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Regenerating tissues must remember or interpret their spatial position, using this information to restore original size and patterning. The external skeleton of the zebrafish caudal fin is composed of 18 rays; after any portion of the fin is amputated, position-dependent regenerative growth restores each ray to its original length. We tested for transcriptional differences during regeneration of proximal versus distal tissues and identified 489 genes that differed in proximodistal expression. Thyroid hormone directs multiple aspects of ray patterning along the proximodistal axis, and we identified 364 transcripts showing a proximodistal expression pattern that was dependent on thyroid hormone context. To test what aspects of ray positional identity are directed by extrinsic environental cues versus remembered identity autonomous to the tissue, we transplanted distal portions of rays to proximal environments and evaluated regeneration within the new location. Native regenerating proximal tissue showed robust expression of scpp7 , a transcript with thyroid-regulated proximal enrichment; in contrast, regenerating rays originating from transplanted distal tissue showed reduced (distal-like) expression during outgrowth. These distal-to-proximal transplants regenerated far beyond the length of the graft itself, indicating that cues from the proximal environment promoted additional growth. Nonetheless, these transplants initiated regeneration at a much slower rate compared to controls, suggesting memory of distal identity was retained by the transplanted tissue. This early growth retardation caused rays that originated from transplants to grow noticeably shorter than neighboring native rays. While several aspects of fin ray morphology (bifurcation, segment length) were found to be determined by the environment, we found that both regeneration speed and ray length are remembered autonomously by tissues, and that persist through multiple rounds of amputation and regeneration. [Display omitted] • Gene expression patterns during fin regeneration correspond to proximodistal location. • Distal portions of rays transplanted to proximal regions retain positional identity that influences growth rate and length during regeneration. • Ray patterning along the proximodistal axis is determined by the regenerative environment, not remembered identity. [ABSTRACT FROM AUTHOR]

The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires. [Display omitted] • Rohon-Beard neurons are primary somatosensory neurons in the dorsal spinal cord of fishes and amphibians. • The entire population of Rohon-Beard neurons survives until free-swimming and juvenile stages in zebrafish. • Concomitant processes may have produced the illusion of disappearance over time. • Canonical cell death markers are absent in Rohon-Beard neurons. • Rohon-Beard neurons co-exist with neurons of the Dorsal Root Ganglia, which form later and are part of the secondary somatosensory system [ABSTRACT FROM AUTHOR]

During neural development, sculpting of early formed circuits by cell death and synaptic pruning is necessary to generate a functional and efficient nervous system. This allows for the establishment of rudimentary circuits which necessitate early organism survival to later undergo subsequent refinement. These changes facilitate additional specificity to stimuli which can lead to increased behavioral complexity. In multiple species, Rohon-Beard neurons (RBs) are the earliest mechanosensory neurons specified and are critical in establishing a rudimentary motor response circuit. Sensory input from RBs gradually becomes redundant as dorsal root ganglion (DRG) neurons develop and integrate into motor circuits. Previous studies demonstrate that RBs undergo a dramatic wave of cell death concurrent with development of the DRG. However, contrary to these studies, we show that neurogenin1 + (ngn1) RBs do not undergo a widespread wave of programmed cell death during early zebrafish development and instead persist until at least 15 days post fertilization (dpf). Starting at 2 dpf, we also observed a dramatic medialization and shrinkage of ngn1 + RB somas along with a gradual downregulation of ngn1 in RBs. This alters a fundamental premise of early zebrafish neural development and opens new avenues to explore mechanisms of RB function, persistence, and circuit refinement. [Display omitted] • Rohon-Beard neurons do not succumb to cell death during early zebrafish development. • The vast majority of Rohon-Beard neurons do not express markers of cell death. • Rohon-Beard neurons persist until 15 days post fertilization. [ABSTRACT FROM AUTHOR]

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen2 1-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG2 11 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG2 1-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications. [Display omitted] • The split-mNG2 system achieves tissue-specific protein labeling while maintaining endogenous expression levels. • mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis. • mNG2 11 is fused to proteins of interest using CRISPR/Cas-directed gene editing. • The feasibility of the split-mNG2 approach is demonstrated for the cytoskeletal genes tubb4b and krt8. • The split-mNG2 system can also be used to direct protein localization. [ABSTRACT FROM AUTHOR]

The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene – kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) – have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2 −/− mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2 −/− . These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects. [Display omitted] • Two KCNG4b mutants differentially affect Kv channel properties. • The C1 mutant represents the milder version of Kcng4b loss-of-function. • The C2 mutant polypeptide has the dominant negative properties. • The number of otoliths in the C1 mutant ear is increased. • The C2 mutant ear contains no otoliths. [ABSTRACT FROM AUTHOR]

In the zebrafish lateral line, non-sensory supporting cells readily re-enter the cell cycle to generate new hair cells and supporting cells during homeostatic maintenance and following damage to hair cells. This contrasts with supporting cells from mammalian vestibular and auditory sensory epithelia which rarely re-enter the cell cycle, and hence loss of hair cells results in permanent sensory deficit. Lateral line supporting cells are derived from multipotent progenitor cells that migrate down the trunk midline as a primordium and are deposited to differentiate into a neuromast. We have found that we can revert zebrafish support cells back to a migratory progenitor state by pharmacologically altering the signaling environment to mimic that of the migratory primordium, with active Wnt signaling and repressed FGF signaling. The reverted supporting cells migrate anteriorly and posteriorly along the horizontal myoseptum and will re-epithelialize to form an increased number of neuromasts along the midline when the pharmacological agents are removed. These data demonstrate that supporting cells can be readily reprogrammed to a migratory multipotent progenitor state that can form new sensory neuromasts, which has important implications for our understanding of how the lateral line system matures and expands in fish and also suggest avenues for returning mammalian supporting cells back to a proliferative state. [Display omitted] • Zebrafish neuromasts can be pharmacologically reprogrammed to a migratory state. • Reprogrammed progenitors migrate along midline. • Migratory progenitors reaggregate into sensory organs following washout. • Reconstituted sensory organs form functional sensory cells and are innervated. [ABSTRACT FROM AUTHOR]

Genome duplications and ploidy transitions have occurred in nearly every major taxon of eukaryotes, but they are far more common in plants than in animals. Due to the conservation of the nuclear:cytoplasmic volume ratio increased DNA content results in larger cells. In plants, polyploid organisms are larger than diploids as cell number remains relatively constant. Conversely, vertebrate body size does not correlate with cell size and ploidy as vertebrates compensate for increased cell size to maintain tissue architecture and body size. This has historically been explained by a simple reduction in cell number that matches the increase in cell size maintaining body size as ploidy increases, but here we show that the compensatory mechanisms that maintain body size in triploid zebrafish are tissue-specific: A) erythrocytes respond in the classical pattern with a reduced number of larger erythrocytes in circulation, B) muscle, a tissue comprised of polynucleated muscle fibers, compensates by reducing the number of larger nuclei such that myofiber and myotome size in unaffected by ploidy, and C) vascular tissue compensates by thickening blood vessel walls, possibly at the expense of luminal diameter. Understanding the physiological implications of ploidy on tissue function requires a detailed description of the specific mechanisms of morphological compensation occurring in each tissue to understand how ploidy changes affect development and physiology. [Display omitted] • Tissue-specific mechanisms compensate for changes in cell size. • Blastomere size, cleavage pattern and rate are not affected by ploidy. • Erythrocytes conform to the canonical pattern of 'fewer but larger cells'. • Multinucleate skeletal muscle cells adjust the number of nuclei to maintain size. • Vasculature is comprised of the same number and pattern of larger endothelial cells. [ABSTRACT FROM AUTHOR]

Despite extensive studies on endogenous heart regeneration within the past 20 years, the players involved in initiating early regeneration events are far from clear. Here, we assessed the function of neutrophils, the first-responder cells to tissue damage, during zebrafish heart regeneration. We detected rapid neutrophil mobilization to the injury site after ventricular amputation, peaking at 1-day post-amputation (dpa) and resolving by 3 dpa. Further analyses indicated neutrophil mobilization coincides with peak epicardial cell proliferation, and recruited neutrophils associated with activated, expanding epicardial cells at 1 dpa. Neutrophil depletion inhibited myocardial regeneration and significantly reduced epicardial cell expansion, proliferation, and activation. To explore the molecular mechanism of neutrophils on the epicardial regenerative response, we performed scRNA-seq analysis of 1 dpa neutrophils and identified enrichment of the FGF and MAPK/ERK signaling pathways. Pharmacological inhibition of FGF signaling indicated its' requirement for epicardial expansion, while neutrophil depletion blocked MAPK/ERK signaling activation in epicardial cells. Ligand-receptor analysis indicated the EGF ligand, hbegfa , is released from neutrophils and synergizes with other FGF and MAPK/ERK factors for induction of epicardial regeneration. Altogether, our studies revealed that neutrophils quickly motivate epicardial cells, which later accumulate at the injury site and contribute to heart regeneration. [Display omitted] • Neutrophil mobilization coincides with peak epicardial cell proliferation. • Neutrophils associated with activated, expanding epicardial cells. • Neutrophil significantly reduced epicardial cell expansion and activation. • FGF signaling is required for epicardial expansion. [ABSTRACT FROM AUTHOR]

The proneural factor Ascl1 is involved in several steps of neurogenesis, from neural progenitor maintenance to initiation of terminal differentiation and neuronal subtype specification. In neural progenitor cells, Ascl1 initiates the cell-cycle exit of progenitors, and contributes to their differentiation into mainly GABAergic neurons. Several catecholaminergic neuron groups in the forebrain of zebrafish use GABA as co-transmitter, but a potential role of the two paralogues Ascl1a and Ascl1b in their neurogenesis is not understood. Here, we show that ascl1a , ascl1b double mutant embryos develop a significantly reduced number of neurons in all GABAergic and catecholaminergic dual transmitter neuron anatomical clusters in the fore- and hindbrain, while glutamatergic catecholaminergic clusters develop normally. However, none of the affected catecholaminergic cell clusters are lost completely, suggesting an impairment in progenitor pools, or a requirement of Ascl1a/b for differentiation of a subset of neurons in each cluster. Early progenitors which are dlx2a +, fezf2 + or emx2 + are not reduced whereas late progenitors and differentiating neurons marked by the expression of dlx5a , isl1 and arxa are severely reduced in ascl1a , ascl1b double mutant embryos. This suggests that Ascl1a and Ascl1b play only a minor or no role in the maintenance of their progenitor pools, but rather contribute to the initiation of terminal differentiation of GABAergic catecholaminergic neurons. [Display omitted] • Ascl1 controls catecholaminergic and GABAergic dual transmitter neuron development. • Dopaminergic dlx2a progenitor populations form normally in Ascl1-deficent embryos. • Ascl1 required for terminal differentiation of dopaminergic GABAergic neurons. • Differentiation of hindbrain noradrenergic neurons depends on Ascl1. • Ascl1 also required for prethalamic Sst1.1 GABAergic neurons. [ABSTRACT FROM AUTHOR]

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However , the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals. [Display omitted] • We develop a new strategy for a knock-in reporter generation utilizing minicircle technology and a minimal promoter. • Minicircle serve as an efficient donor template for knock-in reporter generation in zebrafish. • Addition of an ectopic promoter can resolve the unforeseeable problem to create a knock-in reporter line. [ABSTRACT FROM AUTHOR]

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration. [Display omitted] • Imaging approaches to investigate zebrafish juvenile heart development and adult regeneration. • Novel whole volume, medium throughput imaging optimized for zebrafish heart. • Reliable and convenient tissue clearing and antibody labeling. • Live time-lapse imaging to capture cardiac regenerative processes. • Methods optimized for coronary vessels but applicable to other models/organs. [ABSTRACT FROM AUTHOR]

Transmembrane p24 trafficking protein 10 (TMED10) is a conserved vesicle trafficking protein. It is dysregulated in Alzheimer disease and plays a pivotal role in the pathogenesis of Alzheimer disease. In addition to the brain, TMED10 is highly expressed in the exocrine pancreas; however, its biological functions and underlying mechanisms remain largely unknown. We studied reduced Tmed10 in zebrafish embryos by morpholino oligonucleotide knockdown and CRISPR-Cas9 mutagenesis. Tmed10-deficient embryos showed extensive loss of acinar mass and impaired acinar differentiation. TMED10 has been reported to have an inhibitory effect on γ-secretase. As one of the substrates of γ-secretase, membrane-bound β-catenin was significantly reduced in Tmed10-deficient embryos. Increased γ-secretase activity in wild-type embryos resulted in a phenotype similar to that of tmed10 mutants. And the mutant phenotype could be rescued by treatment with the γ-secretase inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester (DAPT). In addition, the reduced membrane-bound β-catenin was accompanied with up-regulated β-catenin target genes in Tmed10-deficient embryos. Overexpression of β-catenin signaling inhibitor Dickkopf-1 (DKK-1) could rescue the exocrine pancreas defects. Taken together, our study reveals that Tmed10 regulates exocrine pancreatic differentiation through γ-secretase. Reduced membrane-bound β-catenin, accompanied with hyperactivation of β-catenin signaling, is an important cause of exocrine pancreas defects in Tmed10-deficient embryos. Our study reaffirms the importance of appropriate β-catenin signaling in exocrine pancreas development. These findings may provide a theoretical basis for the development of treatment strategies for TMED10 -related diseases. [Display omitted] • Tmed10 is a conserved protein, and its mRNA is expressed in the exocrine pancreas. • Tmed10-deficent embryos exhibited impaired differentiation of exocrine cell. • Inhibition of γ-secretase by DAPT restores exocrine pancreas formation in Tmed10-deficient embryos. • Overexpression of DKK-1 restores exocrine pancreas formation in Tmed10-deficient embryos. [ABSTRACT FROM AUTHOR]

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis. [Display omitted] • LSD1 mutant zebrafish has defects in definitive hematopoiesis. • Zebrafish LSD1 has a critical role in HSPC development. • LSD1 promotes the egress of HSPCs into the bloodstream. [ABSTRACT FROM AUTHOR]

Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes. [Display omitted] • The sclerotome is the embryonic source of distinct fibroblasts in zebrafish. • The sclerotome contributes to the proper development of the axial skeleton. • The sclerotome mostly contains unipotent and bipotent progenitors. • Sclerotome differentiation is biased by the axial position and migratory path. [ABSTRACT FROM AUTHOR]

Animal development and homeostasis depend on precise temporal and spatial intercellular signaling. Components shared between signaling pathways, generally thought to decrease specificity, paradoxically can also provide a solution to pathway coordination. Here we show that the Bone Morphogenetic Protein (BMP) and Wnt signaling pathways share Apcdd1 as a common inhibitor and that Apcdd1 is a taxon-restricted gene with novel domains and signaling functions. Previously, we showed that Apcdd1 inhibits Wnt signaling (Shimomura et al., 2010), here we find that Apcdd1 potently inhibits BMP signaling in body axis formation and neural differentiation in chicken, frog, zebrafish. Furthermore, we find that Apcdd1 has an evolutionarily novel protein domain. Our results from experiments and modeling suggest that Apcdd1 may coordinate the outputs of two signaling pathways that are central to animal development and human disease.

Urp1 and Urp2 are two neuropeptides, members of the Urotensin 2 family, that have been recently involved in the control of body axis morphogenesis in zebrafish. They are produced by a population of sensory spinal neurons, called cerebrospinal fluid contacting neurons (CSF-cNs), under the control of signals relying on the Reissner fiber, an extracellular thread bathing in the CSF. Here, we have investigated further the function of Urp1 and Urp2 (Urp1/2) in body axis formation and maintenance. We showed that urp1;urp2 double mutants develop strong body axis defects during larval growth, revealing the redundancy between the two neuropeptides. These defects were similar to those previously reported in uts2r3 mutants. We observed that this phenotype is not associated with congenital defects in vertebrae formation, but by using specific inhibitors, we found that, at least in the embryo, the action of Urp1/2 signaling depends on myosin II contraction. Finally, we provide evidence that while the Urp1/2 signaling is functioning during larval growth, it is dispensable for embryonic development. Taken together, our results show that Urp1/2 signaling is required in larvae to promote correct vertebral body axis, most likely by regulating muscle tone. [Display omitted] • urp1;urp2 double mutants develop strong spine deformations during larval growth. • Urp1 and Urp2 can act through myosin II contraction. • Urp1 and Urp2 are dispensable for embryonic development. [ABSTRACT FROM AUTHOR]

HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3 / HES3's role in neurogenesis. To investigate HES3's function during development , we generated a zebrafish CRISPR/Cas9 null mutation of her3 , the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants exhibit dysregulated neurog1 expression, a her3 target gene, and the mutant her3 fails to bind the neurog1 promoter sequence. Further, her3 mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 null mutant embryos are enriched for Hox and Sox10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3 -mediated neural development and its misappropriation in cancer contexts. [Display omitted] • Generated zebrafish her3 CRISPR/Cas9 knockouts with loss-of-function mutations. • her3 mutant is unable to bind known target, neurog1 , causing dysregulated expression. • her3 loss causes abnormal expression of core subset of developmental regulatory genes. • Sox10 DNA binding motifs are enriched in her3 knockout differentially expressed genes. • Tumor microenvironment and matrix metalloprotease pathways are enriched in her3 knockout. [ABSTRACT FROM AUTHOR]

The extracellular signal-regulated kinase (ERK1/2) pathway is essential in embryonic development. The scaffold protein Shoc2 is a critical modulator of ERK1/2 signals, and mutations in the shoc2 gene lead to the human developmental disease known as Noonan-like syndrome with loose anagen hair (NSLH). The loss of Shoc2 and the shoc2 NSLH-causing mutations affect the tissues of neural crest (NC) origin. In this study, we utilized the zebrafish model to dissect the role of Shoc2-ERK1/2 signals in the development of NC. These studies established that the loss of Shoc2 significantly altered the expression of transcription factors regulating the specification and differentiation of NC cells. Using comparative transcriptome analysis of NC-derived cells from shoc2 CRISPR/Cas9 mutant larvae, we found that Shoc2-mediated signals regulate gene programs at several levels, including expression of genes coding for the proteins of extracellular matrix (ECM) and ECM regulators. Together, our results demonstrate that Shoc2 is an essential regulator of NC development. This study also indicates that disbalance in the turnover of the ECM may lead to the abnormalities found in NSLH patients. [Display omitted] • Shoc2 loss alters the transcriptional gene regulation of neural crest. • All phases of neural crest development and its derivatives require Shoc2-ERK1/2 signals. • Expression of extracellular matrix proteins is mediated by Shoc2 signals.• Shoc2 controls transcriptional circuits required for chondrogenesis and osteogenesis [ABSTRACT FROM AUTHOR]

The process of morphogenesis carefully crafts cells into complex organ structures which allows them to perform their unique functions. During brain development, the neuroepithelial tissue must go through apical and basal folding which is mediated through the instruction of both intrinsic and extrinsic factors. While much is known about apical folding, the mechanisms that regulate basal folding are less understood. Using the highly conserved zebrafish midbrain-hindbrain boundary (MHB) as an epithelial tissue model, we have identified the basement membrane protein laminin-111 as a key extrinsic factor in basal tissue folding. Laminin-111 is a highly conserved, heterotrimeric protein that lines the basal surface of the neuroepithelium. Laminin-111 is comprised of one alpha, one beta, and one gamma chain encoded by the genes lama1 , lamb1 , and lamc1 , respectively. Human mutations in individual laminin-111 genes result in disparate disease phenotypes; therefore, we hypothesized that each laminin gene would have a distinctive role in tissue morphogenesis. Using zebrafish mutants for laminin-111 genes, we found that each laminin chain has a unique impact on basal folding. We found that lamc1 is the most critical gene for MHB morphogenesis, followed by lama1 , and finally lamb1a. This hierarchy was discovered via three-dimensional single cell shape analysis, localization of myosin regulatory light chain (MRLC), and with analysis of MHB tissue folding later in development. These findings are essential for development of novel techniques in tissue engineering and to elucidate differences in human diseases due to specific chain mutations. [Display omitted] • Each laminin-111 gene is required for MHB basal epithelial tissue folding. • MHB tissue shape and cell shape are dependent on each laminin-111 gene. • Laminin-111 genes affect localization of intracellular cytoskeletal mediators. • A hierarchy exists across laminin-111 genes in regulating MHB morphogenesis. • lamc1 is the most critical for MHB formation, followed by lama1, then lamb1a. [ABSTRACT FROM AUTHOR]

Enhancers and promoters are cis -regulatory elements that control gene expression. Enhancers are activated in a cell type-, tissue-, and condition-specific manner to stimulate promoter function and transcription. Zebrafish have emerged as a powerful animal model for examining the activities of enhancers derived from various species through transgenic enhancer assays, in which an enhancer is coupled with a minimal promoter. However, the efficiency of minimal promoters and their compatibility with multiple developmental and regeneration enhancers have not been systematically tested in zebrafish. Thus, we assessed the efficiency of six minimal promoters and comprehensively interrogated the compatibility of the promoters with developmental and regeneration enhancers. We found that the fos minimal promoter and Drosophila synthetic core promoter (DSCP) yielded high rates of leaky expression that may complicate the interpretation of enhancer assays. Notably, the adenovirus E1b promoter, the zebrafish lepb 0.8-kb (P0.8) and lepb 2-kb (P2) promoters, and a new zebrafish synthetic promoter (ZSP) that combines elements of the E1b and P0.8 promoters drove little or no ectopic expression, making them suitable for transgenic assays. We also found significant differences in compatibility among specific combinations of promoters and enhancers, indicating the importance of promoters as key regulatory elements determining the specificity of gene expression. Our study provides guidelines for transgenic enhancer assays in zebrafish to aid in the discovery of functional enhancers regulating development and regeneration. [Display omitted] • fos and DSCP minimal promoters drive high rates of leaky expression. • Developmental enhancers are preferentially compatible with E1b promoter. • Regeneration enhancers are preferentially compatible with P0.8 and P2 promoters. • ZSP promoter is highly compatible with developmental and regeneration enhancers. [ABSTRACT FROM AUTHOR]

The axonal projections of retinal ganglion cells (RGCs) of the eye are topographically organized so that spatial information from visual images is preserved. This retinotopic organization is established during development by secreted morphogens that pattern domains of transcription factor expression within naso-temporal and dorso-ventral quadrants of the embryonic eye. Poorly understood are the downstream signaling molecules that generate the topographically organized retinal cells and circuits. The secreted signaling molecule Semaphorin 3fa (Sema3fa) belongs to the Sema family of molecules that provide positional information to developing cells. Here, we test a role for Sema3fa in cell genesis of the temporal zebrafish retina. We compare retinal cell genesis in wild type and sema3fa CRISPR zebrafish mutants by in situ hybridization and immunohistochemistry. We find that mRNAs for sema3fa and known receptors, neuropilin2b (nrp2b) and plexina1a (plxna1a), are expressed by progenitors of the temporal, but not nasal zebrafish embryonic retina. In the sema3fa ca304/ca304 embryo, initially the domains of expression for atoh7 and neurod4 , transcription factors necessary for the specification of RGCs and amacrine cells, respectively, are disrupted. Yet, post-embryonically only amacrine cells of the temporal retina are reduced in numbers, with both GABAergic and glycinergic subtypes affected. These data suggest that Sema3fa acts early on embryonic temporal progenitors to control in a spatially-dependent manner the production of amacrine cells, possibly to allow the establishment of neural circuits with domain-specific functions. We propose that spatially restricted extrinsic signals in the neural retina control cell genesis in a domain-dependent manner. [Display omitted] • mRNA for the secreted Semaphorin 3fa is expressed by temporal eye progenitors. • Sema3fa controls the production of amacrine cells in the temporal retina. • Spatial extrinsic cues control domain-restricted retinal cell genesis. [ABSTRACT FROM AUTHOR]

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development. [Display omitted] • Myod and Myf5 are required for dorsal aorta and intersegmental vessel formation. • Myod and Myf5 are required for somitic expression of the VEGF ligand vegfaa. • Restoring vegfaa expression in Myod/Myf5 double mutants rescues aortic fate. • Myod and Myf5 function cooperatively with Hedgehog signaling to induce vegfaa. • vegfaa maintenance requires continued bHLH activity in muscle cells. Summary statement: The myogenic transcription factors Myod and Myf5 have a novel function in inducing the artery through regulation of VEGF. [ABSTRACT FROM AUTHOR]

The vertebrate peripheral nervous system (PNS) is an intricate network that conveys sensory and motor information throughout the body. During development, extracellular cues direct the migration of axons and glia through peripheral tissues. Currently, the suite of molecules that govern PNS axon-glial patterning is incompletely understood. To elucidate factors that are critical for peripheral nerve development, we characterized the novel zebrafish mutant, stl159 , that exhibits abnormalities in PNS patterning. In these mutants, motor and sensory nerves that develop adjacent to axial muscle fail to extend normally, and neuromasts in the posterior lateral line system, as well as neural crest-derived melanocytes, are incorrectly positioned. The stl159 genetic lesion lies in the basic helix-loop-helix (bHLH) transcription factor tcf15 , which has been previously implicated in proper development of axial muscles. We find that targeted loss of tcf15 via CRISPR-Cas9 genome editing results in the PNS patterning abnormalities observed in stl159 mutants. Because tcf15 is expressed in developing muscle prior to nerve extension, rather than in neurons or glia, we predict that tcf15 non-cell-autonomously promotes peripheral nerve patterning in zebrafish through regulation of extracellular patterning cues. Our work underscores the importance of muscle-derived factors in PNS development. The transcription factor tcf15 (also called paraxis) is expressed in embryonic zebrafish paraxial mesoderm, prior to peripheral nervous system (PNS) development. Expression of tcf15 is necessary for proper development of axons and glia in the posterior lateral line nerve (green), neuromasts (yellow), motor nerves (red), and neural crest-derived melanocytes (black) in larval zebrafish. In tcf15 mutants, muscle appears grossly normal, but nerves, neuromasts, and melanocytes that migrate along muscle are mispatterned. [Display omitted] • Neural crest-derived melanocytes and glia develop abnormally in stl159 zebrafish. • The stl159 lesion truncates tcf15, which is required for proper muscle development. • Sensory and motor nerves adjacent to axial muscle require tcf15 for patterning. • Early tcf15 expression in developing muscle precedes peripheral nerve patterning. [ABSTRACT FROM AUTHOR]

Craniofacial skeletal elements are derived from cranial neural crest cells (CNCCs), which migrate along discrete paths and populate distinct pharyngeal arches, structures that are separated by the neighboring endodermal pouches (EPs). Interactions between the CNCCs and the endoderm are critical for proper craniofacial development. In zebrafish, integrin α5 (Itga5) functions in the endoderm to regulate formation of specifically the first EP (EP1) and the development of the hyoid cartilage. Here we show that fibronectin (Fn), a major component of the extracellular matrix (ECM), is also required for these developmental processes, and that the penetrance of defects in mutants is temperature-dependent. fn1a −/− embryos exhibited defects that are similar to, but much more severe than, those of itga5 −/− embryos, and a loss of integrin av (itgav) function enhanced both endoderm and cartilage defects in itga5 −/− embryos, suggesting that Itga5 and Itgav cooperate to transmit signals from Fn to regulate the development of endoderm and cartilage. Whereas the endodermal defects in itga5; itga5v −/− double mutant embryos were comparable to those of fn1a −/− mutants, the cartilage defects were much milder. Furthermore, Fn assembly was detected in migrating CNCCs, and the epithelial organization and differentiation of CNCC-derived arches were impaired in fn1a −/− embryos, indicating that Fn1 exerts functions in arch development that are independent of Itga5 and Itgav. Additionally, reduction of itga5 function in fn1a −/− embryos led to profound defects in body axis elongation, as well as in endoderm and cartilage formation, suggesting that other ECM proteins signal through Itga5 to regulate development of the endoderm and cartilage. Thus, our studies reveal that Fn1a and Itga5 have both overlapping and independent functions in regulating development of the pharyngeal endoderm and cartilage. [Display omitted] • Fn1a is required for morphogenesis of the pharyngeal endoderm and the development of cartilage. • Integrin α5 interacts with integrin αv to regulate endoderm morphogenesis and cartilage formation. • Fn1a likely functions through integrin α5 and integrin αv to orchestrate endoderm development. • Fn1a is critical for the formation of cranial neural crest cell-derived arches. [ABSTRACT FROM AUTHOR]

The vertebrate skeleton changes its shape during development through the activities of chondrocytes, osteoblasts and osteoclasts. Although much is known about the mechanisms for differentiation in these cells, it is less understood how they behave in a region-specific manner to acquire unique bone shapes. To address this question, we investigated the development of the hyomandibular (Hm) system in zebrafish. The Hm originates as cartilage carrying a single foramen (the Hm foramen), through which the facial (VII) nerve passes. We reveal that Schwann cells, which myelinate the VII nerve, regulate rearrangement of the chondrocytes to enlarge the Hm foramen. The Hm cartilage then becomes ossified in the perichondrium, where the marrow chondrocytes are replaced by adipocytes. Then, the bone matrix along the VII nerve is resorbed by osteoclasts, generating a gateway to the bone marrow. Subsequent movement of the VII nerve into the marrow, followed by deposition of new bone matrix, isolates the nerve from the jaw muscle insertion. Genetic ablation of osteoblasts and osteoclasts reveals specific roles of these cells during remodeling processes. Interestingly, the VII nerve relocation does not occur in medaka; instead, bone deposition distinct from those in zebrafish separates the VII nerve from the muscle insertion. Our results define novel mechanisms for skeletal remodeling, by which the bone shapes in a region- and species-specific manner. [Display omitted] • Hyomandibular (Hm) skeleton development was analyzed in zebrafish. • Schwann cell regulation of chondrocyte rearrangement enlarges embryonic Hm foramen. • Osteoclasts degrade bone matrix to relocate the facial nerve into the bone marrow. • Osteoblasts deposit new bone matrix to the Hm, allowing insertion of jaw muscles. • Facial nerve relocation does not occur in the Hm bone of medaka. [ABSTRACT FROM AUTHOR]

During neural development, progenitor cells generate different types of neurons in specific time windows. Despite the characterisation of many of the transcription factor networks involved in these differentiation events, the mechanism behind their temporal regulation is poorly understood. To address this question, we studied the temporal differentiation of the simple lateral floor plate (LFP) domain in the zebrafish spinal cord. LFP progenitors generate both early-born Kolmer-Agduhr" (KA") interneuron and late-born V3 interneuron populations. Analysis using a Notch signalling reporter demonstrates that these cell populations have distinct Notch signalling profiles. Not only do V3 progenitors receive higher total levels of Notch response, but they collect this response over a longer duration compared to KA" progenitors. To test whether the duration of Notch signalling determines the temporal cell fate specification, we combined a transgene that constitutively activates Notch signalling in the ventral spinal cord with a heat shock inducible Notch signalling terminator to switch off Notch response at any given time. Sustained Notch signalling results in expanded LFP progenitors while KA" and V3 interneurons fail to specify. Early termination of Notch signalling leads to exclusively KA" cell fate, despite the high level of Notch signalling, whereas late attenuation of Notch signalling drives only V3 cell fate. This suggests that the duration of Notch signalling, not simply the level, mediates cell fate specification. Interestingly, knockdown experiments reveal a role for the Notch ligand Jag2b in maintaining LFP progenitors and limiting their differentiation into KA" and V3 interneurons. Our results indicate that Notch signalling is required for neural progenitor maintenance while a specific attenuation timetable defines the fate of the postmitotic progeny. [Display omitted] • LFP progenitors generate KA" and V3 interneurons in a sequential manner. • Cells in the LFP domain display distinct temporal profiles of Notch signalling. • A NotchON-NotchOFF system can be applied to manipulate Notch signalling duration. • The duration of Notch signalling controls temporal cell fate specification. [ABSTRACT FROM AUTHOR]

Heterozygous R391 TUBB4B pathogenic variations are responsible for an association of hearing loss and retinal dystrophy in human. With the goal of understanding the functions of TuBB4b and the pathogenic role of R391 variations, we characterized tubB4B in zebrafish and identified the gene regulatory elements necessary and sufficient for expression of TubB4b as in endogenous tissues. Using knock-out and transgenic approaches, we determined that R391 mutations impair neither localization of TubB4B within sensory hair cells (SHC) nor their structure, but induced to a small decrease in SHC number from anterior crista. Expression of R391 mutations in sensory hair cells has no effect on zebrafish audition, suggesting a different equilibrium between various tubulin isotypes in zebrafish possibly due to compensatory mechanisms. The careful expression analysis and transgenic tools generated in this study could help understand how recently described pathogenic variants lead to more severe clinical forms of TUBB4B-related diseases., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Proper organ formation depends on the precise delineation of organ territories containing defined numbers of progenitor cells. Kidney progenitors reside in bilateral stripes of posterior mesoderm that are referred to as the intermediate mesoderm (IM). Previously, we showed that the transcription factors Hand2 and Osr1 act to strike a balance between the specification of the kidney progenitors in the IM and the vessel progenitors in the laterally adjacent territory. Recently, the transcription factor Npas4l - an early and essential driver of vessel and blood progenitor formation - was shown to inhibit kidney development. Here we demonstrate how kidney progenitor specification is coordinated by hand2, osr1, and npas4l. We find that npas4l and the IM marker pax2a are transiently co-expressed in the posterior lateral mesoderm, and npas4l is necessary to inhibit IM formation. Consistent with the expression of npas4l flanking the medial and lateral sides of the IM, our findings suggest roles for npas4l in defining the IM boundaries at each of these borders. At the lateral IM border, hand2 promotes and osr1 inhibits the formation of npas4l-expressing lateral vessel progenitors, and hand2 requires npas4l to inhibit IM formation and to promote vessel formation. Meanwhile, npas4l appears to have an additional role in suppressing IM fate at the medial border: npas4l loss-of-function enhances hand2 mutant IM defects and results in excess IM generated outside of the lateral hand2-expressing territory. Together, our findings reveal that establishment of the medial and lateral boundaries of the IM requires inhibition of kidney progenitor specification by the neighboring drivers of vessel progenitor fate., Competing Interests: Declaration of competing interest Declarations of interest: none., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

PLK4 plays a crucial role in centriole duplication, which is essential for maintaining cellular processes such as cell division, cytoskeletal stability, and cilia formation. However, the mechanisms of PLK4 remain incompletely understood, especially in the embryonic development of vertebrate species. In this study, we observed that Plk4 dysfunction led to abnormal embryonic development in zebrafish, characterized by symptoms such as dark and wrinkled skin, microphthalmia, and body axis curvature. In plk4 mutants, defects in centriole duplication led to abnormal cell division, apoptosis, and ciliogenesis defects. Moreover, overexpression of plk4 in zebrafish embryos caused excessive centrosome amplification, disrupting embryonic gastrulation through abnormal cell division and ultimately resulting in embryonic lethality. Furthermore, we identified the "cryptic" polo box (CPB) domain, consisting of two PBs (PB1 and PB2), as the critical centrosome localization domain of Plk4. Surprisingly, overexpression of these two PB domains alone was sufficient to induce embryonic lethality. Additionally, we discovered a truncated form of CPB that localizes to the centrosome without causing defects in embryonic development. Our results demonstrate that Plk4 tightly controls centriole duplication, which is essential for early embryonic development in zebrafish., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Axon regeneration in response to injury has been documented in many animals over several hundred years. In contrast, how neurons respond to dendrite injury has been examined only in the last decade. So far, dendrite regeneration after injury has been documented in invertebrate model systems, but has not been assayed in a vertebrate. In this study, we use zebrafish motor neurons to track neurons after dendrite injury. We address two major gaps in our knowledge of dendrite regeneration: 1) whether post-synaptic dendrites can regenerate and 2) whether vertebrate dendrites can regenerate. We find that motor neurons survive laser microsurgery to remove one or all dendrites. Outgrowth of new dendrites typically initiated one to three days after injury, and a new, stable dendrite arbor was in place by five days after injury. We conclude that zebrafish motor neurons have the capacity to regenerate a new dendrite arbor. [Display omitted] • Zebrafish motor neuron dendrite shape is largely stable 8 days post-fertilization. • Motor neurons survive dendrite removal in vivo. • A dendrite arbor can regenerate after partial or complete dendrite removal. [ABSTRACT FROM AUTHOR]

Over the past 20 years, various zebrafish injury models demonstrated efficient heart regeneration after cardiac tissue loss. However, no established coronary vessel injury methods exist in the zebrafish model, despite coronary endothelial dysfunction occurring in most patients with acute coronary syndrome. This is due to difficulties performing surgery on small coronary vessels and a lack of genetic tools to precisely manipulate coronary cells in zebrafish. We determined that the Notch ligand gene deltaC regulatory sequences drive gene expression in zebrafish coronary endothelial cells, enabling us to overcome these obstacles. We created a deltaC fluorescent reporter line and visualized robust coronary growth during heart development and regeneration. Importantly, this reporter facilitated the visualization of coronary growth without an endocardial background. Moreover, we visualized robust coronary growth on the surface of juvenile hearts and regrowth in the wounded area of adult hearts ex vivo. With this approach, we observed growth inhibition by reported vascular growth antagonists of the VEGF, EGF and Notch signaling pathways. Furthermore, we established a coronary genetic ablation system and observed that severe coronary endothelial cell loss resulted in fish death, whereas fish survived mild coronary cell loss. Coronary cell depletion triggered regenerative responses, which resulted in the restoration of damaged cardiac tissues within several weeks. Overall, our work demonstrated the efficacy of using deltaC regulatory elements for high-resolution visualization of the coronary endothelium; screening small molecules for coronary growth effects; and revealed complete recovery in adult zebrafish after coronary-induced heart damage. Although several zebrafish cardiac tissue injury and regeneration models have been established, no zebrafish coronary vessel injury methods exist. We established a coronary genetic ablation system utilizing the Notch ligand gene deltaC regulatory sequences which drive gene expression in zebrafish coronary endothelial cells. Severe coronary endothelial cell loss resulted in fish death, whereas fish survived mild coronary cell loss. Coronary cell depletion triggered regenerative responses, which resulted in restoration of damaged cardiac tissues within several weeks. Our work revealed complete cardiac recovery in adult zebrafish after coronary-induced heart damage. [Display omitted] • DeltaC regulatory sequences drive gene expression in coronary endothelial cells in zebrafish ventricle. • High-resolution visualization of zebrafish coronary vasculature with the deltaC:EGFP reporter. • Live visualization of coronary growth in the juvenile heart surface and in wounded area of adult hearts. • Coronary endothelial cell loss triggered rapid regeneration responses. [ABSTRACT FROM AUTHOR]

Adult endochondral bones are prefigured in the embryo as cellular condensations within fields of more loosely distributed skeletal progenitors. How these early condensations are initiated and shaped has remained enigmatic, despite the wealth of research on later stages of cartilage differentiation and endochondral ossification. Using the simple larval zebrafish facial skeleton as a model, we reevaluate the involvement of the master cartilage regulator Sox9 in shaping facial condensations and find it to be largely dispensable. We then use new lineage-tracing tools to definitively show that precartilaginous condensations originate from neighboring clusters of cells termed mesenchymal condensations. These cartilage-generating mesenchymal condensations express a cohort of transcription factors that are also expressed in odontogenic mesenchyme in mammals, including barx1 , lhx6a/8a , and pax9. We hypothesized that the position of each mesenchymal condensation determines the axis of growth of its corresponding precartilaginous condensation, thus influencing its final shape. Consistent with this idea, we find that positive Fgf and inhibitory Jagged-Notch signals intersect to precisely position a mesenchymal condensation in the dorsal half of the second pharyngeal arch, with loss of pathway function leading to predictable shape changes in the resulting cartilage element. Deciphering the full array of signals that control the spatial distribution of mesenchymal condensations and regulate their maturation into precartilaginous condensations thus offers a promising approach for understanding the origins of skeletal form. [Display omitted] • Endochondral bone shape is prefigured in early precartilaginous condensations. • Precartilaginous condensations arise from precursor mesenchymal condensations. • Mesenchymal condensations in the face are positioned by Fgf and Notch signaling. [ABSTRACT FROM AUTHOR]

The formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa had an impact on endothelial cell (EC) migration and proliferation. Surprisingly, mutations in vegfab more strongly affected EC proliferation in distinct blood vessels, such as intersegmental blood vessels in the zebrafish trunk and central arteries in the head. Analysis of downstream signaling pathways revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutant phenotypes in affected blood vessels. Together, these results suggest that extracellular matrix bound Vegfa might act through PI3K signaling to control EC proliferation in a distinct set of blood vessels during angiogenesis. [Display omitted] • Vegfab zebrafish mutants show defects in endothelial cell proliferation in distinct blood vessels. • Inhibition of PI3 kinase signaling leads to similar defects in endothelial cell proliferation. • Overexpression of vegfab leads to an increase in endothelial cell proliferation. • Blood vessel plasticity can rescue vascular phenotypes in vegfab mutants. [ABSTRACT FROM AUTHOR]

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is a key regulator of sex determination and/or gonadal sex differentiation across metazoan animals. This is unusual given that sex determination genes are typically not well conserved. The mechanisms by which zebrafish sex is determined have remained elusive due to the lack of sex chromosomes and the complex polygenic nature of sex determination in domesticated strains. To investigate the role of dmrt1 in zebrafish sex determination and gonad development, we isolated mutations disrupting this gene. We found that the majority of dmrt1 mutant fish develop as fertile females suggesting a complete male-to-female sex reversal in mutant animals that would have otherwise developed as males. A small percentage of mutant animals became males, but were sterile and displayed testicular dysgenesis. Therefore zebrafish dmrt1 functions in male sex determination and testis development. Mutant males had aberrant gonadal development at the onset of gonadal sex-differentiation, displaying reduced oocyte apoptosis followed by development of intersex gonads and failed testis morphogenesis and spermatogenesis. By contrast, female ovaries developed normally. We found that Dmrt1 is necessary for normal transcriptional regulation of the amh (anti-Müllerian hormone) and foxl2 (forkhead box L2) genes, which are thought to be important for male or female sexual development respectively. Interestingly, we identified one dmrt1 mutant allele that co-operates with a linked segregation distorter locus to generate an apparent XY sex determination mechanism. We conclude that dmrt1 is dispensable for ovary development but necessary for testis development in zebrafish, and that dmrt1 promotes male development by transcriptionally regulating male and female genes as has been described in other animals. Furthermore, the strong sex-ratio bias caused by dmrt1 reduction-of-function points to potential mechanisms through which sex chromosomes may evolve.

Tbx20 is a T-box transcription factor that plays essential roles in the development and maintenance of the heart. Although it is expressed by cardiac progenitors in all species examined, an involvement of Tbx20 in cardiac progenitor formation in vertebrates has not been previously described. Here we report the identification of a zebrafish tbx20 mutation that results in an inactive, truncated protein lacking any functional domains. The cardiac progenitor population is strongly diminished in this mutant, leading to the formation of a small, stretched-out heart. We found that overexpression of Tbx20 results in an enlarged heart with significantly more cardiomyocytes. Interestingly, this increase in cell number is caused by both enhanced cardiac progenitor cell formation and the proliferation of differentiated cardiomyocytes, and is dependent upon the activity of Tbx20's T-box and transcription activation domains. Together, our findings highlight a previously unappreciated role for Tbx20 in promoting cardiac progenitor formation in vertebrates and reveal a novel function for its activation domain in cardiac cell proliferation during embryogenesis.

Targeted mutagenesis in zebrafish, fruit flies, and C. elegans has been significantly improved over the years through CRISPR technology. CRISPR enables researchers to efficiently examine cellular pathways by inducing small, targeted mutations in vivo. Though these mutations are commonly random insertions or deletions (indels), they often result in functionally disrupted alleles of a target gene if the CRISPR components are appropriately designed. However, current protocols used to identify the presence of CRISPR-generated indels are often labor intensive, time-consuming, or expensive. Here, we describe a straightforward, high-throughput method for identifying the presence of mutations by using a fragment analyzer platform which allows for DNA fragment sizing through high-resolution capillary gel-electrophoresis. Following this protocol, small indels—down to 2 base pairs—can be quickly and reliably identified, thus allowing for large-scale genotyping of newly-generated or stable mutant lines. [Display omitted] • Workflow of fragment analysis to identify and genotype CRISPR-generated indels. • Identify and track a novel zebrafish indel with the Agilent 5300 Fragment Analyzer. • Resolve indels as small as 2 base pairs in DNA fragments from standard PCR products. • Steps to target, mutate, genotype, and verify alleles in zebrafish mutants. [ABSTRACT FROM AUTHOR]

Glia are a diverse and essential cell type in the vertebrate nervous system. Transgenic tools and fluorescent reporter lines are critical resources to investigate how glial subtypes develop and function. However, despite the many lines available in zebrafish, the community still lacks the ability to label all unique stages of glial development and specific subpopulations of cells. To address this issue, we screened zebrafish gene and enhancer trap lines to find a novel reporter for peripheral glial subtypes. From these, we generated the gSAIzGFFD37A transgenic line that expresses GFP in neural crest cells and central and peripheral glia. We found that the gene trap construct is located within an intron of erbb3b , a gene essential for glial development. Additionally, we confirmed that GFP+ ​cells express erbb3b along with sox10 , a known glial marker. From our screen, we have identified the gSAIzGFFD37A line as a novel and powerful tool for studying glia in the developing zebrafish, as well as a new resource to manipulate erbb3b + ​cells. [Display omitted] • Novel zebrafish line gSAIzGFFD37A labels neural crest and glial cells. • gSAIzGFFD37A transgene is inserted in the erbb3b locus. • gSAIzGFFD37A shares spatiotemporal expression patterns with erbb3b. • gSAIzGFFD37A can be used with existing UAS transgenic tools. Summary: This manuscript characterizes the zebrafish gene trap line gSAIzGFFD37A, which drives GFP expression in erbb3b ​+ ​cells. This line is a novel and valuable tool for studying neural crest cells and glia. [ABSTRACT FROM AUTHOR]

Zebrafish can achieve scar-free healing of heart injuries, and robustly replace all cardiomyocytes lost to injury via dedifferentiation and proliferation of mature cardiomyocytes. Previous studies suggested that Wnt/β-catenin signaling is active in the injured zebrafish heart, where it induces fibrosis and prevents cardiomyocyte cell cycling. Here, via targeting the destruction complex of the Wnt/β-catenin pathway with pharmacological and genetic tools, we demonstrate that Wnt/β-catenin activity is required for cardiomyocyte proliferation and dedifferentiation, as well as for maturation of the scar during regeneration. Using cardiomyocyte-specific conditional inhibition of the pathway, we show that Wnt/β-catenin signaling acts cell-autonomously to promote cardiomyocyte proliferation. Our results stand in contrast to previous reports and rather support a model in which Wnt/β-catenin signaling plays a positive role during heart regeneration in zebrafish. [Display omitted] • Wnt/β-catenin signaling is active in cardiomyocytes at the wound border. • Wnt signaling is required for cardiomyocyte dedifferentiation and proliferation. • Wnt signaling is required for scar maturation and resolution. • Wnt signaling acts cell autonomously in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Spear-like collagen complexes, known as actinotrichia, underlie the epidermal cell layer in the tip of teleost fins and are known to contribute toward fin formation; however, their specific role remains largely unclear. In this study, we investigated of actinotrichia in the role of caudal fin formation by generating collagen9a1c (col9a1c)-knockout zebrafish. Although actinotrichia were initially produced normally and aligned correctly in the knockout fish, the number of actinotrichia decreased as the fish grew and their alignment became disordered. Simultaneously, the fin tip gradually shortened in the dorsal-ventral direction and the entire fin became oval-shaped, while the fin-rays rarely bifurcated and instead underwent fusion, suggesting that actinotrichia are essential for spreading fins dorsoventrally. Furthermore, the epithelial cells that are usually thinly spread in normal fish became spherical in the knockout fish, reducing the area covered by each cell and thus the area of the fin tip. Together, these findings suggest that the tight alignment of actinotrichia provides physical support in the dorsal-ventral direction that allows caudal fins to expand in a triangular-shape. • The physical role of collagen fibers called actinotrichia in caudal fin shape formation was investigated. • Actinotrichia are arranged in parallel to form a two-dimensional scaffold. • When the scaffold is lost, an extreme contraction of epithelial cells occurs. • The contraction of the epithelial cells is thought to shorten the fins along dorsal-ventral axis. [ABSTRACT FROM AUTHOR]

The corpuscles of Stannius (CS) represent a unique endocrine organ of teleostean fish that secrets stanniocalcin-1 (Stc1) to maintain calcium homeostasis. Appearing at 20–25 somite stage in the distal zebrafish pronephros, stc1 -expressing cells undergo apical constriction, and are subsequently extruded to form a distinct gland on top of the distal pronephric tubules at 50 ​h post fertilization (hpf). Several transcription factors (e.g. Hnf1b, Irx3b, Tbx2a/b) and signaling pathways (e.g. Notch) control CS development. We report now that Fgf signaling is required to commit tubular epithelial cells to differentiate into stc1 -expressing CS cells. Inhibition of Fgf signaling by SU5402, dominant-negative Fgfr1, or depletion of fgf8a prevented CS formation and stc1 expression. Ablation experiments revealed that CS have the ability to partially regenerate via active cell migration involving extensive filopodia and lamellipodia formation. Activation of Wnt signaling curtailed stc1 expression, but had no effect on CS formation. Thus, our observations identify Fgf signaling as a crucial component of CS cell fate commitment. [Display omitted] • The corpuscles of Stannius (CS) can regenerate after laser-induced ablation. • The inhibition of Fgf signaling blocks the CS development. • Activation of Wnt signaling represses stc1 expression at early developmental stages. [ABSTRACT FROM AUTHOR]

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo , we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry. [Display omitted] • Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). • CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. • Towards understanding CARMIL function during embryogenesis in vivo , we analyzed zebrafish lines carrying mutations of carmil3. • Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). • This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. [ABSTRACT FROM AUTHOR]

Cardiac trabeculae are muscular ridge-like structures within the ventricular wall that are crucial for cardiac function. In zebrafish, these structures first form primarily through the delamination of compact wall cardiomyocytes (CMs). Although defects in proteasomal degradation have been associated with decreased cardiac function, whether they also affect cardiac development has not been extensively analyzed. Here we report a role during cardiac wall morphogenesis in zebrafish for the E3 ubiquitin-protein ligase Rbx1, which has been shown to regulate the degradation of key signaling molecules. Although development is largely unperturbed in zebrafish rbx1 mutant larvae, they exhibit CM multi-layering. This phenotype is not affected by blocking ErbB signaling, but fails to manifest itself in the absence of blood flow/cardiac contractility. Surprisingly, rbx1 mutants display ErbB independent Notch reporter expression in the myocardium. We generated tissue-specific rbx1 overexpression lines and found that endothelial, but not myocardial, specific rbx1 expression normalizes the cardiac wall morphogenesis phenotype. In addition, we found that pharmacological activation of Hedgehog signaling ameliorates the multi-layered myocardial wall phenotype in rbx1 mutants. Collectively, our data indicate that endocardial activity of Rbx1 is essential for cardiac wall morphogenesis. [Display omitted] • rbx1 mutants exhibit a multi-layered myocardial wall phenotype. • rbx1 mutants exhibit reduced cardiomyocyte proliferation in their ventricle. • rbx1 regulates endocardial morphology. • Overexpression of rbx1 in endothelial cells can rescue multi-layered myocardial wall phenotype in rbx1 mutants. • Activation of Hedgehog signaling normalizes the multi-layered myocardial wall phenotype in rbx1 mutants. [ABSTRACT FROM AUTHOR]

The onset of circulation in a developing embryo requires intact blood vessels to prevent hemorrhage. The development of endothelial cells, and their subsequent recruitment of perivascular mural cells are important processes to establish and maintain vascular integrity. These processes are genetically controlled during development, and mutations that affect endothelial cell specification, pattern formation, or maturation through the addition of mural cells can result in early developmental hemorrhage. We created a strong loss of function allele of the zebrafish GDP-mannose 4,6 dehydratase (gmds) gene that is required for the de novo synthesis of GDP-fucose, and homozygous embryos display cerebral hemorrhages. Our data demonstrate that gmds mutants have early defects in vascular patterning with ectopic branches observed at time of hemorrhage. Subsequently, defects in the number of mural cells that line the vasculature are observed. Moreover, activation of Notch signaling rescued hemorrhage phenotypes in gmds mutants, highlighting a potential downstream pathway that requires protein fucosylation for vascular integrity. Finally, supplementation with fucose can rescue hemorrhage frequency in gmd s mutants, demonstrating that synthesis of GDP-fucose via an alternative (salvage) pathway may provide an avenue toward therapeutic correction of phenotypes observed due to defects in de novo GDP-fucose synthesis. Together, these data are consistent with a novel role for the de novo and salvage protein fucosylation pathways in regulating vascular integrity through a Notch dependent mechanism. • Mutation of gmds affects vascular integrity, as evidenced by cerebral hemorrhages. • Inhibition of gmds results in ectopic endothelial cell branches. • Loss of gmds causes defects in vascular maturation. • The fucosylation salvage pathway plays a role in vascular integrity. • Increasing Notch signalling rescues the cerebral hemorrhage phenotype in gmds mutants. [ABSTRACT FROM AUTHOR]

The neural crest (NC) is a transient multipotent cell population that migrates extensively to produce a remarkable array of vertebrate cell types. NC cell specification progresses in an anterior to posterior fashion, resulting in distinct, axial-restricted subpopulations. The anterior-most, cranial, population of NC is specified as gastrulation concludes and neurulation begins, while more posterior populations become specified as the body elongates. The mechanisms that govern development of the more posterior NC cells remain incompletely understood. Here, we report a key role for zebrafish Cdx4, a homeodomain transcription factor, in the development of posterior NC cells. We demonstrate that cdx4 is expressed in trunk NC cell progenitors, directly binds NC cell-specific enhancers in the NC GRN, and regulates expression of the key NC development gene foxd3 in the posterior body. Moreover, c dx4 mutants show disruptions to the segmental pattern of trunk NC cell migration due to loss of normal leader/follower cell dynamics. Finally, using cell transplantation to generate chimeric specimens, we show that Cdx4 does not function in the paraxial mesoderm—the environment adjacent to which crest migrates—to influence migratory behaviors. We conclude that cdx4 plays a critical, and likely tissue autonomous, role in the establishment of trunk NC migratory behaviors. Together, our results indicate that cdx4 functions as an early NC specifier gene in the posterior body of zebrafish embryos. [Display omitted] • Zebrafish cdx4 is expressed in premigratory trunk neural crest (NC) cells. • Cdx4 regulates expression of foxd3 and other NC specifier genes. • Segmental NC migration patterns are disrupted in cdx4 −/− embryos. • cdx4 −/− trunk NC cells resemble 'follower' cells, with 'leader' cells absent. • NC migration is independent of somitic Cdx4 function and is likely cell-autonomous. [ABSTRACT FROM AUTHOR]

Vertebrate skin appendages - particularly avian feathers and mammalian hairs, glands and teeth - are perennially useful systems for investigating fundamental mechanisms of development. The most common type of skin appendage in teleost fishes is the elasmoid scale, yet this structure has received much less attention than the skin appendages of tetrapods. Elasmoid scales are thin, overlapping plates of partially mineralized extracellular matrices, deposited in the skin in a hexagonal pattern by a specialized population of dermal cells in cooperation with the overlying epidermis. Recent years have seen rapid progress in our understanding of elasmoid scale development and regeneration, driven by the deployment of developmental genetics, live imaging and transcriptomics in larval and adult zebrafish. These findings are reviewed together with histological and ultrastructural approaches to understanding scale development and regeneration., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects. [Display omitted] [ABSTRACT FROM AUTHOR]

Ventral spinal cord progenitor cells, which express the basic helix loop helix transcription factor Olig2, sequentially produce motor neurons and oligodendrocyte precursor cells (OPCs). Following specification some OPCs differentiate as myelinating oligodendrocytes while others persist as OPCs. Though a considerable amount of work has described the molecular profiles that define motor neurons, OPCs, and oligodendrocytes, less is known about the progenitors that produce them. To identify the developmental origins and transcriptional profiles of motor neurons and OPCs, we performed single-cell RNA sequencing on isolated pMN cells from embryonic zebrafish trunk tissue at stages that encompassed motor neurogenesis, OPC specification, and initiation of oligodendrocyte differentiation. Downstream analyses revealed two distinct pMN progenitor populations: one that appears to produce neurons and one that appears to produce OPCs. This latter population, called Pre-OPCs, is marked by expression of GS Homeobox 2 (gsx2), a gene that encodes a homeobox transcription factor. Using fluorescent in situ hybridizations, we identified gsx2 -expressing Pre-OPCs in the spinal cord prior to expression of canonical OPC marker genes. Our data therefore reveal heterogeneous gene expression profiles among pMN progenitors, supporting prior fate mapping evidence. [Display omitted] • Single-cell RNA sequencing reveals trajectories of neurons and glia produced by pMN progenitor cells in zebrafish embryos. • Distinct pMN progenitors are the apparent sources of neurons or oligodendrocytes, consistent with fate mapping data. • gsx2 expression marks pMN progenitors that produce oligodendrocyte lineage cells. [ABSTRACT FROM AUTHOR]

Thyroid hormone is a key regulator of post-embryonic vertebrate development. Skin is a biomedically important thyroid hormone target organ, but the cellular and molecular mechanisms underlying skin pathologies associated with thyroid dysfunction remain obscure. The transparent skin of zebrafish is an accessible model system for studying vertebrate skin development. During post-embryonic development of the zebrafish, scales emerge in the skin from a hexagonally patterned array of dermal papillae, like other vertebrate skin appendages such as feathers and hair follicles. We show here that thyroid hormone regulates the rate of post-embryonic dermal development through interaction with nuclear hormone receptors. This couples skin development with body growth to generate a well ordered array of correctly proportioned scales. This work extends our knowledge of thyroid hormone actions on skin by providing in-vivo evidence that thyroid hormone regulates multiple aspects of dermal development. [Display omitted] • Thyroid hormone (TH) is necessary for normal squamation patterning in zebrafish. • Stratified dermis develops by migration of primary hypodermal cells. • Dermis stratifies in an invariant wave. • TH regulates the rates of multiple aspects of dermis development. • Scale size and density are sensitive to skin size at onset of squamation. [ABSTRACT FROM AUTHOR]

Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.

Cardiac neural crest cells are essential for outflow tract remodeling in animals with divided systemic and pulmonary circulatory systems, but their contributions to cardiac development in animals with a single-loop circulatory system are less clear. Here we genetically labeled neural crest cells and examined their contribution to the developing zebrafish heart. We identified two populations of neural crest cells that contribute to distinct compartments of zebrafish cardiovascular system at different developmental stages. A stream of neural crest cells migrating through pharyngeal arches 1 and 2 integrates into the myocardium of the primitive heart tube between 24 and 30 h post fertilization and gives rise to cardiomyocytes. A second wave of neural crest cells migrating along aortic arch 6 envelops the endothelium of the ventral aorta and invades the bulbus arteriosus after three days of development. Interestingly, while inhibition of FGF signaling has no effect on the integration of neural crest cells to the primitive heart tube, it prevents these cells from contributing to the outflow tract, demonstrating disparate responses of neural crest cells to FGF signaling. Furthermore, neural crest ablation in zebrafish leads to multiple cardiac defects, including reduced heart rate, defective myocardial maturation and a failure to recruit progenitor cells from the second heart field. These findings add to our understanding of the contribution of neural crest cells to the developing heart and provide insights into the requirement for these cells in cardiac maturation.