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Background: The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F 1 fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single-insertion transgenic lines and the need to complete experiments within a reasonable timeframe., Results: In this article, we introduce a simple competitive dilution strategy for rapid generation of single-insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single-insertion F 1 generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single-insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects., Conclusions: Wide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research., (© 2024 American Association for Anatomy.)

Background: The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord., Results: We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans., Conclusions: Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression., (© 2024 The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: During neurogenesis, growing axons must navigate through the complex extracellular environment and make correct synaptic connections for the proper functioning of neural circuits. The mechanisms underlying the formation of functional neural networks are still only partially understood., Results: Here we analyzed the role of a novel gene si:ch73-364h19.1/drish in the neural and vascular development of zebrafish embryos. We show that drish mRNA is expressed broadly and dynamically in multiple cell types including neural, glial, retinal progenitor and vascular endothelial cells throughout the early stages of embryonic development. To study Drish function during embryogenesis, we generated drish genetic mutant using CRISPR/Cas9 genome editing. drish loss-of-function mutant larvae displayed defects in early retinal ganglion cell, optic nerve and the retinal inner nuclear layer formation, as well as ectopic motor axon branching. In addition, drish mutant adults exhibited deficient retinal outer nuclear layer and showed defective light response and locomotory behavior. However, vascular patterning and blood circulation were not significantly affected., Conclusions: Together, these data demonstrate important roles of zebrafish drish in the retinal ganglion cell, optic nerve and interneuron development and in spinal motor axon branching., (© 2024 American Association for Anatomy.)

Background: The two-pore domain potassium (K2P) channels are a major type of potassium channels that maintain the cell membrane potential by conducting passive potassium leak currents independent of voltage change. They play prominent roles in multiple physiological processes, including neuromodulation, perception of pain, breathing and mood control, and response to volatile anesthetics. Mutations in K2P channels have been linked to many human diseases, such as neuronal and cardiovascular disorders and cancers. Significant progress has been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and function during embryonic development remain largely unknown., Results: We employed the zebrafish model and identified 23 k2p genes using BLAST search and gene cloning. We first analyzed vertebrate K2P channel evolution by phylogenetic and syntenic analyses. Our data revealed that the six subtypes of the K2P genes have already evolved in invertebrates long before the emergence of vertebrates. Moreover, the vertebrate K2P gene number increased, most likely due to two whole-genome duplications. Furthermore, we examined zebrafish k2p gene expression during early embryogenesis by in situ hybridization. Each subgroup's genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues consistent with their known function of neural excitability regulation. However, a few k2p genes were expressed temporarily in specific tissues or organs, suggesting that these K2P channels may be needed for embryonic development., Conclusions: Our phylogenetic and developmental analyses of K2P channels shed light on their evolutionary history and potential roles during embryogenesis related to their physiological functions and human channelopathies., (© 2024 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Elongation of the spinal cord is dependent on neural development from neuromesodermal progenitors in the tail bud. We previously showed the involvement of the Oct4-type gene, pou5f3, in this process in zebrafish mainly by dominant-interference gene induction, but, to compensate for the limitation of this transgene approach, mutant analysis was indispensable. pou5f3 involvement in the signaling pathways was another unsolved question., Results: We examined the phenotypes of pou5f3 mutants and the effects of Pou5f3 activation by the tamoxifen-ERT2 system in the posterior neural tube, together confirming the involvement of pou5f3. The reporter assays using P19 cells implicated tail bud-related transcription factors in pou5f3 expression. Regulation of tail bud development by retinoic acid (RA) signaling was confirmed by treatment of embryos with RA and the synthesis inhibitor, and in vitro reporter assays further showed that RA signaling regulated pou5f3 expression. Importantly, the expression of the RA degradation enzyme gene, cyp26a1, was down-regulated in embryos with disrupted pou5f3 activity., Conclusions: The involvement of pou5f3 in spinal cord extension was supported by using mutants and the gain-of-function approach. Our findings further suggest that pou5f3 regulates the RA level, contributing to neurogenesis in the posterior neural tube., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Noncanonical Wnts are morphogens that can elevate intracellular Ca 2+ , activate the Ca 2+ /calmodulin-dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation., Results: Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1-3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant-negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes., Conclusions: Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Essential patterning processes transform the heart tube into a compartmentalized organ with distinct chambers separated by an atrioventricular canal (AVC). This transition involves the refinement of expression of genes that are first found broadly throughout the heart tube and then become restricted to the AVC. Despite the importance of cardiac patterning, we do not fully understand the mechanisms that limit gene expression to the AVC., Results: We show that the zebrafish gene smarcc1a, encoding a BAF chromatin remodeling complex subunit homologous to mammalian BAF155, is critical for cardiac patterning. In smarcc1a mutants, myocardial differentiation and heart tube assembly appear to proceed normally. Subsequently, the smarcc1a mutant heart fails to exhibit refinement of gene expression patterns to the AVC, and the persistence of broad gene expression is accompanied by failure of chamber expansion. In addition to their cardiac defects, smarcc1a mutants lack pectoral fins, indicating similarity to tbx5a mutants. However, comparison of smarcc1a and tbx5a mutants suggests that perturbation of tbx5a function is not sufficient to cause the smarcc1a mutant phenotype., Conclusions: Our data indicate an important role for Smarcc1a-containing chromatin remodeling complexes in regulating the changes in gene expression and morphology that distinguish the AVC from the cardiac chambers., (© 2023 American Association for Anatomy.)

Background: Zebrafish possess a remarkable regenerative capacity, which is mediated by the induction of various genes upon injury. Injury-dependent transcription is governed by the tissue regeneration enhancer elements (TREEs). Here, we utilized leptin b (lepb), an injury-specific factor, and its TREE to dissect heterogeneity of noncardiomyocytes (CMs) in regenerating hearts., Results: Our single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the endothelium/endocardium(EC) is activated to induce distinct subpopulations upon injury. We demonstrated that lepb can be utilized as a regeneration-specific marker to subset injury-activated ECs. lepb + ECs robustly induce pro-regenerative factors, implicating lepb + ECs as a signaling center to interact with other cardiac cells. Our scRNA-seq analysis identified that lepb is also produced by subpopulation of epicardium (Epi) and epicardium-derived cells (EPDCs). To determine whether lepb labels injury-emerging non-CM cells, we tested the activity of lepb-linked regeneration enhancer (LEN) with chromatin accessibility profiles and transgenic lines. While nondetectable in uninjured hearts, LEN directs EC and Epi/EPDC expression upon injury. The endogenous LEN activity was assessed using LEN deletion lines, demonstrating that LEN deletion abolished injury-dependent expression of lepb, but not other nearby genes., Conclusions: Our integrative analyses identify regeneration-emerging cell-types and factors, leading to the discovery of regenerative features of hearts., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Phenotypic variation is of paramount importance in development, evolution, and human health; however, the molecular mechanisms that influence organ shape and shape variability are not well understood. During craniofacial development, the behavior of skeletal precursors is regulated by both biochemical and environmental inputs, and the primary cilia play critical roles in transducing both types of signals. Here, we examine a gene that encodes a key constituent of the ciliary rootlets, crocc2, and its role in cartilage morphogenesis in larval zebrafish., Results: Geometric morphometric analysis of crocc2 mutants revealed altered craniofacial shapes and expanded variation. At the cellular level, we observed altered chondrocyte shapes and planar cell polarity across multiple stages in crocc2 mutants. Notably, cellular defects were specific to areas that experience direct mechanical input. Cartilage cell number, apoptosis, and bone patterning were not affected in crocc2 mutants., Conclusions: Whereas "regulatory" genes are widely implicated in patterning the craniofacial skeleton, genes that encode "structural" aspects of the cell are increasingly implicated in shaping the face. Our results add crocc2 to this list, and demonstrate that it affects craniofacial geometry and canalizes phenotypic variation. We propose that it does so via mechanosensing, possibly through the ciliary rootlet. If true, this would implicate a new organelle in skeletal development and evolution., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: RERE is a highly conserved transcriptional co-regulator that is associated with a human neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH, OMIM: 616975)., Results: We show that the zebrafish rerea mutant (babyface) robustly recapitulates optic fissure closure defects resulting from loss of RERE function, as observed in humans. These defects result from expansion of proximal retinal optic stalk (OS) and reduced expression of some of the ventral retinal fate genes due to deregulated protein signaling. Using zebrafish and cell-based assays, we determined that NEDBEH-associated human RERE variants function as hypomorphs in their ability to repress shh signaling and some exhibit abnormal nuclear localization. Inhibiting shh signaling by the protein inhibitor HPI-1 rescues coloboma, confirming our observation that coloboma in rerea mutants is indeed due to deregulation of shh signaling., Conclusions: Zebrafish rerea mutants exhibit OS and optic fissure closure defects. The optic fissure closure defect was rescued by an shh signaling inhibitor, suggesting that this defect could arise due to deregulated shh signaling., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Homeobox transcription factor encoding genes, genomic screen homeobox 1 and 2 (gsx1 and gsx2), are expressed during neurodevelopment in multiple vertebrates. However, we have limited knowledge of the dynamic expression of these genes through developmental time and the gene networks that they regulate in zebrafish., Results: We confirmed that gsx1 is expressed initially in the hindbrain and diencephalon and later in the optic tectum, pretectum, and cerebellar plate. gsx2 is expressed in the early telencephalon and later in the pallium and olfactory bulb. gsx1 and gsx2 are co-expressed in the hypothalamus, preoptic area, and hindbrain, however, rarely co-localize in the same cells. gsx1 and gsx2 mutant zebrafish were made with TALENs. gsx1 mutants exhibit stunted growth, however, they survive to adulthood and are fertile. gsx2 mutants experience swim bladder inflation failure that prevents survival. We also observed significantly reduced expression of multiple forebrain patterning distal-less homeobox genes in mutants, and expression of foxp2 was not significantly affected., Conclusions: This work provides novel tools with which other target genes and functions of Gsx1 and Gsx2 can be characterized across the central nervous system to better understand the unique and overlapping roles of these highly conserved transcription factors., (© 2022 American Association for Anatomy.)

Background: In the zebrafish midbrain, GABAergic neurons develop from precursors located in the nucleus of the medial longitudinal fasciculus (nMLF). However, the precise mechanisms that underline generation of the nMLF GABAergic neuron are poorly understood., Results: GABAergic neurons in the nMLF co-express transcription factors tal2, gata2a, gata3, and nkx1.2lb. The Nodal-related gene and shh signaling are required for differentiation of nMLF GABAergic neuron precursors. Tal2 is important for nMLF GABAergic neurogenesis. Disruption of Tal2, embryos completely lack the GABA-synthesizing enzyme glutamic acid decarboxylase 67 gene (gad67) expressing cells in the nMLF, and the whole nkx1.2lb expressing cells in the midbrain. Although almost all tal2-expressing cells in the diencephalon and/or nMLF are gata2a- and gata3-positive, simultaneous knockdown of gata2a and gata3 does not affect either tal2 or gad67 expression., Conclusions: In the zebrafish midbrain, expression of tal2, gata2a, and/or gata3 is independent of each other. The function of gata2a and gata3 is dispensable for generation of GABAergic neuron in the nMLF. This suggests that the functional connections of the regulatory genes leading to generation of nMLF GABAergic neurons have diverged between mouse and zebrafish., (© 2022 American Association for Anatomy.)

Background: The vitamin A derivative all-trans retinoic acid (RA) regulates early stages of inner ear development. As the early disruption of the RA pathway results in profound mispatterning of the developing inner ear, this confounds analyses of specific roles in later stages. Therefore, we used the temporal-specific exposure of all-trans RA or diethylaminobenzaldehyde to evaluate RA functions in late otic development., Results: Perturbing late RA signaling causes behavioral defects analogous to those expected in larvae suffering from vestibular dysfunction. These larvae also demonstrate malformations of the semi-circular canals, as visualized through (a) use of the transgenic strain nkhspdmc12a, a fluorescent reporter expressed in otic epithelium; and (b) injection of the fluorescent lipophilic dye DiI. We also noted the altered expression of genes encoding ECM proteins or modifying enzymes. Other malformations of the inner ear observed in our work include the loss or reduced size of the utricular and saccular otoliths, suggesting a role for RA in otolith maintenance., Conclusion: Our work has identified a previously undescribed late phase of RA activity in otic development, demonstrating that vestibular defects observed in human patients in relation to perturbed RA signaling are not solely due to its early disruption in otic development., (© 2022 American Association for Anatomy.)

Background: MLLT3 (AF9) is a nuclear transcription factor crucial for hematopoietic stem cell and progenitor cell maintenance, but its role during embryonic hematopoiesis remains uncertain. Here, we examine the role of mllt3 in developmental hematopoiesis during embryogenesis using zebrafish., Results: Cloning, sequencing, phylogenetic, and synteny analyses showed high evolutionary conservation between important functional domains of the zebrafish orthologue of mllt3 and MLLT3 in humans. Quantitative reverse transcription-PCR and in situ hybridization analyses revealed that mllt3 is maternally supplied and zygotically expressed throughout embryonic development, and that expression is highest between 10 and 24 hours post-fertilization (hpf) coincident with enrichment in the intermediate cell mass (ICM) and posterior blood island, which are the sites of the primitive and transient definitive hematopoiesis in zebrafish, respectively. Further, we found co-expression of mllt3 with the early hematopoietic progenitor markers tal1, gata2, and gata1a in the posterior ICM. By investigating zebrafish hematopoietic mutants, we discovered that mllt3 is involved in erythroid precursor formation. By 48-72 hpf, mllt3 expression proved to be restricted to non-hematopoietic tissues including head structures, pronephric tubules, and liver primordium., Conclusions: These findings establish a link between mllt3 and primitive erythropoiesis and provide the basis for future functional investigations., (© 2022 American Association for Anatomy.)

Background: Migrating muscle progenitors delaminate from the somite and subsequently form muscle tissue in distant anatomical regions such as the paired appendages, or limbs. In amniotes, this process requires a signaling cascade including the transcription factor paired box 3 (Pax3)., Results: In this study, we found that, unlike in mammals, pax3a/3b double mutant zebrafish develop near to normal appendicular muscle. By analyzing numerous mutant combinations of pax3a, pax3b and pax7a, and pax7b, we determined that there is a feedback system and a compensatory mechanism between Pax3 and Pax7 in this developmental process, even though Pax7 alone is not required for appendicular myogenesis. pax3a/3b/7a/7b quadruple mutant developed muscle-less pectoral fins., Conclusions: We found that Pax3 and Pax7 are redundantly required during appendicular myogenesis in zebrafish, where Pax7 is able to activate the same developmental programs as Pax3 in the premigratory progenitor cells., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Cohesinopathies is a term that refers to/covers rare genetic diseases caused by mutations in the cohesin complex proteins. The cohesin complex is a multiprotein complex that facilitates different aspects of cell division, gene transcription, DNA damage repair, and chromosome architecture. Shugoshin proteins prevent the cohesin complex from premature dissociation from chromatids during cell division. Patients with a homozygous missense mutation in SGO1, which encodes for Shugoshin1, have problems with normal pacing of the heart and gut., Results: To study the role of shugoshin during embryo development, we mutated the zebrafish sgo1 gene. Homozygous sgo1 mutant embryos display various phenotypes related to different organs, including a reduced heart rate accompanied by reduced cardiac function. In addition, sgo1 mutants are vision-impaired as a consequence of structurally defective and partially non-functional photoreceptor cells. Furthermore, the sgo1 mutants display reduced food intake and early lethality., Conclusion: We have generated a zebrafish model of Sgo1 that showed its importance during organ development and function., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival., Results: Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damage-induced apoptosis., Conclusion: Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53-dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo-injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo-injury procedures are common in mouse and zebrafish., Results: In the present study, we induced cryo-damage on the ventricle in Iberian ribbed newts using a liquid nitrogen-chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo-injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures., Conclusions: Notably, we established a cryo-injury procedure for the newt and confirmed that regeneration of the cryo-damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo-injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models., (© 2021 American Association of Anatomists.)

Background: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis., Results: To overcome these challenges, we adapted the chromatin profiling methods Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and CUT&Tag for use in zebrafish and applied these methods to generate high-resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z using tissue isolated from whole, mid-gastrula stage embryos. Using this data, we identify a subset of genes that may be bivalently regulated during both zebrafish and mouse gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the effectiveness of CUT&RUN for detecting H3K9me3 enrichment at repetitive sequences., Conclusions: Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis., (© 2021 American Association for Anatomy.)

Background: Lamin A/C gene (LMNA) mutations frequently cause cardiac and/or skeletal muscle diseases called striated muscle laminopathies. We created a zebrafish muscular laminopathy model using CRISPR/Cas9 technology to target the zebrafish lmna gene., Results: Heterozygous and homozygous lmna mutants present skeletal muscle damage at 1 day post-fertilization (dpf), and mobility impairment at 4 to 7 dpf. Cardiac structure and function analyses between 1 and 7 dpf show mild and transient defects in the lmna mutants compared to wild type (WT). Quantitative RT-PCR analysis of genes implicated in striated muscle laminopathies show a decrease in jun and nfκb2 expression in 7 dpf homozygous lmna mutants compared to WT. Homozygous lmna mutants have a 1.26-fold protein increase in activated Erk 1/2, kinases associated with striated muscle laminopathies, compared to WT at 7 dpf. Activated Protein Kinase C alpha (Pkc α), a kinase that interacts with lamin A/C and Erk 1/2, is also upregulated in 7 dpf homozygous lmna mutants compared to WT., Conclusions: This study presents an animal model of skeletal muscle laminopathy where heterozygous and homozygous lmna mutants exhibit prominent skeletal muscle abnormalities during the first week of development. Furthermore, this is the first animal model that potentially implicates Pkc α in muscular laminopathies., (© 2021 American Association for Anatomy.)

Background: Pax2 is required for optic fissure development in many organisms, including humans and zebrafish. Zebrafish loss-of-function mutations in pax2a display coloboma, yet the etiology of the morphogenetic defects is unclear. Further, pax2 is duplicated in zebrafish, and a role for pax2b in optic fissure development has not been examined., Results: Using a combination of imaging and molecular genetics, we interrogated a potential role for pax2b and examined how loss of pax2 affects optic fissure development. Although optic fissure formation appears normal in pax2 mutants, an endothelial-specific subset of periocular mesenchyme (POM) fails to initially localize within the optic fissure, yet both neural crest and endothelial-derived POM ectopically accumulate at later stages in pax2a and pax2a; pax2b mutants. Apoptosis is not up-regulated within the optic fissure in pax2 mutants, yet cell death is increased in tissues outside of the optic fissure, and when apoptosis is inhibited, coloboma is partially rescued. In contrast to pax2a, loss of pax2b does not appear to affect optic fissure morphogenesis., Conclusions: Our results suggest that pax2a, but not pax2b, supports cell survival outside of the optic fissure and POM abundance within it to facilitate optic fissure closure., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Inwardly rectifying potassium channels are essential for normal potassium homeostasis, maintaining the cellular resting membrane potential, and regulating electrolyte transportation. Mutations in Kir channels have been known to cause debilitating diseases ranging from neurological abnormalities to renal and cardiac failures. Many efforts have been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and functions during embryonic development remain largely unknown., Results: Using zebrafish as a model, we identified and renamed 31 kir genes. We also analyzed Kir gene evolution by phylogenetic and syntenic analyses. Our data indicated that the four subtypes of the Kir genes might have already evolved out in chordates. These vertebrate Kir genes most likely resulted from both whole-genome duplications and tandem duplications. In addition, we examined zebrafish kir gene expression during early embryogenesis. Each subgroup's genes showed similar but distinct gene expression domains. The gene expression of ohnologous genes from teleost-specific whole-genome duplication indicated subfunctionalization. Varied temporal gene expression domains suggest that Kir channels may be needed for embryonic patterning or regulation., Conclusions: Our phylogenetic and developmental analyses of Kir channels shed light on their evolutionary history and potential functions during embryogenesis related to congenital diseases and human channelopathies., (© 2021 American Association for Anatomy.)

Background: The Transforming Growth Factor β (TGFβ) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFβ family that participates in heart, liver and kidney development as revealed by the tgfbr3-null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish., Results: Here, we present CRISPR/Cas9-derived zebrafish Tgfbr3-null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3-null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFβ signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process., Conclusion: Betaglycan's "ligand presentation" function contributes to the optimal TGFβ signaling required for zebrafish chordacentra mineralization., (© 2021 American Association for Anatomy.)

Background: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus., Results: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap., Conclusions: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes., (© 2021 American Association for Anatomy.)

Background: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression., Results: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation., Conclusions: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Although the cell cycle and cell differentiation should be coordinately regulated to generate a variety of neurons in the brain, the molecules that are involved in this coordination still remain largely unknown. In this study, we analyzed the roles of a nuclear protein Cfdp1, which is thought to be involved in chromatin remodeling, in zebrafish neurogenesis., Results: Zebrafish cfdp1 mutants maintained the progenitors of granule cells (GCs) in the cerebellum, but showed defects in their differentiation to GCs. cfdp1 mutants showed an increase in phospho-histone 3 (pH 3)-positive cells and apoptotic cells, as well as a delayed cell cycle transition from the G2 to the M phase in the cerebellum. The inhibition of tp53 prevented apoptosis but not GC differentiation in the cfdp1 mutant cerebellum. A similar increase in apoptotic cells and pH 3-positive cells, and defective cell differentiation, were observed in the cfdp1 mutant retina. Although mitotic spindles formed, mitosis was blocked before anaphase in both the cerebellum and retina of cfdp1 mutant larvae. Furthermore, expression of the G2/mitotic-specific cyclin B1 gene increased in the cfdp1 mutant cerebellum., Conclusions: Our findings suggest that Cfdp1 regulates the cell cycle of neural progenitors, thereby promoting neural differentiation in the brain., (© 2021 American Association for Anatomy.)

Progress in understanding mechanisms of inner ear development has been remarkably rapid in recent years. The research community has benefited from the availability of several diverse model organisms, including zebrafish, chick, and mouse. The complexity of the inner ear has proven to be a challenge, and the complexity of the mammalian cochlea in particular has been the subject of intense scrutiny. Zebrafish lack a cochlea and exhibit a number of other differences from amniote species, hence they are sometimes seen as less relevant for inner ear studies. However, accumulating evidence shows that underlying cellular and molecular mechanisms are often highly conserved. As a case in point, consideration of the diverse functions of Fgf and its downstream effectors reveals many similarities between vertebrate species, allowing meaningful comparisons the can benefit the entire research community. In this review, I will discuss mechanisms by which Fgf controls key events in early otic development in zebrafish and provide direct comparisons with chick and mouse., (© 2021 American Association of Anatomists.)

Background: Calcium-activated potassium channels (KCa) are a specific type of potassium channel activated by intracellular calcium concentration changes. This group of potassium channels plays fundamental roles ranging from regulating neuronal excitability to immune cell activation. Many human diseases such as schizophrenia, hypertension, epilepsy, and cancers have been linked to mutations in this group of potassium channels. Although the KCa channels have been extensively studied electrophysiologically and pharmacologically, their spatiotemporal gene expression during embryogenesis remains mostly unknown., Results: Using zebrafish as a model, we identified and renamed 14 KCa genes. We further performed phylogenetic and syntenic analyses on vertebrate KCa genes. Our data revealed that the number of KCa genes in zebrafish was increased, most likely due to teleost-specific whole-genome duplication. Moreover, we examined zebrafish KCa gene expression during early embryogenesis. The duplicated ohnologous genes show distinct and overlapped gene expression. Furthermore, we found that zebrafish KCa genes are expressed in various tissues and organs (somites, fins, olfactory regions, eye, kidney, and so on) and neuronal tissues, suggesting that they may play important roles during zebrafish embryogenesis., Conclusions: Our phylogenetic and developmental analyses shed light on the potential functions of the KCa genes during embryogenesis related to congenital diseases and human channelopathies., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association of Anatomists.)

Background: Vitamin D is an essential nutrient that has long been known to regulate skeletal growth and integrity. In models of major appendage regeneration, treatment with vitamin D analogs has been reported to improve aspects of zebrafish fin regeneration in specific disease or gene misexpression contexts, but also to disrupt pattern in regenerating salamander limbs. Recently, we reported strong mitogenic roles for vitamin D signaling in several zebrafish tissues throughout life stages, including epidermal cells and osteoblasts of adult fins. To our knowledge, molecular genetic approaches to dissect vitamin D function in appendage regeneration have not been described., Results: Using a knock-in GFP reporter for the expression of the vitamin D target gene and negative regulator cyp24a1, we identified active vitamin D signaling in adult zebrafish fins during tissue homeostasis and regeneration. Transgenic expression of cyp24a1 or a dominant-negative vitamin D receptor (VDR) inhibited regeneration of amputated fins, whereas global vitamin D treatment accelerated regeneration. Using tissue regeneration enhancer elements, we found that local enhancement of VDR expression could improve regeneration with low doses of a vitamin D analog., Conclusions: Vitamin D signaling enhances the efficacy of fin regeneration in zebrafish., (© 2020 American Association of Anatomists.)

Background: The nuclear progesterone receptor (Pgr) is a ligand-dependent transcription factor primarily responsible for mediating progesterone actions relevant for reproduction across vertebrates. Information on the cellular localization of Pgr expression in the reproductive system is required for developing a comprehensive approach to elucidate the role of Pgr in reproduction., Results: We generated transgenic zebrafish Tg(pgr:eGFP) that express enhanced green fluorescent protein (eGFP) driven by promoter sequence of pgr gene. The tissue distribution pattern of egfp mRNA is consistent with the pgr mRNA expression in Tg(pgr:eGFP). In the pituitary, GFP signals are found in the proximal pars distalis. In order to better discern the cellular localization of GFP signals in gonads, Tg(pgr:eGFP) line was crossed with Tg(gsdf:nfsB-mCherry) line, specifically expressing nitroreductase-mCherry fusion protein in granulosa and Sertoli cells in ovary and testis, respectively. Imaging of testis tissue showed that GFP expression was confined to Leydig cells. In the ovary, GFP expression colocalized with the mCherry signal in granulosa cells. Intriguingly, we also identified some non-granulosa cells close to where blood vessels branched, expressing stronger GFP signals than granulosa cells., Conclusions: Analyzing Tg(pgr:eGFP) expression in zebrafish provided leads toward new routes to study the role of Pgr in reproduction., (© 2020 Wiley Periodicals LLC.)

Background: With the goal of labeling and manipulating the zebrafish hypothalamus, we sought to target a green fluorescent protein (gfp) transgene to the expression domains of nkx2.4b, a gene expressed during hypothalamic and thyroid development. We combined transcription activator-like effector nucleases (TALENs)-mediated mutagenesis with a targeting construct to enable insertion of a gfp transgene into the endogenous nkx2.4b genomic locus., Results: Injection of TALENs targeted to the first exon of nkx2.4b created a predicted null allele, and homozygous mutant embryos displayed loss of thyroid markers. From embryos injected with both TALENs and a targeting construct carrying a gfp transgene, we recovered a line in which GFP was expressed specifically in the hypothalamus and thyroid. Fish homozygous for this allele lacked exon 1 of nkx2.4b and exhibited hypothyroid phenotypes., Conclusions: By combining TALENs injections with a targeting construct that contained a gfp transgene, we were able to recover an allele in which GFP is expressed in the nkx2.4b expression domains, with homozygous phenotypes suggesting the creation of a loss-of-function transgenic line. These results demonstrate the creation of a useful tool for studying hypothalamus and thyroid development., (© 2020 Wiley Periodicals LLC.)

Background: The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown., Results: By generating novel alleles in the zebrafish PRPS paralogs, prps1a and prps1b, we gained new insight into the role of PRPS filaments during eye development. We found that mutations in prps1a alone are sufficient to generate abnormally small eyes along with defects in head size, pigmentation, and swim bladder inflation. Furthermore, a loss-of-function mutation that truncates the Prps1a protein resulted in the failure of PRPS filament assembly. Lastly, in mutants that fail to assemble PRPS filaments, we observed disorganization of the actin network in the lens fibers., Conclusions: The truncation of Prps1a blocked PRPS filament formation and resulted in a disorganized lens fiber actin network. Altogether, these findings highlight a potential role for PRPS filaments during lens fiber organization in zebrafish., (© 2020 Wiley Periodicals, Inc.)

A hierarchically organized lymphatic vascular system extends throughout the vertebrate body for tissue fluid homeostasis, immune trafficking, and the absorption of dietary fats. Intralymphatic dye injection and serial sectioning have been the main tools for visualizing lymphatic vessels. Specific markers for identifying the lymphatic vasculature in zebrafish and medaka have appeared as new tools that enable the study of lymphangiogenesis using genetic and experimental manipulation. Transgenic fishes have become excellent organisms for visualizing the lymphatic vasculature in living embryos, but this method has limited usefulness, especially in later developmental stages. The functional lymphatic endothelium predominantly takes up foreign particles in zebrafish and medaka. We utilized this physiological activity and lymph flow to label lymphatic vessels. Intraperitoneal injection of trypan blue is useful for temporal observations of the lymphatic ducts, which are essential for large-scale genetic screening, while cinnabar (HgS) injection allows identification of the lymphatic endothelium under electron microscopy, avoiding artefactual damage. This injection method, which is not high in cost and does not require high skill or special devices, is applicable to any live fish with functioning lymphatic vessels, even mutants, with high reproducibility for visualizing the entire lymphatic vascular system., (© 2019 Wiley Periodicals, Inc.)

Background: Atrioventricular valve development relies upon the precisely defined dimensions of the atrioventricular canal (AVC). Current models suggest that Wnt signaling plays an important role atop a pathway that promotes AVC development. The factors that confine AVC differentiation to the appropriate location, however, are less well understood., Results: Transmembrane protein 2 (Tmem2) is a key player in restricting AVC differentiation: in zebrafish, tmem2 mutants display an expansion of AVC characteristics, but the molecular mechanism of Tmem2 function in this context remains unclear. Through structure-function analysis, we demonstrate that the extracellular portion of Tmem2 is crucial for its role in restricting AVC boundaries. Importantly, the Tmem2 ectodomain contains regions implicated in the depolymerization of hyaluronic acid (HA). We find that tmem2 mutant hearts exhibit excess HA deposition alongside broadened distribution of Wnt signaling. Moreover, addition of ectopic hyaluronidase can restore the restriction of AVC differentiation in tmem2 mutants. Finally, we show that alteration of a residue important for HA depolymerization impairs the efficacy of Tmem2 function during AVC development., Conclusions: Taken together, our data support a model in which HA degradation, regulated by Tmem2, limits the distribution of Wnt signaling and thereby confines the differentiation of the AVC., (© 2019 Wiley Periodicals, Inc.)

Background: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR., Methods: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses., Results: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination., Conclusion: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans., (© 2019 Wiley Periodicals, Inc.)

Background: Astyanax mexicanus is a well-established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems., Results: Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, a Cntnap2-mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish., Conclusion: The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype-phenotype gap., (© 2019 Wiley Periodicals, Inc.)

Background: NOTCH1 plays a key role in the differentiation of ventricles, and mutations are strongly associated with both sporadic and familial bicuspid aortic valves. However, few heart-specific enhancers have been identified to date., Results: In this study, we investigated evolutionary conserved regions (ECRs) that might act as potential enhancers within the region approximately 150-kb upstream and downstream of the NOTCH1 gene. Functional validation revealed that one 127-bp ECR located ~85-kb downstream of the NOTCH1 gene drives green fluorescent protein expression in the zebrafish embryo heart. Transcription factor (TF) prediction and core TF distribution analyses were performed to identify the core region. Dissection of ECR3 was performed to identify the 42-bp sequence, which is sufficient for heart-specific expression. In situ hybridization experiments showed that notch1b is expressed in the heart. Overexpression experiments in cells indicated that NKX2-5 is critical for enhancer activity. Mutation of the NKX-5 binding site significantly decreased reporter gene expression. Next, compared with the commonly used myocardium-labeled zebrafish transgenic strain Tg(cmlc2: mCherry), this 42-bp enhancer-labeled stable line mediated a similar expression pattern but with a smaller core region., Conclusion: This study identified a 42-bp heart-specific enhancer near the NOTCH1 gene and further verified its functional targeting by NKX2-5., (© 2019 Wiley Periodicals, Inc.)

Background: In zebrafish and many other organisms, specification of primordial germ cells (PGCs) requires the transmission of maternally-derived germ plasm. Zebrafish germ plasm ribonucleoparticles (RNPs) aggregate along the cleavage furrows during the first several cell cycles, segregate asymmetrically during the cleavage stages, and undergo cytoplasmic dispersal in the late blastula., Results: For all tested germ plasm RNAs [carbonic anhydrase 15b (ca15b), deleted in azoospermia-like (dazl), dead end (dnd), nanos 3 (nos3), regulator of G-protein signaling14a (rgs14a), and vasa/DEAD box polypeptide 4 (vasa/ddx4)], RNPs are homotypic (containing a single RNA type), with RNPs packing tightly yet remaining distinct within germ plasm aggregates. Homotypic clustering of RNAs within RNPs is observed before aggregation in the cortex and is maintained through germ plasm recruitment, asymmetric segregation and RNP dispersal. We also identify a step of germ plasm fragmentation during the cleavage stages that precedes RNP dispersal., Conclusions: Our findings suggest that germ plasm aggregates act as subcellular compartments that temporarily collect and carry single RNA-type RNPs from fertilization until their cytoplasmic dispersal in PGCs at the end of the blastula period, and describe a previously unknown fragmentation step that allows for an increase in the pool of germ plasm-carrying cells, presumably PGCs. Developmental Dynamics 248:306-318, 2019. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Background: Transient heat shock during early development is an established experimental paradigm for doubling the genome of the zebrafish zygote, which has practical applications in expedited identification of recessive mutations in genetic screens. Despite the simplicity of the strategy and the genetic tractability of zebrafish, heat shock has not been used for genome doubling since the proof-of-principle experiments done in the 1980s. This is because of poor survival of embryos that ensue from transient heat shocks and gross developmental abnormalities in the few survivors, which is incompatible with phenotype driven screens., Results: We show that heat shocks during early zebrafish development uncouple the second cycle of DNA and centrosome duplication. Interestingly, the developmental time of the heat shock that triggers the dissociation between DNA and centrosome duplication cycles significantly affect the potential of embryos to survive and attain normal morphology. The potential to develop normally after a heat shock alters in a developmental time span of 2 min in zebrafish embryos, a phenomenon that has not been reported in any species., Conclusions: The existence of heat resilient developmental windows and reduced heat teratogenicity during these windows could be an effective step forward in practical application of transient heat for experimental manipulation of ploidy in zebrafish. More broadly, heat resilience before zygotic genome activation suggests that metazoan embryos may possess innate protective features against heat beyond the canonical heat shock response. Developmental Dynamics 247:992-1004, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 The Authors Developmental Dynamics published by Wiley Periodicals, Inc.)

Background: Opsins are a group of light-sensitive proteins present in photoreceptor cells, which convert the energy of photons into electrochemical signals, thus allowing vision. Given their relevance, we aimed to visualize the two red opsins at subcellular scale in photoreceptor cells., Results: We generated a novel Zebrafish BAC transgenic line, which express fluorescently tagged, full-length Opsin 1 long-wave-sensitive 1 (Opn1lw1) and full-length Opsin 1 long-wave-sensitive 2 (Opn1lw2) under the control of their endogenous promoters. Both fusion proteins are localized in the outer segments of photoreceptor cells. During development, Opn1lw2-mKate2 is detected from the initial formation of outer segments onward. In contrast, Opn1lw1-mNeonGreen is first detected in juvenile Zebrafish at about 2 weeks postfertilization, and both opsins continue to be expressed throughout adulthood. It is important to note that the presence of the transgene did not significantly alter the size of outer segments., Conclusions: We have generated a transgenic line that mimics the endogenous expression pattern of Opn1lw1 and Opn1lw2 in the developing and adult retina. In contrast to existing lines, our transgene design allows to follow protein localization. Hence, we expect that these lines could act as useful real-time reporters to directly measure phenomena in retinal development and disease models. Developmental Dynamics 247:951-959, 2018. © 2018 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists., (© 2018 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

Background: Mustn1 is a specific musculoskeletal protein that plays a critical role in myogenesis and chondrogenesis in vertebrates. Whole-mount in situ hybridization revealed that mustn1b mRNAs are specifically expressed in skeletal and cardiac muscles in Zebrafish embryos. However, the precise function and the regulatory elements required for its muscle-specific expression are largely unknown., Results: The purpose of this study was to explore and uncover the target genomic regions that regulate mustn1b gene expression by in vivo functional characterization of the mustn1b promoter. We report here stable expression analyses of eGFP from fluorescent transgenic reporter Zebrafish line containing a 0.8kb_mustn1b-Tol2-eGFP construct. eGFP expression was specifically found in the skeletal and cardiac muscle tissues. We show that reporter Zebrafish lines generated replicate the endogenous mustn1b expression pattern in early Zebrafish embryos. Specific site directed-mutagenesis analysis revealed that promoter activity resides in two annotated genomic regulatory regions, each one corresponding to a specific functional transcription factor binding site., Conclusions: Our data indicate that mustn1b is specifically expressed in skeletal and cardiac muscle tissues and its muscle specificity is controlled by the 0.2-kb promoter and flanking sequences and in vivo regulated by the action of two sequence-specific families of transcription factors. Developmental Dynamics 246:992-1000, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Zebrafish is a model organism that affords experimental advantages toward investigating the normal function of genes associated with congenital birth defects. Here we summarize zebrafish studies of genes implicated in orofacial cleft (OFC). The most common use of zebrafish in this context has been to explore the normal function an OFC-associated gene product in craniofacial morphogenesis by inhibiting expression of its zebrafish ortholog. The most frequently deployed method has been to inject embryos with antisense morpholino oligonucleotides targeting the desired transcript. However, improvements in targeted mutagenesis strategies have led to widespread adoption of CRISPR/Cas9 technology. A second application of zebrafish has been for functional assays of gene variants found in OFC patients; such in vivo assays are valuable because the success of in silico methods for testing allele severity has been mixed. Finally, zebrafish have been used to test the tissue specificity of enhancers that harbor single nucleotide polymorphisms associated with risk for OFC. We review examples of each of these approaches in the context of genes that are implicated in syndromic and non-syndromic OFC. Developmental Dynamics 246:897-914, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: The transcriptional co-repressor Sin3 is highly conserved from yeast to vertebrates and has multiple roles controlling cell fate, cell cycle progression, and senescence programming. Sin3 proteins recruit histone deacetylases and other chromatin modifying factors to specific loci through interactions with transcription factors including Myc, Rest, p53 and E2F. Most vertebrates have two Sin3 family members (sin3a and sin3b), but zebrafish have a second sin3a paralogue. In mice, sin3a and sin3b are essential for embryonic development. Sin3b knockout mice show defects in growth as well as bone and blood differentiation., Results: To study the requirement for Sin3b during development, we disrupted zebrafish sin3b using CRISPR-Cas9, and studied the effects on early development and locomotor behavior., Conclusions: Surprisingly, Sin3b is not essential in zebrafish. sin3b mutants show a decrease in fitness, small size, changes to locomotor behavior, and delayed bone development. We did not detect a role for Sin3b in cell proliferation. Our analysis of the sin3b mutant revealed a more nuanced requirement for zebrafish Sin3b than would be predicted from analysis of mutants in other species. Developmental Dynamics 246:946-955, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: Zebrafish visual function depends on quality optics. An F3 screen for developmental mutations in the Zebrafish nervous system was conducted in wild-type (wt) AB Zebrafish exposed to 3 mM of N-ethyl-N-nitrosourea (ENU)., Results: Mutant offspring, identified in an F3 screen, were characterized by a small pupil, resulting from retinal hypertrophy or hyperplasia and a small lens. Deficits in visual function made feeding difficult after hatching at approximately 5-6 days postfertilization (dpf). Special feeding conditions were necessary for survival of the occhiolino (occ) mutants after 6 dpf. Optokinetic response (OKR) tests measured defects in visual function in the occ mutant, although electroretinograms (ERGs) were normal in the mutant and wt. Consistent with the ERGs, histology found normal retinal structure in the occ mutant and wt Zebrafish. However, lens development was abnormal. Multiphoton imaging of the developmental stages of live embryos confirmed the formation of a secondary mass of lens cells in the developing eye of the mutant Zebrafish at 3-4 dpf, and laminin immunohistochemistry indicated the lens capsule was thin and disorganized in the mutant Zebrafish., Conclusions: The occ Zebrafish is a novel disease model for visual defects associated with abnormal lens development. Developmental Dynamics 246:915-924, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: With the exception of the head, the vertebrate embryonic body is formed progressively in an anterior-posterior direction, originating from a posteriorly located bipotential neural-mesodermal progenitor population. The T-box transcription factor Brachyury is expressed within the progenitors and is essential for the formation of the posterior mesoderm. A novel cold-sensitive mutant of Zebrafish Brachyury (ntla(cs) ) is described that allows exploration of the temporal role of this key factor., Results: The ntla(cs) mutant is used to show that Ntla has an essential role during early gastrulation, but as gastrulation proceeds the importance of Ntla declines as Ntlb acquires a capacity to form the posterior mesoderm. Remarkably, ntla(cs) embryos held at the nonpermissive temperature just during the gastrula stages show recovery of normal levels of mesodermal gene expression, demonstrating the plasticity of the posterior progenitors., Conclusion: ntla(cs) is a valuable tool for exploring the processes forming the posterior body since it allows temporally specific activation and inactivation of Brachyury function. It is used here to show the changing roles of Ntla during early development and the dynamics of the neuromesodermal progenitors. Developmental Dynamics 245:874-880, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: One of the most prevalent congenital birth defects is cleft palate. The palatal skeleton is derived from the cranial neural crest and platelet-derived growth factors (Pdgf) are critical in palatogenesis. Of the two Pdgf receptors, pdgfra is required for neural crest migration and palatogenesis. However, the role pdgfrb plays in the neural crest, or whether pdgfra and pdgfrb interact during palatogenesis is unclear., Results: We find that pdgfrb is dispensable for craniofacial development in zebrafish. However, the palatal defect in pdgfra;pdgfrb double mutants is significantly more severe than in pdgfra single mutants. Data in mouse suggest this interaction is conserved and that neural crest requires both genes. In zebrafish, pdgfra and pdgfrb are both expressed by neural crest within the pharyngeal arches, and pharmacological analyses demonstrate Pdgf signaling is required at these times. While neither proliferation nor cell death appears affected, time-lapsed confocal analysis of pdgfra;pdgfrb mutants shows a failure of proper neural crest condensation during palatogenesis., Conclusions: We provide data showing that pdgfra and pdgfrb interact during palatogenesis in both zebrafish and mouse. In zebrafish, this interaction affects proper condensation of maxillary neural crest cells, revealing a previously unknown interaction between Pdgfra and Pdgfrb during palate formation. Developmental Dynamics 245:641-652, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus-renal failure). The zebrafish insertional mutant scarb2a(hi1463Tg) has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates., Results: There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di-leucine motif. We found that scarb2a(hi1463Tg) homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole-mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short-lived in the presumptive notochord and precedes the complete differentiation of the notochord., Conclusions: This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development., (© 2016 Wiley Periodicals, Inc.)

Background: Roberts syndrome (RBS) is a rare genetic disorder characterized by craniofacial abnormalities, limb malformation, and often severe mental retardation. RBS arises from mutations in ESCO2 that encodes an acetyltransferase and modifies the cohesin subunit SMC3. Mutations in SCC2/NIPBL (encodes a cohesin loader), SMC3 or other cohesin genes (SMC1, RAD21/MCD1) give rise to a related developmental malady termed Cornelia de Lange syndrome (CdLS). RBS and CdLS exhibit overlapping phenotypes, but RBS is thought to arise through mitotic failure and limited progenitor cell proliferation while CdLS arises through transcriptional dysregulation. Here, we use the zebrafish regenerating fin model to test the mechanism through which RBS-type phenotypes arise., Results: esco2 is up-regulated during fin regeneration and specifically within the blastema. esco2 knockdown adversely affects both tissue and bone growth in regenerating fins-consistent with a role in skeletal morphogenesis. esco2-knockdown significantly diminishes cx43/gja1 expression which encodes the gap junction connexin subunit required for cell-cell communication. cx43 mutations cause the short fin (sof(b123) ) phenotype in zebrafish and oculodentodigital dysplasia (ODDD) in humans. Importantly, miR-133-dependent cx43 overexpression rescues esco2-dependent growth defects., Conclusions: These results conceptually link ODDD to cohesinopathies and provide evidence that ESCO2 may play a transcriptional role critical for human development., (© 2015 Wiley Periodicals, Inc.)

Background: Cdx factors expressed in caudal regions of vertebrate embryos regulate hox patterning gene transcription. While loss of Cdx function is known to shift hox spatial expression domains posteriorly, the mechanism underlying the shift is not understood. We addressed this problem by analyzing the spatiotemporal expression profile of all 49 zebrafish hox genes in wild-type and Cdx4-deficient embryos., Results: Loss of Cdx4 had distinct effects on hox spatial expression in a paralogous group-dependent manner: in the head, group 4 expression was expanded posteriorly; in the trunk, group 5-10 expression was shifted posteriorly; and in the tail, group 11-13 genes were expressed in the tail bud but not in more differentiated tissues. In the trunk neural tissue, loss of Cdx4 severely delayed both transcriptional activation of hox genes during the initiation phase, and the anterior-ward expansion of hox expression domains during the establishment phase. In contrast, in the trunk mesoderm, loss of Cdx4 only delayed the hox initiation phase., Conclusions: These results indicate that Cdx4 differentially regulates the transcription of head, trunk and tail hox genes. In the trunk, Cdx4 conveys spatial positional information to axial tissues primarily by regulating the time of hox gene transcriptional activation during the initiation phase., (© 2015 Wiley Periodicals, Inc.)

Background: Functions for the early embryonic vasculature in regulating development of central nervous system tissues, such as the retina, have been suggested by in vitro studies and by in vivo manipulations that caused additional ocular vessels to develop. Here, we use an avascular zebrafish embryo, cloche-/- (clo-/-), to begin to identify necessary developmental functions of the ocular vasculature in regulating development and patterning of the neural retina, in vivo. These studies are possible in zebrafish embryos, which do not yet rely upon the vasculature for tissue oxygenation., Results: clo-/- embryos lacked early ocular vasculature and were microphthalmic, with reduced retinal cell proliferation and cell survival. Retinas of clo mutants were disorganized, with irregular synaptic layers, mispatterned expression domains of retinal transcription factors, morphologically abnormal Müller glia, reduced differentiation of specific retinal cell types, and sporadically distributed cone photoreceptors. Blockade of p53-mediated cell death did not completely rescue this phenotype and revealed ectopic cones in the inner nuclear layer. clo-/- embryos did not upregulate a molecular marker for hypoxia., Conclusions: The disorganized retinal phenotype of clo-/- embryos is consistent with a neural and glial developmental patterning role for the early ocular vasculature that is independent of its eventual function in gas exchange., (© 2015 Wiley Periodicals, Inc.)