A zebrafish gephyrinb mutant distinguishes synaptic and enzymatic functions of Gephyrin.

Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinbvo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinbvo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinbvo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinbvo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinbvo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin.

Somatostatin signalling coordinates energy metabolism allocation to reproduction in zebrafish.

BACKGROUND: Energy allocation between growth and reproduction determines puberty onset and fertility. In mammals, peripheral hormones such as leptin, insulin and ghrelin signal metabolic information to the higher centres controlling gonadotrophin-releasing hormone neurone activity. However, these observations could not be confirmed in lower vertebrates, suggesting that other factors may mediate the energetic trade-off between growth and reproduction. A bioinformatic and experimental study suggested co-regulation of the circadian clock, reproductive axis and growth-regulating genes in zebrafish. While loss-of-function of most of the identified co-regulated genes had no effect or only had mild effects on reproduction, no such information existed about the co-regulated somatostatin, well-known for its actions on growth and metabolism. RESULTS: We show that somatostatin signalling is pivotal in regulating fecundity and metabolism. Knock-out of zebrafish somatostatin 1.1 (sst1.1) and somatostatin 1.2 (sst1.2) caused a 20-30% increase in embryonic primordial germ cells, and sst1.2-/- adults laid 40% more eggs than their wild-type siblings. The sst1.1-/- and sst1.2-/- mutants had divergent metabolic phenotypes: the former had 25% more pancreatic α-cells, were hyperglycaemic and glucose intolerant, and had increased adipocyte mass; the latter had 25% more pancreatic ß-cells, improved glucose clearance and reduced adipocyte mass. CONCLUSIONS: We conclude that somatostatin signalling regulates energy metabolism and fecundity through anti-proliferative and modulatory actions on primordial germ cells, pancreatic insulin and glucagon cells and the hypothalamus. The ancient origin of the somatostatin system suggests it could act as a switch linking metabolism and reproduction across vertebrates. The results raise the possibility of applications in human and animal fertility.

Leucine-Rich Repeat Kinase-2 Controls the Differentiation and Maturation of Oligodendrocytes in Mice and Zebrafish.

Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson's disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of other brain cells and oligodendrocytes (OLs) in PD. Altogether, these observations suggest crucial function(s) of LRRK2 in OPCs/Ols, which would be interesting to explore. In this study, we investigated the role of LRRK2 in OLs. We showed that LRRK2 knock-out (KO) OPC cultures displayed defects in the transition of OPCs into OLs, suggesting a role of LRRK2 in OL differentiation. Consistently, we found an alteration of myelin basic protein (MBP) striosomes in LRRK2 KO mouse brains and reduced levels of oligodendrocyte transcription factor 2 (Olig2) and Mbp in olig2:EGFP and mbp:RFP transgenic zebrafish embryos injected with lrrk2 morpholino (MO). Moreover, lrrk2 knock-down zebrafish exhibited a lower amount of nerve growth factor (Ngf) compared to control embryos, which represents a potent regulator of oligodendrogenesis and myelination. Overall, our findings indicate that LRRK2 controls OL differentiation, affecting the number of mature OLs.

Glutamate transporters are involved in direct inhibitory synaptic transmission in the vertebrate retina.

In the central nervous system of vertebrates, glutamate serves as the primary excitatory neurotransmitter. However, in the retina, glutamate released from photoreceptors causes hyperpolarization in post-synaptic ON-bipolar cells through a glutamate-gated chloride current, which seems paradoxical. Our research reveals that this current is modulated by two excitatory glutamate transporters, EAAT5b and EAAT7. In the zebrafish retina, these transporters are located at the dendritic tips of ON-bipolar cells and interact with all four types of cone photoreceptors. The absence of these transporters leads to a decrease in ON-bipolar cell responses, with eaat5b mutants being less severely affected than eaat5b/eaat7 double mutants, which also exhibit altered response kinetics. Biophysical investigations establish that EAAT7 is an active glutamate transporter with a predominant anion conductance. Our study is the first to demonstrate the direct involvement of post-synaptic glutamate transporters in inhibitory direct synaptic transmission at a central nervous system synapse.

MRAP2a Binds and Modulates Activity and Localisation of Prokineticin Receptor 1 in Zebrafish.

The prokineticin system plays a role in hypothalamic neurons in the control of energy homeostasis. Prokineticin receptors (PKR1 and PKR2), like other G-protein-coupled receptors (GPCRs) are involved in the regulation of energy intake and expenditure and are modulated by the accessory membrane protein 2 of the melanocortin receptor (MRAP2). The aim of this work is to characterise the interaction and regulation of the non-melanocortin receptor PKR1 by MRAP2a in zebrafish (zMRAP2a) in order to use zebrafish as a model for the development of drugs targeting accessory proteins that can alter the localisation and activity of GPCRs. To this end, we first showed that zebrafish PKR1 (zPKR1) is able to interact with both zMRAP2a and human MRAP2 (hMRAP2). This interaction occurs between the N-terminal region of zPKR1 and the C-terminal domain of zMRAP2a, which shows high sequence identity with hMRAP2 and a similar propensity for dimer formation. Moreover, we demonstrated that in Chinese hamster ovary (CHO) cells, zMRAP2a or hMRAP2 are able to modulate zPKR1 activation induced by zebrafish PK2 (zPK2) resulting in an impaired ERK and STAT3 activation.

Histone lactylation-ROS loop contributes to light exposure-exacerbated neutrophil recruitment in zebrafish.

Light serves as a crucial external zeitgeber for maintaining and restoring physiological homeostasis in most organisms. Disrupting of light rhythms often leads to abnormal immune function, characterized by excessive inflammatory responses. However, the underlying regulatory mechanisms behind this phenomenon remain unclear. To address this concern, we use in vivo imaging to establish inflammation models in zebrafish, allowing us to investigate the effects and underlying mechanisms of light disruption on neutrophil recruitment. Our findings reveal that under sustained light conditions (LL), neutrophil recruitment in response to caudal fin injury and otic vesicle inflammation is significantly increased. This is accompanied by elevated levels of histone (H3K18) lactylation and reactive oxygen species (ROS) content. Through ChIP-sequencing and ChIP‒qPCR analysis, we discover that H3K18 lactylation regulates the transcriptional activation of the duox gene, leading to ROS production. In turn, ROS further promote H3K18 lactylation, forming a positive feedback loop. This loop, driven by H3K18 lactylation-ROS, ultimately results in the over recruitment of neutrophils to inflammatory sites in LL conditions. Collectively, our study provides evidence of a mutual loop between histone lactylation and ROS, exacerbating neutrophil recruitment in light disorder conditions, emphasizing the significance of maintaining a proper light-dark cycle to optimize immune function.

Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.

The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.

Regulation of MicroRNAs After Spinal Cord Injury in Adult Zebrafish.

Spinal cord injury (SCI) is a central nerve injury that often leads to loss of motor and sensory functions at or below the level of the injury. Zebrafish have a strong ability to repair after SCI, but the role of microRNAs (miRNAs) after SCI remains unclear. Locomotor behavior analysis showed that adult zebrafish recovered about 30% of their motor ability at 2 weeks and 55% at 3 weeks after SCI, reflecting their strong ability to repair SCI. Through miRNA sequencing, mRNA sequencing, RT-qPCR experiment verification, and bioinformatics predictive analysis, the key miRNAs and related genes in the repair of SCI were screened. A total of 38 miRNAs were significantly different, the top ten miRNAs were verified by RT-qPCR. The prediction target genes were verified by the mRNAs sequencing results at the same time point. Finally, 182 target genes were identified as likely to be networked regulated by the 38 different miRNAs. GO and KEGG enrichment analysis found that miRNAs targeted gene regulation of many key pathways, such as membrane tissue transport, ribosome function, lipid binding, and peroxidase activity. The PPI network analysis showed that miRNAs were involved in SCI repair through complex network regulation, among which dre-miR-21 may enhance cell reversibility through nop56, and that dre-miR-125c regulates axon growth through kpnb1 to repair SCI.

Meningeal lymphatic supporting cells govern the formation and maintenance of zebrafish mural lymphatic endothelial cells.

The meninges are critical for the brain functions, but the diversity of meningeal cell types and intercellular interactions have yet to be thoroughly examined. Here we identify a population of meningeal lymphatic supporting cells (mLSCs) in the zebrafish leptomeninges, which are specifically labeled by ependymin. Morphologically, mLSCs form membranous structures that enwrap the majority of leptomeningeal blood vessels and all the mural lymphatic endothelial cells (muLECs). Based on its unique cellular morphologies and transcriptional profile, mLSC is characterized as a unique cell type different from all the currently known meningeal cell types. Because of the formation of supportive structures and production of pro-lymphangiogenic factors, mLSCs not only promote muLEC development and maintain the dispersed distributions of muLECs in the leptomeninges, but also are required for muLEC regeneration after ablation. This study characterizes a newly identified cell type in leptomeninges, mLSC, which is required for muLEC development, maintenance, and regeneration.

FBLN2 is associated with Goldenhar syndrome and is essential for cranial neural crest cell development.

Goldenhar syndrome, a rare craniofacial malformation, is characterized by developmental anomalies in the first and second pharyngeal arches. Its etiology is considered to be heterogenous, including both genetic and environmental factors that remain largely unknown. To further elucidate the genetic cause in a five-generation Goldenhar syndrome pedigree and exploit the whole-exome sequencing (WES) data of this pedigree, we generated collapsed haplotype pattern markers based on WES and employed rare variant nonparametric linkage analysis. FBLN2 was identified as a candidate gene via analysis of WES data across the significant linkage region. A fbln2 knockout zebrafish line was established by CRISPR/Cas9 to examine the gene's role in craniofacial cartilage development. fbln2 was expressed specifically in the mandible during the zebrafish early development, while fbln2 knockout zebrafish exhibited craniofacial malformations with abnormal chondrocyte morphologies. Functional studies revealed that fbln2 knockout caused abnormal chondrogenic differentiation, apoptosis, and proliferation of cranial neural crest cells (CNCCs), and downregulated the bone morphogenic protein (BMP) signaling pathway in the zebrafish model. This study demonstrates the role of FBLN2 in CNCC development and BMP pathway regulation, and highlights FBLN2 as a candidate gene for Goldenhar syndrome, which may have implications for the selection of potential screening targets and the development of treatments for conditions like microtia-atresia.

Comparative Study of Condensed and Hydrolysable Tannins during the Early Stages of Zebrafish Development.

In this study, we present data on the effects of condensed tannins (CTs) and hydrolysable tannins (HTs), polyphenols extracted from plants, at different concentrations on zebrafish development to identify the range of concentrations with toxic effects. Zebrafish embryos were exposed to CTs and HTs at two different concentration ranges (5.0-20.0 µgL-1 and 5.0-20.0 mgL-1) for 72 h. The toxicity parameters were observed up to 72 h of treatment. The uptake of CTs and HTs by the zebrafish larvae was assessed via HPLC analysis. A qRT-PCR analysis was performed to evaluate the expressions of genes cd63, zhe1, and klf4, involved in the hatching process of zebrafish. CTs and HTs at 5.0, 10.0, and 20.0 µgL-1 were not toxic. On the contrary, at 5.0, 10.0, and 20.0 mgL-1, HTs induced a delay in hatching starting from 48 h of treatment, while CTs showed a delay in hatching mainly at 48 h. The analysis of gene expression showed a downregulation in the group exposed to HTs, confirming the hatching data. We believe that this study is important for defining the optimal doses of CTs and HTs to be employed in different application fields such as the chemical industry, the animal feed industry, and medical science.

Transcriptomic Characterization of Key Factors and Signaling Pathways for the Regeneration of Partially Hepatectomized Liver in Zebrafish.

Liver regeneration induced by partial hepatectomy (PHx) has attracted intensive research interests due to the great significance for liver resection and transplantation. The zebrafish (Danio rerio) is an excellent model to study liver regeneration. In the fish subjected to PHx (the tip of the ventral lobe was resected), the lost liver mass could be fully regenerated in seven days. However, the regulatory mechanisms underlying the liver regeneration remain largely unknown. In this study, gene expression profiles during the regeneration of PHx-treated liver were explored by RNA sequencing (RNA-seq). The genes responsive to the injury of PHx treatment were identified and classified into different clusters based on the expression profiles. Representative gene ontology (GO) enrichments for the early responsive genes included hormone activity, ribosome biogenesis and rRNA processing, etc., while the late responsive genes were enriched in biological processes such as glutathione metabolic process, antioxidant activity and cellular detoxification. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments were also identified for the differentially expressed genes (DEGs) between the time-series samples and the sham controls. The proteasome was overrepresented by the up-regulated genes at all of the sampling time points. Inhibiting proteasome activity by the application of MG132 to the fish enhanced the expression of Pcna (proliferating cell nuclear antigen), an indicator of hepatocyte proliferation after PHx. Our data provide novel insights into the molecular mechanisms underlying the regeneration of PHx-treated liver.

Hypomyelinated vps16 Mutant Zebrafish Exhibit Systemic and Neurodevelopmental Pathologies.

Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE.

Combined Pituitary Hormone Deficiency in lhx4-Knockout Zebrafish.

LIM homeobox 4 (LHX4) is a transcription factor crucial for anterior pituitary (AP) development. Patients with LHX4 mutation suffer from combined pituitary hormone deficiency (CPHD), short statures, reproductive and metabolic disorders and lethality in some cases. Lhx4-knockout (KO) mice fail to develop a normal AP and die shortly after birth. Here, we characterize a zebrafish lhx4-KO model to further investigate the importance of LHX4 in pituitary gland development and regulation. At the embryonic and larval stages, these fish express lower levels of tshb mRNA compared with their wildtype siblings. In adult lhx4-KO fish, the expressions of pituitary hormone-encoding transcripts, including growth hormone (gh), thyroid stimulating hormone (tshb), proopiomelanocortin (pomca) and follicle stimulating hormone (fshb), are reduced, the pomca promoter-driven expression in corticotrophs is dampened and luteinizing hormone (lhb)-producing gonadotrophs are severely depleted. In contrast to Lhx4-KO mice, Lhx4-deficient fish survive to adulthood, but with a reduced body size. Importantly, lhx4-KO males reach sexual maturity and are reproductively competent, whereas the females remain infertile with undeveloped ovaries. These phenotypes, which are reminiscent of those observed in CPHD patients, along with the advantages of the zebrafish for developmental genetics research, make this lhx4-KO fish an ideal vertebrate model to study the outcomes of LHX4 mutation.

Piezo 1 and Piezo 2 in the Chemosensory Organs of Zebrafish (Danio rerio).

The ion channels Piezo 1 and Piezo 2 have been identified as membrane mechano-proteins. Studying mechanosensitive channels in chemosensory organs could help in understanding the mechanisms by which these channels operate, offering new therapeutic targets for various disorders. This study investigates the expression patterns of Piezo proteins in zebrafish chemosensory organs. For the first time, Piezo protein expression in adult zebrafish chemosensory organs is reported. In the olfactory epithelium, Piezo 1 immunolabels kappe neurons, microvillous cells, and crypt neurons, while Calretinin is expressed in ciliated sensory cells. The lack of overlap between Piezo 1 and Calretinin confirms Piezo 1's specificity for kappe neurons, microvillous cells, and crypt neurons. Piezo 2 shows intense immunoreactivity in kappe neurons, one-ciliated sensory cells, and multi-ciliated sensory cells, with overlapping Calretinin expression, indicating its olfactory neuron nature. In taste buds, Piezo 1 immunolabels Merkel-like cells at the bases of cutaneous and pharyngeal taste buds and the light and dark cells of cutaneous and oral taste buds. It also marks the dark cells of pharyngeal taste buds and support cells in oral taste buds. Piezo 2 is found in the light and dark cells of cutaneous and oral taste buds and isolated chemosensory cells. These findings provide new insights into the distribution of Piezo channels in zebrafish chemosensory organs, enhancing our understanding of their sensory processing and potential therapeutic applications.

Localisation and function of key axonemal microtubule inner proteins and dynein docking complex members reveal extensive diversity among vertebrate motile cilia.

Vertebrate motile cilia are classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition. The extent to which these features are conserved in (9+0) cilia remains unclear. CFAP53, a key axonemal filamentous microtubule inner protein (fMIP) and a centriolar satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, CFAP53 functions redundantly with a paralogous fMIP, MNS1. MNS1 localises to ciliary axonemes, and combined loss of both proteins in zebrafish and mice caused severe outer dynein arm loss from (9+2) cilia, significantly affecting their motility. Using immunoprecipitation, we demonstrate that, whereas MNS1 can associate with itself and CFAP53, CFAP53 is unable to self-associate. We also show that additional axonemal dynein-interacting proteins, two outer dynein arm docking (ODAD) complex members, show differential localisation between types of motile cilia. Together, our findings clarify how paralogous fMIPs, CFAP53 and MNS1, function in regulating (9+2) versus (9+0) cilia motility, and further emphasise extensive structural diversity among these organelles.

Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq.

Currently, the dynamic accessible elements that determine regulatory programs responsible for the unique identity and function of each cell type during zebrafish embryogenesis lack detailed study. Here we present SPATAC-seq: a split-pool ligation-based assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 800,000 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stage. Using this chromatin accessibility map, we identified 604 cell states and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineated development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, enhancer reporter assays confirmed that the majority of tested cCREs exhibited robust enhanced green fluorescent protein expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a valuable open resource for exploring driver regulators of cell fate decisions in zebrafish embryogenesis.

Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvß3.

In zebrafish, brain lymphatic endothelial cells (BLECs) are essential for meningeal angiogenesis and cerebrovascular regeneration. Although epidermal growth factor-like domain 7 (Egfl7) has been reported to act as a pro-angiogenic factor, its roles in lymphangiogenesis remain unclear. Here, we show that Egfl7 is expressed in both blood and lymphatic endothelial cells. We generate an egfl7 cq180 mutant with a 13-bp-deletion in exon 3 leading to reduced expression of Egfl7. The egfl7 cq180 mutant zebrafish exhibit defective formation of BLEC bilateral loop-like structures, although trunk and facial lymphatic development remains unaffected. Moreover, while the egfl7 cq180 mutant displays normal BLEC lineage specification, the migration and proliferation of these cells are impaired. Additionally, we identify integrin αvß3 as the receptor for Egfl7. αvß3 is expressed in the CVP and sprouting BLECs, and blocking this integrin inhibits the formation of BLEC bilateral loop-like structures. Thus, this study identifies a role for Egfl7 in BLEC development that is mediated through the integrin αvß3.

In Vivo Monitoring of Fabp7 Expression in Transgenic Zebrafish.

In zebrafish, like in mammals, radial glial cells (RGCs) can act as neural progenitors during development and regeneration in adults. However, the heterogeneity of glia subpopulations entails the need for different specific markers of zebrafish glia. Currently, fluorescent protein expression mediated by a regulatory element from the glial fibrillary acidic protein (gfap) gene is used as a prominent glia reporter. We now expand this tool by demonstrating that a regulatory element from the mouse Fatty acid binding protein 7 (Fabp7) gene drives reliable expression in fabp7-expressing zebrafish glial cells. By using three different Fabp7 regulatory element-mediated fluorescent protein reporter strains, we reveal in double transgenic zebrafish that progenitor cells expressing fluorescent proteins driven by the Fabp7 regulatory element give rise to radial glia, oligodendrocyte progenitors, and some neuronal precursors. Furthermore, Bergmann glia represent the almost only glial population of the zebrafish cerebellum (besides a few oligodendrocytes), and the radial glia also remain in the mature cerebellum. Fabp7 regulatory element-mediated reporter protein expression in Bergmann glia progenitors suggests their origin from the ventral cerebellar proliferation zone, the ventricular zone, but not from the dorsally positioned upper rhombic lip. These new Fabp7 reporters will be valuable for functional studies during development and regeneration.