The brain ventricle system, composed of the ventricular cavities and the cerebral spinal fluid within, performs critical functions including circulation of nutrients, removal of wastes, and cushioning of neural tissues. Development of the hindbrain ventricle requires a series of factors that coordinate its initial formation and subsequent inflation. Previous work has demonstrated that the transcriptional co-activator Taz (also known as WW domain-containing transcription regulator protein 1, Wwtr1), a component of Hippo signalling, is active at hindbrain rhombomere boundaries where it is regulated by mechanotransduction and promotes proliferation. Here, we demonstrate that Taz is also a critical regulator of hindbrain ventricle development. Zebrafish embryos that lack Taz protein fail to undergo initial midline separation of the hindbrain ventricle. Furthermore, the ventricle phenotype is a result of disorganized cytoskeletal F-actin and apicobasal polarity components. In addition, we have demonstrated that the hindbrain rhombomere boundaries are a location of active Wnt-Hippo crosstalk. Through our work, we propose a model where Taz protein is stabilized at rhombomere boundaries and promotes proper cell polarity necessary for formation of the brain ventricle.

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld–Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes.

Humans and other jawed vertebrates rely heavily on their craniofacial skeleton for eating, breathing, and communicating. As such, it is vital that the elements of the craniofacial skeleton develop properly during embryogenesis to ensure a high quality of life and evolutionary fitness. Indeed, craniofacial abnormalities, including cleft palate and craniosynostosis, represent some of the most common congenital abnormalities in newborns. Like many other organ systems, the development of the craniofacial skeleton is complex, relying on specification and migration of the neural crest, patterning of the pharyngeal arches, and morphogenesis of each skeletal element into its final form. These processes must be carefully coordinated and integrated. One way this is achieved is through the spatial and temporal deployment of cell signaling pathways. Recent studies conducted using the zebrafish model underscore the importance of the Transforming Growth Factor Beta (TGF-β) and Bone Morphogenetic Protein (BMP) pathways in craniofacial development. Although both pathways contain similar components, each pathway results in unique outcomes on a cellular level. In this review, we will cover studies conducted using zebrafish that show the necessity of these pathways in each stage of craniofacial development, starting with the induction of the neural crest, and ending with the morphogenesis of craniofacial elements. We will also cover human skeletal and craniofacial diseases and malformations caused by mutations in the components of these pathways (e.g., cleft palate, craniosynostosis, etc.) and the potential utility of zebrafish in studying the etiology of these diseases. We will also briefly cover the utility of the zebrafish model in joint development and biology and discuss the role of TGF-β/BMP signaling in these processes and the diseases that result from aberrancies in these pathways, including osteoarthritis and multiple synostoses syndrome. Overall, this review will demonstrate the critical roles of TGF-β/BMP signaling in craniofacial development and show the utility of the zebrafish model in development and disease.

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, these data suggest that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

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.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, this data suggests that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

Precise spatiotemporal expression of the Nodal-Lefty-Pitx2 cascade in the lateral plate mesoderm establishes the left–right axis, which provides vital cues for correct organ formation and function. Mutations of one cascade constituent PITX2 and, separately, the Forkhead transcription factor FOXC1 independently cause a multi-system disorder known as Axenfeld–Rieger syndrome (ARS). Since cardiac involvement is an established ARS phenotype and because disrupted left–right patterning can cause congenital heart defects, we investigated in zebrafish whether foxc1 contributes to organ laterality or situs. We demonstrate that CRISPR/Cas9-generated foxc1a and foxc1b mutants exhibit abnormal cardiac looping and that the prevalence of cardiac situs defects is increased in foxc1a−/−; foxc1b−/− homozygotes. Similarly, double homozygotes exhibit isomerism of the liver and pancreas, which are key features of abnormal gut situs. Placement of the asymmetric visceral organs relative to the midline was also perturbed by mRNA overexpression of foxc1a and foxc1b. In addition, an analysis of the left–right patterning components, identified in the lateral plate mesoderm of foxc1 mutants, reduced or abolished the expression of the NODAL antagonist lefty2. Together, these data reveal a novel contribution from foxc1 to left–right patterning, demonstrating that this role is sensitive to foxc1 gene dosage, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld–Rieger syndrome patients.

The vertebrate inner ear is a labyrinthine sensory organ responsible for perceiving sound and body motion. While a great deal of research has been invested in understanding the auditory system, a growing body of work has begun to delineate the complex developmental program behind the apparatuses of the inner ear involved with vestibular function. These animal studies have helped identify genes involved in inner ear development and model syndromes known to include vestibular dysfunction, paving the way for generating treatments for people suffering from these disorders. This review will provide an overview of known inner ear anatomy and function and summarize the exciting discoveries behind inner ear development and the evolution of its vestibular apparatuses.

Ocular coloboma is a congenital disorder of the eye where a gap exists in the inferior retina, lens, iris, or optic nerve tissue. With a prevalence of 2-19 per 100,000 live births, coloboma, and microphthalmia, an associated ocular disorder, represent up to 10% of childhood blindness. It manifests due to the failure of choroid fissure closure during eye development, and it is a part of a spectrum of ocular disorders that include microphthalmia and anophthalmia. Use of genetic approaches from classical pedigree analyses to next generation sequencing has identified more than 40 loci that are associated with the causality of ocular coloboma. As we have expanded studies to include singleton cases, hereditability has been very challenging to prove. As such, researchers over the past 20 years, have unraveled the complex interrelationship amongst these 40 genes using vertebrate model organisms. Such research has greatly increased our understanding of eye development. These genes function to regulate initial specification of the eye field, migration of retinal precursors, patterning of the retina, neural crest cell biology, and activity of head mesoderm. This review will discuss the discovery of loci using patient data, their investigations in animal models, and the recent advances stemming from animal models that shed new light in patient diagnosis.

Normal body situs requires precise spatiotemporal expression of the Nodal-Lefty-Pitx2 cascade in the lateral plate mesoderm. The ultimate output of this patterning is establishment of the left-right axis, which provides vital cues for correct organ formation and function. Mutations, deletions and duplications in PITX2 and FOXC1 lead to the rare genetic disease Axenfeld-Rieger syndrome (ARS). While situs defects are not a recognised feature of ARS, partial penetrance of cardiac septal defects and valve incompetence is observed; both of these congenital heart defects (CHDs) also occur following disruption of left-right patterning. Here we investigated whether foxc1 genes have a critical role in specifying organ situs. We demonstrate that CRISPR/Cas9 generated mutants for the zebrafish paralogs foxc1a and foxc1b recapitulate ARS phenotypes including craniofacial dysmorphism, hydrocephalus and intracranial haemorrhage. Furthermore, foxc1a -/-; foxc1b -/- mutant animals display cardiac and gut situs defects. Modelling FOXC1 duplication by transient mRNA overexpression revealed that increased foxc1 dosage also results in organ situs defects. Analysis of known left-right patterning genes revealed a loss in expression of the NODAL antagonist lefty2 in the lateral plate mesoderm. Consistently, LEFTY2 mutations are known to cause human cardiac situs defects. Our data reveal a novel role for the forkhead-box transcription factor foxc1 in patterning of the left-right axis, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld-Rieger syndrome patients. Author Summary This manuscript investigates the functional consequences of abrogating the activity of Foxc1 (Forkhead Box C1). We demonstrate that loss of zebrafish foxc1a and foxc1b results in phenotypes that resemble human patients with deletions in the FOXC1 locus. Notably, such phenotypes include alterations to the morphology of the heart. Investigations into the mechanisms underlying this phenotype led to the discovery that Foxc1 functions as a regulator of left-right patterning. Most components of left-right specification function normally in foxc1a/b mutants, but there is a pronounced loss of lefty2 , a known inhibitor of Nodal signaling. This supports a model in which Foxc1 regulates situs of the heart via the regulation of Lefty2.

Purpose:Analysis of photoreceptor morphology and gene expression in mispatterned eyes of zebrafish growth differentiation factor 6a (gdf6a) mutants.Methods:Rod and cone photoreceptors were compared between gdf6a mutant and control zebrafish from larval to late adult stages using transgenic labels, immunofluorescence, and confocal microscopy, as well as by transmission electron microscopy. To compare transcriptomes between larval gdf6a mutant and control zebrafish, RNA-Seq was performed on isolated eyes.Results:Although rod and cone photoreceptors differentiate in gdf6a mutant zebrafish, the cells display aberrant growth and morphology. The cone outer segments, the light-detecting sensory endings, are reduced in size in the mutant larvae and fail to recover to control size at subsequent stages. In contrast, rods form temporarily expanded outer segments. The inner segments, which generate the required energy and proteins for the outer segments, are shortened in both rods and cones at all stages. RNA-Seq analysis provides a set of misregulated genes associated with the observed abnormal photoreceptor morphogenesis.Conclusions:GDF6 mutations were previously identified in patients with Leber congenital amaurosis. Here, we reveal a unique photoreceptor phenotype in the gdf6a mutant zebrafish whereby rods and cones undergo abnormal maturation distinct for each cell type. Further, subsequent development shows partial recovery of cell morphology and maintenance of the photoreceptor layer. By conducting a transcriptomic analysis of the gdf6a larval eyes, we identified a collection of genes that are candidate regulators of photoreceptor size and morphology.

Cryptochromes (Cry) are ancient flavoproteins known to regulate circadian rhythms. In plants and some animals, Cry is sensitive to blue light due to its ability to bind the chromophore flavin adenine dinucleotide (FAD). Cry is also suggested to function in magnetoreception, since it can create light-dependent radical pairs with FAD that are sensitive to magnetic fields (Ritz2000; Liedvogel et al., 2007; Solov'yov et al., 2014). Cry is expressed in the visual system of various animals and specifically co-localizes with both short– and long–wavelength cone photoreceptors in birds (Bischof et al., 2011; Günther et al., 2018). However, magnetoreception is not limited to birds and the expression of cry genes in the photoreceptors of other vertebrates is unknown. Here, we use zebrafish to examine the retinal expression pattern of cry family genes. Zebrafish have seven cry genes and while most are known regulators of the circadian clock, relatively little is known about cry2 and cry4 (Haug et al., 2015; Liu et al., 2015). Therefore, we explored cry2 and cry4 expression in the larval and adult zebrafish retina. We demonstrate that cry4 is predominantly expressed in the short-wavelength ultraviolet (UV)-sensitive cone photoreceptors, while cry2 is expressed in UV cones and additional retinal photoreceptors during the day. Using Nitroreductase (NTZ)-mediated cell ablation and qRT-PCR, we find that cry4 expression significantly decreases when UV cones are ablated, but not when the neighboring short-wavelength sensitive blue cones are ablated. cry2 expression decreases after UV cone ablation but is still significantly detectable, while blue cone ablation does not alter cry2 expression. This study provides a more detailed annotation of cry2 and cry4 expression in the zebrafish retina and highlights the feasibility of a well-established ablation paradigm to test if photoreceptors are required for magnetoreception in fish. Although evidence of magnetoreception in adult zebrafish has gained considerable evidence over the last decade (Shcherbakov et al., 2005; Takebe et al., 2012; Krylov et al., 2016; Myklatun et al., 2018) the mediating mechanism(s) remain unknown. Additionally, despite limited evidence that larval zebrafish are magnetoreceptive, many other larval fish have a characterized magnetic sense; sockeye salmon fry, larval coral reef fish, larval medaka and larval Atlantic haddock have been shown to be responsive to magnetic fields (Quinn; 1980; Bottesch et al., 2016; O'Connor and Muheim. 2017; Myklatun et al., 2018; Cresci, et al. 2019). If cry-cone interactions are conserved within fish, our findings may suggest one potential mechanism, such that UV cones appear poised for light-dependent magnetoreception via photoreceptor subtype-specific expression of cry.

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrβ positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter's needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

The vertebrate hindbrain is composed of a series of lineage-restricted segments termed rhombomeres. Segment-specific gene expression drives unique programs of neuronal differentiation. Two critical embryonic signaling pathways, Fibroblast Growth Factor (FGF) and Retinoic Acid (RA), regulate early embryonic rhombomere patterning. The earliest expressed hox genes, hoxb1b and hoxb1a in zebrafish, are logical candidates for establishing signaling networks that specify segmental identity. We sought to determine the mechanism by which hox genes regulate hindbrain patterning in zebrafish. We demonstrate that hoxb1a regulates r4-specific patterning, while hoxb1b regulates rhombomere segmentation and size. Hoxb1a and hoxb1b redundantly regulate vhnf1 expression. Loss of hoxb1b together with pbx4 reverts the hindbrain to a groundstate identity, demonstrating the importance of hox genes in patterning nearly the entire hindbrain, and a key requirement for Pbx in this process. Additionally, we provide evidence that while pbx genes regulate RA signaling, hoxb1b regulates hindbrain identity through complex regulation of FGF signaling.

NODAL is a morphogen essential for early embryonic development in vertebrates. Since much of our understanding of NODAL comes from model organisms, we aimed to directly assess post-transcriptional regulation of human NODAL with specific attention to a newly discovered human-specific NODAL splice variant. Selective depletion of the NODAL variant in human embryonic stem cells resulted in increased LIFR levels, while total NODAL knockdown resulted in a decrease of several markers of pluripotency. The NODAL variant did not transmit a canonical NODAL signal in zebrafish embryos, but may share some functional capability with canonical NODAL in cancer cells. At the protein level, disruption of disulfide bond formation dramatically enhanced proteolytic processing of NODAL. Disruption of NODAL N-glycosylation decreased its secretion but not extracellular stability, and a novel N-glycosylation in the NODAL variant contributed to enhanced secretion. Collectively, this work offers a direct and precise account of post-transcriptional regulation of human NODAL.

Despite the critical role of vascular mural cells (smooth muscle cells and pericytes) in supporting the endothelium of blood vessels, we know little of their early morphogenesis and differentiation. foxc1b:EGFP expressing cells in zebrafish associate with the vascular endothelium ( kdrl ) and co-express a smooth muscle marker ( acta2 ), but not a pericyte marker ( pdgfrβ ). The expression of foxc1b in early peri-endothelial mesenchymal cells allows us to follow the morphogenesis of mesenchyme into acta2 expressing vascular smooth muscle cells. We show that mural cells expressing different markers associate with vessels of different diameters, depending on their embryonic location and developmental timing, suggesting marker expression is predictive of functional differences. We identify gene expression signatures for an enriched vascular smooth muscle cell population ( foxc1b + acta2) and all smooth muscle (acta2) using fluorescence-activated cell sorting and RNA-Seq. Finally, we demonstrate that progressive loss of foxc1a / foxc1b results in decreased smooth muscle cell coverage. Together, our data highlight the early cellular dynamics and transcriptome profiles of smooth muscle cells in vivo, using foxc1b as a unique tool to probe vascular smooth muscle cell differentiation.Summary Statement Tracing the morphogenesis and transcriptome of early vascular smooth muscle cells using foxc1b