofacial development. Misexpression of wt (wt) or mutant hDVL1 variants in vivo caused upper beak shortening (wtDVL1 n=8/14; DVL11519{Delta}T 12/13). At early stages of development, the DVL11519{Delta}T inhibited frontonasal mass narrowing, chondrogenesis, and proliferation. To test whether the phenotypes were caused due to the abnormal C-terminal peptide in DVL11519{Delta}T, we designed two additional constructs. The DVL11519* (DVL1507*) retains first 30 amino acids of the C-terminus while DVL11431* (DVL1477*) removes the entire C-terminus. DVL11519* injected embryos had normal beaks while DVL11431* caused high mortality and the phenotypes were like the DVL11519{Delta}T. In frontonasal micromass cultures, both DVL11519{Delta}T and DVL11431* inhibited skeletogenesis while the DVL11519* resembled wtDVL1 and GFP cultures. In luciferase assays DVL11519{Delta}T, DVL11519*and DVL11431* weakly activated the WNT canonical and non-canonical JNK-PCP pathways compared to wtDVL1. Furthermore, we observed that variant DVL1507*fs is stalled in the nucleus similar to hDVL1477*, possibly due to the abnormal C-terminus interfering with the nuclear export sequence. wtDVL1 and DVL1507* were distributed in nucleus and the cytoplasm. Our RS-DVL11519{Delta}T avian model recapitulates the broad face and jaw hypoplasia and demonstrates defects in both branches of WNT signaling. This is the first study to clarify the role of abnormal C-terminus in ADRS and to recognize the importance of an uncharacterized C-terminal sequence. Summary StatementFunctional and biochemical studies on chicken embryos with the Robinow syndrome (RS) DVL1 variant demonstrate defects in skeletogenesis and both branches of WNT signaling. This is the first study to establish a link between the RS facial defects and the mutated C-terminal sequence. We identified first 30 amino acids of the DVL1 C-terminus are sufficient for normal development.

Emerging evidence implicates retinal microglia and inflammation as important components impacting the outcome of retinal regeneration, which is spontaneously achieved in zebrafish retina following acute damage but is limited or blocked in mammals. In this paper, we describe the regenerative response in the larval zebrafish retina following ablation of cone photoreceptors. To investigate the role of microglia in the regenerative response, we used both irf8st95 heterozygote (microglia-sufficient) and irf8st95 homozygous mutant (microglia-deficient) zebrafish. We compared multiple aspects of the regenerative response in irf8+/- and irf8-/- larval retinas, including entry of the Muller glia (MG) into the cell cycle, the amplification of MG-derived progenitor cell (MGPC) proliferation, inflammatory and glial reactivity-associated gene expression, and the regeneration of cones. We found only modest impacts to early and late stages of MGPC proliferation and to inflammatory gene expression in irf8 mutants, with no obvious impacts to the regeneration of cones. Notably, we detected a population of immune cells in irf8 mutants that emerged following cone ablation, which expanded in number then were reduced over time, following a trajectory similar to microglia-sufficient siblings but at markedly reduced abundance. The immune cells detected in irf8 mutants included a subset with L-plastin/4C4 antibody staining patterns different than those in microglia-sufficient siblings, suggesting distinct origins and/or phenotype compared to resident microglia in controls. Though strong conclusions about the role of microglia were limited due to the presence of such immune cell populations in irf8 mutants, our results are consistent with several reports that indicate a role for microglia and inflammation in regulating MGPC proliferation in the regenerating retina. Collectively considered with other reports, our results further indicate that compensatory responses, which may include different immune cells and/or signaling from other retinal cell types such as the Muller glia, are elicited in microglia-deficient retinas upon neuronal damage.

ABSTRACTOrofacial clefts are among the most common human congenital malformations. Irf6 and Esrp1 are two key genes important for palate development, conserved across vertebrates. In the zebrafish, we found that irf6 regulates the expression of esrp1. Using RNAscope, we detailed overlapping Irf6 and Esrp1/2 gene expression in the mouse frontonasal prominence ectoderm, lambda joint periderm, palate and lip epithelium. In the zebrafish, irf6 and esrp1/2 share expression in the pre-gastrulation periderm and the embryonic frontonasal ectoderm, oral epithelium ventral to the anterior neurocranium (ANC), and the developing stomodeum. Genetic disruption of irf6 and esrp1/2 in the zebrafish resulted in cleft of the ANC. In the esrp1/2 zebrafish mutant, cleft of the mouth opening formed and appeared to tether into the ANC cleft. Lineage tracing of the anterior cranial neural crest cells revealed that cleft of the ANC resulted not from migration defect, but from impaired chondrogenesis. Molecular analysis of the aberrant cells localized within the ANC cleft revealed that this cell population espresses sox10, col1a1 and irf6 and is adjacent to cells expressing epithelial krt4. Detailed morphogenetic analysis of mouse Irf6 mutant revealed mesenchymal defects not observed in the Esrp1/2 mutant. Analysis of breeding compound Irf6;Esrp1;Esrp2 mutant suggests that these genes interact where the triple mutant is not observed. Taken together, these studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish models and captured an unique aberrant embryonic cell population that contributes to cleft pathogenesis. Future work characterizing this unqiue sox10+, col1a1+, irf6+ cell population will yield additional insight into cleft pathogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

BackgroundElp1, a subunit of the Elongator complex, is essential for tRNA modification and neuronal development. Mutations in ELP1 underlie familial dysautonomia (FD), a disorder marked by sensory and autonomic neuropathy. While loss of Elp1 disrupts trigeminal ganglion formation and survival, the downstream molecular consequences remain poorly defined. ResultsWe performed quantitative proteomic profiling of trigeminal ganglia from Elp1 conditional knockout (CKO) and control embryos at E13.5. Across 5,650 detected proteins, 25 were significantly upregulated and 26 downregulated in Elp1 CKO embryos. EnrichR analysis revealed enrichment of upregulated proteins in amino acid transport and tRNA aminoacylation pathways, with links to neuromuscular and neuropathic diseases. Downregulated proteins were associated with RNA modification, cholesterol biosynthesis, and synaptic organization. Validation by immunohistochemistry confirmed decreased expression of the neurotrophic receptor Gfra3 and the neuropeptide Galanin, and increased levels of the chromatin regulator Chd1, in Elp1 CKO embryos relative to controls. ConclusionsThese findings demonstrate that Elp1 loss disrupts metabolic, RNA modification, and neurotrophic signaling pathways in the developing trigeminal ganglion. Proteomic analysis thus provides new insight into the molecular consequences of Elp1 deficiency and highlights candidate mechanisms contributing to sensory neuron vulnerability in FD.

IntroductionRobinow syndrome has characteristic craniofacial and dental features and can be caused by gain- or loss-of-function variants in Wnt family member 5A (WNT5A) non-canonical signaling. The craniofacial and dental manifestation of Robinow syndrome is heterogenous, as is the effect of altered Wnt5a in animal models. The relationship between Wnt5a and craniofacial and dental phenotypes is not fully understood. MethodsTo investigate the role of Wnt5a in craniofacial and dental development, we utilized a Wnt5a conditional loss-of-function (LOF: Wnt5afl/fl;Ctskcre) and a Wnt5a conditional gain-of-function (GOF: Rosa26-LSL-Wnt5a;Ctskcre) model to determine the effect of both LOF and GOF of Wnt5a in bone cells during craniofacial and dental development. Postnatal day 10 conditional LOF Wnt5a, GOF Wnt5a, and control skulls were scanned by micro-computed tomography and assessed using traditional and geometric morphometrics. Mandibular bone apoptosis was further assessed by TUNEL staining. ResultsConditional Wnt5a LOF resulted in midface hypoplasia, increased maxillary intermolar width, increased rostral basisphenoid width, and delayed molar eruption. Wnt5a LOF mandibles did not have altered bone mineral density or bone microarchitecture unlike our previous study examining Wnt5a LOF femurs. In contrast, conditional Wnt5a GOF results in macrocephaly, shortened hard palate, increased zygomatic length, micrognathia, and mandibular process morphology changes. The micrognathia and mandibular process morphology changes in the Wnt5a GOF mice were not due to increased apoptosis. A partially penetrant snout deviation was present in both the Wnt5a LOF and GOF mice. ConclusionsCraniofacial and dental phenotype differed between mice with conditional GOF and LOF of Wnt5a, consistent with the craniofacial phenotype heterogeneity in Wnt5a-associated Robinow syndrome. We detected tooth eruption delay, mandibular condyle dysmorphology, and facial asymmetry in mice with altered Wnt5a that have not been previously reported in patients. Our data suggest precise regulation of Wnt5a is essential for proper craniofacial and dental development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/665966v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@971284org.highwire.dtl.DTLVardef@40d5f6org.highwire.dtl.DTLVardef@9f0caforg.highwire.dtl.DTLVardef@1f9ba9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Objective and approachesAberrantly proliferating cells are linked to a number of diseases including cancers and developmental defects.To determine the extent to which local extrinsic signals contribute to or ameliorate mutant cell behaviors, we examined survival and differentiation of mutant cells in wild-type retinal environments by generating chimeric zebrafish embryos comprised of unlabeled host cells and GFP-labeled neural progenitor donor cells. In addition, we examined the fate of retinal progenitor cells when cdkn1c, a cyclin dependent kinase inhibitor, was induced in clones within wild-type and hdac1 mutant retinae.\n\nResultsWe found that seven of the ten mutants examined exhibited apoptosis when grafted into wild-type tissue, with cells from two slowly cycling mutants, elys and emi1, noticeably differentiating in a wild-type environment. Observations of the one hyperproliferative mutant, hdac1, revealed that these mutant cells did not appear to die or differentiate but instead survived and formed tumor-like rosettes in a wild-type environment. Ectopic expression of cdkn1c was unable to force cell cycle exit and differentiation of the majority of hdac1 mutant cells.\n\nConclusionsTogether, these results suggest that although a wild-type environment rarely encourages cell cycle exit and differentiation of neural progenitors with cell cycle defects, wild-type survival signals may enable hyperproliferative progenitor cells to persist instead of die.

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural fold borders into multiple cell lineages that differentiate into bone, cartilage, neurons, and glial cells. We previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicates that TWIST1 interacts with /{beta}/{gamma}-CATENINS during neural tube closure, and Irf6 is involved in the structural integrity of the neural tube. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects, and CNCC-derived structural abnormalities. Altogether, this study describes an uncharacterized function of Twist1 and Irf6 in the neural tube and CNCCs and provides new target genes of Twist1 involved in cytoskeletal remodeling. Furthermore, the association between DNA variations within TWIST1 putative enhancers and human facial morphology is also investigated. SUMMARY STATEMENTThis study uncovers a new function of Twist1 in neural tube development and epithelial-to-mesenchymal transition in cranial neural crest cells. Data further shows that Twist1-interacting Irf6 is involved in regulating neural tube integrity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy caused by aberrant expression of the DUX4 retrogene, and it affects skeletal muscles primarily in the face, shoulder, and limbs. In healthy individuals, DUX4 is expressed in early development and is subsequently silenced in most somatic tissues. The spatiotemporal pattern of DUX4 mis-expression beyond the cleavage stage in FSHD is poorly understood because DUX4 is not well conserved beyond primates. Here, we generated Cre reporter mouse lines with human DUX4 regulatory elements to investigate the cell lineages derived from DUX4-expressing cells in embryos and adults. Intriguingly, we found that DUX4-expressing cell lineages were present in embryonic forelimb, hindlimb, and face. In adults, the reporter was expressed strongly in testis and to a lesser extent in other tissues, including weak, sporadic expression in skeletal muscles, reminiscent of mosaic DUX4 expression in FSHD. Within skeletal muscles, DUX4 lineage cells include pericytes, an interstitial cell that contributes to muscle regeneration and repair. Overall, this study introduces a new research tool for the field, and provides new insight into potential developmental mechanisms underlying FSHD pathophysiology. Summary statementCre reporter mouse lines with human DUX4 regulatory elements are discovery tools for developmental processes and mechanisms underlying FSHD pathophysiology.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. We use the sea urchin embryo to achieve a comprehensive understanding of miR-1s function in a developing embryo. Results indicate that miR-1 regulates gut contractions, specification, and positioning of serotonergic neurons, as well as mesodermally-derived muscles, pigment cells, and skeletogenic cells. Gain-of-function of miR-1 generally leads to more severe developmental defects than its loss-of-function. We identified that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Notch, Nodal, and Wnt1 signaling components. We found that miR-1s direct suppression of Nodal may indirectly regulate FoxQ2 to impact serotonergic neurons. Excess miR-1 may lead to decreased Nodal and Notch that result in decreased circumpharnygeal muscle fibers and the number of pigment cells. The striking ectopic skeletal branching induced by miR-1 mimic injections may be explained by miR-1s direct suppression of Nodal that leads to expression changes of Vegf3, and Fgfa that mediate skeletogenesis. This work demonstrates that miR-1 plays a diverse regulatory role that impacts tissues derived from all germ layers. Summary statementThis study identifies wide-ranging regulatory roles and regulatory mechanisms of miR-1 that impact structures derived from all three germ layers during embryonic development.

Neural crest (NC) cells are a transient, multipotent cell population that contributes to the formation of diverse tissues during vertebrate development. While numerous transcription factors and signalling pathways regulate NC specification and differentiation, the role of microRNAs (miRNAs) in these processes remains underexplored. In this study, we employed a double-transgenic zebrafish model (Tg(sox10:eGFP, sox10:mRFP)), in combination with fluorescence-activated cell sorting (FACS) and RNA sequencing, to identify miRNAs enriched in NC cells and assess their role in the development of NC derivatives. Given the parallels between NC development and tumorigenesis, we focused on a subset of miRNAs previously implicated in cancer progression. Functional assays revealed that overexpression of miR-133a and miR-338-3p resulted in significant craniofacial cartilage malformations and a reduction in melanophore numbers, correlating with the downregulation of key developmental genes, such as sox9b, sox10, and runx3. Reporter assays further demonstrated that miR-133a and miR-338-3p directly target the 3 untranslated region (3UTR) of sox9b, supporting their role in translational repression and mRNA degradation. Additionally, miR-338-3p overexpression increased the overall NC cell population, suggesting a regulatory function in cell proliferation. These findings offer new insights into the molecular mechanisms governing NC development and highlight functional parallels between miRNA-mediated NC regulation and tumour biology. Given that miR-133a and miR-338-3p are known tumour suppressors, their roles in NC differentiation and proliferation may reflect conserved pathways underlying both embryogenesis and oncogenesis. This study enhances our understanding of miRNA function in vertebrate embryogenesis and underscores their potential relevance in neurocristopathies and NC-derived cancers.