Cohesin composition and dosage independently affect early development in zebrafish.

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

Mechanical force regulates Sox9 expression at the developing enthesis.

Entheses transmit force from tendons and ligaments to the skeleton. Regional organization of enthesis extracellular matrix (ECM) generates differences in stiffness required for force transmission. Two key transcription factors co-expressed in entheseal tenocytes, scleraxis (Scx) and Sox9, directly control production of enthesis ECM components. Formation of embryonic craniofacial entheses in zebrafish coincides with onset of jaw movements, possibly in response to the force of muscle contraction. We show dynamic changes in scxa and sox9a mRNA levels in subsets of entheseal tenocytes that correlate with their roles in force transmission. We also show that transcription of a direct target of Scxa, Col1a, in enthesis ECM is regulated by the ratio of scxa to sox9a expression. Eliminating muscle contraction by paralyzing embryos during early stages of musculoskeletal differentiation alters relative levels of scxa and sox9a in entheses, primarily owing to increased sox9a expression. Force-dependent TGF-ß (TGFß) signaling is required to maintain this balance of scxa and sox9a expression. Thus, force from muscle contraction helps establish a balance of transcription factor expression that controls specialized ECM organization at the tendon enthesis and its ability to transmit force.

The non-canonical Wnt receptor Ror2 is required for cartilage cell polarity and morphogenesis of the craniofacial skeleton in zebrafish.

Non-canonical/ß-catenin-independent Wnt signaling plays crucial roles in tissue/cell polarity in epithelia, but its functions have been less well studied in mesenchymal tissues, such as the skeleton. Mutations in non-canonical Wnt signaling pathway genes cause human skeletal diseases such as Robinow syndrome and Brachydactyly Type B1, which disrupt bone growth throughout the endochondral skeleton. Ror2 is one of several non-canonical Wnt receptor/co-receptors. Here, we show that ror2-/- mutant zebrafish have craniofacial skeletal defects, including disruptions of chondrocyte polarity. ror1-/- mutants appear to be phenotypically wild type, but loss of both ror1 and ror2 leads to more severe cartilage defects, indicating partial redundancy. Skeletal defects in ror1/2 double mutants resemble those of wnt5b-/- mutants, suggesting that Wnt5b is the primary Ror ligand in zebrafish. Surprisingly, the proline-rich domain of Ror2, but not its kinase domain, is required to rescue its function in mosaic transgenic experiments in ror2-/- mutants. These results suggest that endochondral bone defects in ROR-related human syndromes reflect defects in cartilage polarity and morphogenesis.

Zebrafish endochondral growth zones as they relate to human bone size, shape and disease.

Research on the genetic mechanisms underlying human skeletal development and disease have largely relied on studies in mice. However, recently the zebrafish has emerged as a popular model for skeletal research. Despite anatomical differences such as a lack of long bones in their limbs and no hematopoietic bone marrow, both the cell types in cartilage and bone as well as the genetic pathways that regulate their development are remarkably conserved between teleost fish and humans. Here we review recent studies that highlight this conservation, focusing specifically on the cartilaginous growth zones (GZs) of endochondral bones. GZs can be unidirectional such as the growth plates (GPs) of long bones in tetrapod limbs or bidirectional, such as in the synchondroses of the mammalian skull base. In addition to endochondral growth, GZs play key roles in cartilage maturation and replacement by bone. Recent studies in zebrafish suggest key roles for cartilage polarity in GZ function, surprisingly early establishment of signaling systems that regulate cartilage during embryonic development, and important roles for cartilage proliferation rather than hypertrophy in bone size. Despite anatomical differences, there are now many zebrafish models for human skeletal disorders including mutations in genes that cause defects in cartilage associated with endochondral GZs. These point to conserved developmental mechanisms, some of which operate both in cranial GZs and limb GPs, as well as others that act earlier or in parallel to known GP regulators. Experimental advantages of zebrafish for genetic screens, high resolution live imaging and drug screens, set the stage for many novel insights into causes and potential therapies for human endochondral bone diseases.

How vertebrates got their bite.

A newly discovered enhancer region may have allowed vertebrates to evolve the ability to open and close their jaws.

In vivo macromolecular crowding is differentially modulated by aquaporin 0 in zebrafish lens: Insights from a nanoenvironment sensor and spectral imaging.

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presbyopia. Here, we combine the use of the nanoenvironmental sensor [6-acetyl-2-dimethylaminonaphthalene (ACDAN)] to visualize lens macromolecular crowding with in vivo studies of aquaporin 0 zebrafish mutants that disrupt its regulation. Spectral phasor analysis of ACDAN fluorescence reveals water dipolar relaxation and demonstrates that mutations in two zebrafish aquaporin 0s, Aqp0a and Aqp0b, alter water state and macromolecular crowding in living lenses. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This evidence reveals previously unidentified spatial regulation of macromolecular crowding and spatially distinct roles for Aqp0 in the lens.

Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

Single-cell transcriptomic analysis of zebrafish cranial neural crest reveals spatiotemporal regulation of lineage decisions during development.

Neural crest (NC) cells migrate throughout vertebrate embryos to give rise to a huge variety of cell types, but when and where lineages emerge and their regulation remain unclear. We have performed single-cell RNA sequencing (RNA-seq) of cranial NC cells from the first pharyngeal arch in zebrafish over several stages during migration. Computational analysis combining pseudotime and real-time data reveals that these NC cells first adopt a transitional state, becoming specified mid-migration, with the first lineage decisions being skeletal and pigment, followed by neural and glial progenitors. In addition, by computationally integrating these data with RNA-seq data from a transgenic Wnt reporter line, we identify gene cohorts with similar temporal responses to Wnts during migration and show that one, Atp6ap2, is required for melanocyte differentiation. Together, our results show that cranial NC cell lineages arise progressively and uncover a series of spatially restricted cell interactions likely to regulate such cell-fate decisions.

Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s.

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

Multiple morphogens and rapid elongation promote segmental patterning during development.

The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development.

Endochondral growth zone pattern and activity in the zebrafish pharyngeal skeleton.

BACKGROUND: Endochondral ossification is a major bone forming mechanism in vertebrates, defects in which can result in skeletal dysplasia or craniofacial anomalies in humans. The zebrafish holds great potential to advance our understanding of endochondral growth zone development and genetics, yet several important aspects of its biology remain unexplored. Here we provide a comprehensive description of endochondral growth zones in the pharyngeal skeleton, including their developmental progression, cellular activity, and adult fates. RESULTS: Postembryonic growth of the pharyngeal skeleton is supported by endochondral growth zones located either at skeletal epiphyses or synchondroses. Col2a1a and col10a1a in situ hybridization and anti-PCNA immunostaining identify resting-, hypertrophic- and proliferative zones, respectively, in pharyngeal synchondroses. Cellular hypertrophy and matrix deposition contribute little, if at all, to axial growth in most skeletal elements. Zebrafish endochondral growth zones develop during metamorphosis and arrest in adults. CONCLUSIONS: Two endochondral growth zone configurations in the zebrafish pharyngeal skeleton produce either unidirectional (epiphyses) or bidirectional (synchondroses) growth. Cell proliferation drives endochondral growth and its modulation, in contrast to mammalian long bones in which bone length depends more on cell enlargement during hypertrophy and intramembranous ossification is the default mechanism of bone growth in zebrafish adults.

Tendon Cell Regeneration Is Mediated by Attachment Site-Resident Progenitors and BMP Signaling.

The musculoskeletal system is a striking example of how cell identity and position is coordinated across multiple tissues to ensure function. However, it is unclear upon tissue loss, such as complete loss of cells of a central musculoskeletal connecting tendon, whether neighboring tissues harbor progenitors capable of mediating regeneration. Here, using a zebrafish model, we genetically ablate all embryonic tendon cells and find complete regeneration of tendon structure and pattern. We identify two regenerative progenitor populations, sox10+ perichondrial cells surrounding cartilage and nkx2.5+ cells surrounding muscle. Surprisingly, laser ablation of sox10+ cells, but not nkx2.5+ cells, increases tendon progenitor number in the perichondrium, suggesting a mechanism to regulate attachment location. We find BMP signaling is active in regenerating progenitor cells and is necessary and sufficient for generating new scxa+ cells. Our work shows that muscle and cartilage connective tissues harbor progenitor cells capable of fully regenerating tendons, and this process is regulated by BMP signaling.

Optical development in the zebrafish eye lens.

The optics of the eye is the key to a functioning visual system. The exact nature of the correlation between ocular optics and eye development is not known because of the paucity of knowledge about the growth of a key optical element, the eye lens. The sophisticated optics of the lens and its gradient of refractive index provide the superior optical quality that the eye needs and which, it is thought, has a major influence on the development of proper visual function. The nature of a gradient refractive index lens, however, renders accurate measurements of its development difficult to make and has been the reason why the influence of lens growth on visual function remains largely unknown. Novel imaging techniques have made it possible to investigate growth of the eye lens in the zebrafish. This study shows measurements using X-ray Talbot interferometry of three-dimensional gradient index profiles in eye lenses of zebrafish from late larval to adult stages. The zebrafish lens shows evidence of a gradient of refractive index from the earliest stages measured and its growth suggests an apparent coincidence between periods of rapid increase in refractive index in the lens nucleus and increased expression of a particular crystallin protein group.

Bar, stripe and spot development in sand-dwelling cichlids from Lake Malawi.

BACKGROUND: Melanic patterns such as horizontal stripes, vertical bars and spots are common among teleost fishes and often serve roles in camouflage or mimicry. Extensive research in the zebrafish model has shown that the development of horizontal stripes depends on complex cellular interactions between melanophores, xanthophores and iridophores. Little is known about the development of horizontal stripes in other teleosts, and even less is known about bar or spot development. Here, we compare chromatophore composition and development of stripes, bars and spots in two cichlid species of sand-dwellers from Lake Malawi-Copadichromis azureus and Dimidiochromis compressiceps. RESULTS: (1) In D. compressiceps, stripes are made of dense melanophores underlaid by xanthophores and overlaid by iridophores. Melanophores and xanthophores are either loose or absent in interstripes, and iridophores are dense. In C. azureus, spots and bars are composed of a chromatophore arrangement similar to that of stripes but are separated by interbars where density of melanophores and xanthophores is only slightly lower than in stripes and iridophore density appears slightly greater. (2) Stripe, bar and spot chromatophores appear in the skin at metamorphosis. Stripe melanophores directly differentiate along horizontal myosepta into the adult pattern. In contrast, bar number and position are dynamic throughout development. As body length increases, new bars appear between old ones or by splitting of old ones through new melanophore appearance, not migration. Xanthophore and iridophore distributions follow melanophore patterns. (3) Metamorphic pigmentation arises in cichlids in a fashion similar to that described in zebrafish: melanophore progenitors derived from the medial route of neural crest migration migrate from the vicinity of the neural tube to the skin during metamorphosis. CONCLUSION: The three pigment cell types forming stripes, bars and spots arise in the skin at metamorphosis. Stripes develop by differentiation of melanophores along horizontal myosepta, while bars do not develop along patent anatomical boundaries and increase in number in relation with body size. We propose that metamorphic melanophore differentiation and migratory arrest upon arrival to the skin lead to stripe formation, while bar formation must be supported by extensive migration of undifferentiated melanophores in the skin.

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity.

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

Transcriptomics reveals complex kinetics of dorsal-ventral patterning gene expression in the mandibular arch.

The mandibular or first pharyngeal arch forms the upper and lower jaws in all gnathostomes. A gene regulatory network that defines ventral, intermediate, and dorsal domains along the dorsal-ventral (D-V) axis of the arch has emerged from studies in zebrafish and mice, but the temporal dynamics of this process remain unclear. To define cell fate trajectories in the arches we have performed quantitative gene expression analyses of D-V patterning genes in pharyngeal arch primordia in zebrafish and mice. Using NanoString technology to measure transcript numbers per cell directly we show that, in many cases, genes expressed in similar D-V domains and induced by similar signals vary dramatically in their temporal profiles. This suggests that cellular responses to D-V patterning signals are likely shaped by the baseline kinetics of target gene expression. Furthermore, similarities in the temporal dynamics of genes that occupy distinct pathways suggest novel shared modes of regulation. Incorporating these gene expression kinetics into our computational models for the mandibular arch improves the accuracy of patterning, and facilitates temporal comparisons between species. These data suggest that the magnitude and timing of target gene expression help diversify responses to patterning signals during craniofacial development.

Modeling craniofacial development reveals spatiotemporal constraints on robust patterning of the mandibular arch.

How does pattern formation occur accurately when confronted with tissue growth and stochastic fluctuations (noise) in gene expression? Dorso-ventral (D-V) patterning of the mandibular arch specifies upper versus lower jaw skeletal elements through a combination of Bone morphogenetic protein (Bmp), Endothelin-1 (Edn1), and Notch signaling, and this system is highly robust. We combine NanoString experiments of early D-V gene expression with live imaging of arch development in zebrafish to construct a computational model of the D-V mandibular patterning network. The model recapitulates published genetic perturbations in arch development. Patterning is most sensitive to changes in Bmp signaling, and the temporal order of gene expression modulates the response of the patterning network to noise. Thus, our integrated systems biology approach reveals non-intuitive features of the complex signaling system crucial for craniofacial development, including novel insights into roles of gene expression timing and stochasticity in signaling and gene regulation.

Aqp0a Regulates Suture Stability in the Zebrafish Lens.

Purpose: To investigate the roles of Aquaporin 0a (Aqp0a) and Aqp0b in zebrafish lens development and transparency. Methods: CRISPR/Cas9 gene editing was used to generate loss-of-function deletions in zebrafish aqp0a and/or aqp0b. Wild type (WT), single mutant, and double mutant lenses were analyzed from embryonic to adult stages. Lens transparency, morphology, and growth were assessed. Immunohistochemistry was used to map protein localization as well as to assess tissue organization and distribution of cell nuclei. Results: aqp0a-/- and/or aqp0b-/- cause embryonic cataracts with variable penetrance. While lenses of single mutants of either gene recover transparency in juveniles, double mutants consistently form dense cataracts that persist in adults, indicating partially redundant functions. Double mutants also reveal redundant Aqp0 functions in lens growth. The nucleus of WT lenses moves from the anterior pole to the lens center with age. In aqp0a-/- mutants, the nucleus fails to centralize as it does in WT or aqp0b-/- lenses, and in double mutant lenses there is no consistent lens nuclear position. In addition, the anterior sutures of aqp0a-/-, but not aqp0b-/- mutants, are unstable resulting in failure of suture maintenance at older stages and anterior polar opacity. Conclusions. Zebrafish Aqp0s have partially redundant functions, but only Aqp0a promotes suture stability, which directs the lens nucleus to centralize, failure of which results in anterior polar opacity. These studies support the hypothesis that the two Aqp0s subfunctionalized during fish evolution and that Aqp0-dependent maintenance of the anterior suture is essential for lens transparency.

Cell-type heterogeneity in the early zebrafish olfactory epithelium is generated from progenitors within preplacodal ectoderm.

The zebrafish olfactory epithelium comprises a variety of neuronal populations, which are thought to have distinct embryonic origins. For instance, while ciliated sensory neurons arise from preplacodal ectoderm (PPE), previous lineage tracing studies suggest that both Gonadotropin releasing hormone 3 (Gnrh3) and microvillous sensory neurons derive from cranial neural crest (CNC). We find that the expression of Islet1/2 is restricted to Gnrh3 neurons associated with the olfactory epithelium. Unexpectedly, however, we find no change in Islet1/2+ cell numbers in sox10 mutant embryos, calling into question their CNC origin. Lineage reconstruction based on backtracking in time-lapse confocal datasets, and confirmed by photoconversion experiments, reveals that Gnrh3 neurons derive from the anterior PPE. Similarly, all of the microvillous sensory neurons we have traced arise from preplacodal progenitors. Our results suggest that rather than originating from separate ectodermal populations, cell-type heterogeneity is generated from overlapping pools of progenitors within the preplacodal ectoderm.

An ongoing role for Wnt signaling in differentiating melanocytes in vivo.

A role for Wnt signaling in melanocyte specification from neural crest is conserved across vertebrates, but possible ongoing roles in melanocyte differentiation have received little attention. Using a systems biology approach to investigate the gene regulatory network underlying stable melanocyte differentiation in zebrafish highlighted a requirement for a positive-feedback loop involving the melanocyte master regulator Mitfa. Here, we test the hypothesis that Wnt signaling contributes to that positive feedback. We show firstly that Wnt signaling remains active in differentiating melanocytes and secondly that enhanced Wnt signaling drives elevated transcription of mitfa. We show that chemical activation of the Wnt signaling pathway at early stages of melanocyte development enhances melanocyte specification as expected, but importantly that at later (differentiation) stages, it results in altered melanocyte morphology, although melanisation is not obviously affected. Downregulation of Wnt signaling also results in altered melanocyte morphology and organization. We conclude that Wnt signaling plays a role in regulating ongoing aspects of melanocyte differentiation in zebrafish.