RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Cohesinas , Mutación , Proteínas de Pez Cebra , Pez Cebra , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Mutación/genética , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Wnt/genética , Desarrollo Embrionario/genética , Dosificación de Gen , Mesodermo/metabolismo , Mesodermo/embriologíaRESUMEN
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.
Asunto(s)
Tendones , Pez Cebra , Animales , Pez Cebra/genética , Tendones/metabolismo , Huesos , Transducción de Señal , LigamentosRESUMEN
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.
Asunto(s)
Condrocitos , Pez Cebra , Animales , Huesos/metabolismo , Cartílago/metabolismo , Polaridad Celular/genética , Condrocitos/metabolismo , Morfogénesis/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores Wnt/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez CebraRESUMEN
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.
Asunto(s)
Osteogénesis , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Cráneo/metabolismo , Animales , Condrocitos/citología , Condrocitos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/genética , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Transducción de Señal , Cráneo/embriología , Estrés Mecánico , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Tipificación del Cuerpo/fisiología , Modelos Biológicos , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/genética , Biología Computacional , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/fisiología , Rombencéfalo/citología , Rombencéfalo/embriología , Transducción de Señal , Procesos Estocásticos , Tretinoina/fisiología , Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Desarrollo Óseo , Esqueleto/crecimiento & desarrollo , Pez Cebra/crecimiento & desarrollo , Animales , Condrocitos/fisiología , Faringe/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Córnea/citología , Córnea/fisiología , Cristalino/citología , Cristalino/fisiología , Óptica y Fotónica , Animales , Cómputos Matemáticos , Refractometría , Pez CebraRESUMEN
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.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mandíbula/embriología , Transcriptoma , Animales , Tipificación del Cuerpo , Mandíbula/metabolismo , Ratones , Organogénesis , Pez CebraRESUMEN
Heightened phenotypic variation among mutant animals is a well-known, but poorly understood phenomenon. One hypothetical mechanism accounting for mutant phenotypic variation is progenitor cells variably choosing between two alternative fates during development. Zebrafish mef2cab1086 mutants develop tremendously variable ectopic bone in their hyoid craniofacial skeleton. Here, we report evidence that a key component of this phenotype is variable fate switching from ligament to bone. We discover that a 'track' of tissue prone to become bone cells is a previously undescribed ligament. Fate-switch variability is heritable, and comparing mutant strains selectively bred to high and low penetrance revealed differential mef2ca mutant transcript expression between high and low penetrance strains. Consistent with this, experimental manipulation of mef2ca mutant transcripts modifies the penetrance of the fate switch. Furthermore, we discovered a transposable element that resides immediately upstream of the mef2ca locus and is differentially DNA methylated in the two strains, correlating with differential mef2ca expression. We propose that variable transposon epigenetic silencing underlies the variable mef2ca mutant bone phenotype, and could be a widespread mechanism of phenotypic variability in animals.
Asunto(s)
Hueso Hioides/crecimiento & desarrollo , Ligamentos/crecimiento & desarrollo , Factores de Transcripción MEF2/genética , Osteogénesis/fisiología , Cráneo/crecimiento & desarrollo , Células Madre/citología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Diferenciación Celular/fisiología , Metilación de ADN/genética , Elementos Transponibles de ADN/genética , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica , Osteoblastos/citología , Penetrancia , Pez Cebra/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mandíbula/embriología , Mandíbula/fisiología , Animales , Región Branquial , Adhesión Celular , Simulación por Computador , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Procesos Estocásticos , Transgenes , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Tendons and ligaments are extracellular matrix (ECM)-rich structures that interconnect muscles and bones. Recent work has shown how tendon fibroblasts (tenocytes) interact with muscles via the ECM to establish connectivity and strengthen attachments under tension. Similarly, ECM-dependent interactions between tenocytes and cartilage/bone ensure that tendon-bone attachments form with the appropriate strength for the force required. Recent studies have also established a close lineal relationship between tenocytes and skeletal progenitors, highlighting the fact that defects in signals modulated by the ECM can alter the balance between these fates, as occurs in calcifying tendinopathies associated with aging. The dynamic fine-tuning of tendon ECM composition and assembly thus gives rise to the remarkable characteristics of this unique tissue type. Here, we provide an overview of the functions of the ECM in tendon formation and maturation that attempts to integrate findings from developmental genetics with those of matrix biology.
Asunto(s)
Matriz Extracelular/metabolismo , Tendones/crecimiento & desarrollo , Tendones/metabolismo , Animales , Huesos/metabolismo , Modelos Biológicos , Transducción de Señal , Cicatrización de HeridasRESUMEN
Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development.
Asunto(s)
Proteínas de Microfilamentos/genética , Cresta Neural/crecimiento & desarrollo , Seudópodos/genética , Proteínas de Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Animales , Tipificación del Cuerpo/genética , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación del Desarrollo de la Expresión Génica , Mutación , Cresta Neural/citología , Transducción de Señal , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesisRESUMEN
Cell migration requires dynamic regulation of cell-cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand-receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial-mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Endocitosis/genética , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Cresta Neural/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular , Comunicación Celular , Diferenciación Celular , Movimiento Celular , Clonación Molecular , Embrión no Mamífero , Endosomas/metabolismo , Transición Epitelial-Mesenquimal , Microinyecciones , Morfolinos/genética , Morfolinos/metabolismo , Cresta Neural/citología , Cresta Neural/crecimiento & desarrollo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vía de Señalización Wnt , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismoRESUMEN
Organogenesis requires coordinated regulation of cellular differentiation and morphogenesis. Cartilage cells in the vertebrate skeleton form polarized stacks, which drive the elongation and shaping of skeletal primordia. Here we show that an atypical cadherin, Fat3, and its partner Dachsous-2 (Dchs2), control polarized cell-cell intercalation of cartilage precursors during craniofacial development. In zebrafish embryos deficient in Fat3 or Dchs2, chondrocytes fail to stack and misregulate expression of sox9a. Similar morphogenetic defects occur in rerea/atr2a-/- mutants, and Fat3 binds REREa, consistent with a model in which Fat3, Dchs2 and REREa interact to control polarized cell-cell intercalation and simultaneously control differentiation through Sox9. Chimaeric analyses support such a model, and reveal long-range influences of all three factors, consistent with the activation of a secondary signal that regulates polarized cell-cell intercalation. This coordinates the spatial and temporal morphogenesis of chondrocytes to shape skeletal primordia and defects in these processes underlie human skeletal malformations. Similar links between cell polarity and differentiation mechanisms are also likely to control organ formation in other contexts.
Asunto(s)
Cadherinas/genética , Cartílago/crecimiento & desarrollo , Diferenciación Celular/genética , Polaridad Celular/genética , Morfogénesis/genética , Proteínas de Pez Cebra/genética , Animales , Cadherinas/biosíntesis , Comunicación Celular , Condrocitos/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Humanos , Factor de Transcripción SOX9/biosíntesis , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.
Asunto(s)
Endotelina-1/biosíntesis , Cresta Neural/crecimiento & desarrollo , Vía de Señalización Wnt/genética , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/genética , Región Branquial/crecimiento & desarrollo , Región Branquial/metabolismo , Endotelina-1/genética , Regulación del Desarrollo de la Expresión Génica , Biosíntesis de Proteínas , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS), the most common "cohesinopathy". It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb), knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.
Asunto(s)
Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Genes Homeobox , Haploinsuficiencia/genética , Ratones , Ratones Noqueados , Fenotipo , Unión Proteica , Factores de Transcripción/deficiencia , Pez Cebra , Proteínas de Pez Cebra/deficienciaRESUMEN
We have examined lateral line hair cell and support cell maintenance in adult zebrafish when growth is largely complete. We demonstrate that adult zebrafish not only replenish hair cells after a single instance of hair cell damage, but also maintain hair cells and support cells after multiple rounds of damage and regeneration. We find that hair cells undergo continuous turnover in adult zebrafish in the absence of damage. We identify mitotically-distinct support cell populations and show that hair cells regenerate from underlying support cells in a region-specific manner. Our results demonstrate that there are two distinct support cell populations in the lateral line, which may help explain why zebrafish hair cell regeneration is extremely robust, retained throughout life, and potentially unlimited in regenerative capacity.
Asunto(s)
Sistema de la Línea Lateral/citología , Sistema de la Línea Lateral/fisiología , Mecanorreceptores/fisiología , Regeneración/fisiología , Pez Cebra/fisiología , Animales , Bromodesoxiuridina , Fluorescencia , Inmunohistoquímica , NeomicinaRESUMEN
Cornelia de Lange Syndrome (CdLS) is a multisystem birth defects disorder that affects every tissue and organ system in the body. Understanding the factors that contribute to the origins, prevalence, and severity of these developmental defects provides the most direct approach for developing screens and potential treatments for individuals with CdLS. Since the majority of cases of CdLS are caused by haploinsufficiency for NIPBL (Nipped-B-like, which encodes a cohesin-associated protein), we have developed mouse and zebrafish models of CdLS by using molecular genetic tools to create Nipbl-deficient mice and zebrafish (Nipbl(+/-) mice, zebrafish nipbl morphants). Studies of these vertebrate animal models have yielded novel insights into the developmental etiology and genes/gene pathways that contribute to CdLS-associated birth defects, particularly defects of the gut, heart, craniofacial structures, nervous system, and limbs. Studies of these mouse and zebrafish CdLS models have helped clarify how deficiency for NIPBL, a protein that associates with cohesin and other transcriptional regulators in the nucleus, affects processes important to the emergence of the structural and physiological birth defects observed in CdLS: NIPBL exerts chromosome position-specific effects on gene expression; it influences long-range interactions between different regulatory elements of genes; and it regulates combinatorial and synergistic actions of genes in developing tissues. Our current understanding is that CdLS should be considered as not only a cohesinopathy, but also a "transcriptomopathy," that is, a disease whose underlying etiology is the global dysregulation of gene expression throughout the organism. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Síndrome de Cornelia de Lange/genética , Discapacidades del Desarrollo/genética , Redes Reguladoras de Genes , Animales , Proteínas de Ciclo Celular , Anomalías Congénitas/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas/genética , Pez CebraRESUMEN
The vitamin A derivative retinoic acid (RA) is a morphogen that patterns the anterior-posterior axis of the vertebrate hindbrain. Cellular retinoic acid-binding proteins (Crabps) transport RA within cells to both its nuclear receptors (RARs) and degrading enzymes (Cyp26s). However, mice lacking Crabps are viable, suggesting that Crabp functions are redundant with those of other fatty acid-binding proteins. Here we show that Crabps in zebrafish are essential for posterior patterning of the hindbrain and that they provide a key feedback mechanism that makes signaling robust as they are able to compensate for changes in RA production. Of the four zebrafish Crabps, Crabp2a is uniquely RA inducible and depletion or overexpression of Crabp2a makes embryos hypersensitive to exogenous RA. Computational models confirm that Crabp2a improves robustness within a narrow concentration range that optimizes a 'robustness index', integrating spatial information along the RA morphogen gradient. Exploration of signaling parameters in our models suggests that the ability of Crabp2a to transport RA to Cyp26 enzymes for degradation is a major factor in promoting robustness. These results demonstrate a previously unrecognized requirement for Crabps in RA signaling and hindbrain development, as well as a novel mechanism for stabilizing morphogen gradients despite genetic or environmental fluctuations in morphogen availability.
Asunto(s)
Tipificación del Cuerpo/genética , Receptores de Ácido Retinoico/metabolismo , Rombencéfalo/embriología , Transducción de Señal/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Tipificación del Cuerpo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Modelos Biológicos , Receptores de Ácido Retinoico/genética , Rombencéfalo/efectos de los fármacos , Rombencéfalo/metabolismo , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Proteínas de Pez Cebra/genéticaRESUMEN
T-cell factor/lymphoid enhancer factor (TCF/LEF) proteins regulate transcription by recruiting ß-catenin and its associated co-regulators. Whether TCF/LEFs also recruit more factors through independent, direct interactions is not well understood. Here we discover Ring Finger Protein 14 (RNF14) as a new binding partner for all TCF/LEF transcription factors. We show that RNF14 positively regulates Wnt signalling in human cancer cells and in an in vivo zebrafish model by binding to target promoters with TCF and stabilizing ß-catenin recruitment. RNF14 depletion experiments demonstrate that it is crucial for colon cancer cell survival. Therefore, we have identified a key interacting factor of TCF/ß-catenin complexes to regulate Wnt gene transcription.