RESUMO
BACKGROUND: Vertebrate eye formation requires coordinated inductive interactions between different embryonic tissue layers, first described in amphibians. A network of transcription factors and signaling molecules controls these steps, with mutations causing severe ocular, neuronal, and craniofacial defects. In eyeless mutant axolotls, eye morphogenesis arrests at the optic vesicle stage, before lens induction, and development of ventral forebrain structures is disrupted. RESULTS: We identified a 5-bp deletion in the rax (retina and anterior neural fold homeobox) gene, which was tightly linked to the recessive eyeless (e) axolotl locus in an F2 cross. This frameshift mutation, in exon 2, truncates RAX protein within the homeodomain (P154fs35X). Quantitative RNA analysis shows that mutant and wild-type rax transcripts are equally abundant in E/e embryos. Translation appears to initiate from dual start codons, via leaky ribosome scanning, a conserved feature among gnathostome RAX proteins. Previous data show rax is expressed in the optic vesicle and diencephalon, deeply conserved among metazoans, and required for eye formation in other species. CONCLUSION: The eyeless axolotl mutation is a null allele in the rax homeobox gene, with primary defects in neural ectoderm, including the retinal and hypothalamic primordia.
Assuntos
Ambystoma mexicanum/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Mutação , Fatores de Transcrição/genética , Ambystoma mexicanum/metabolismo , Animais , Desenvolvimento Embrionário/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SEdel/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Neurogênese/fisiologia , Nervo Óptico/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Retina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Interkinetic nuclear migration (IKNM) is the process in which pseudostratified epithelial nuclei oscillate from the apical to basal surface and in phase with the mitotic cycle. In the zebrafish retina, neuroepithelial retinal progenitor cells (RPCs) increase Notch activity with apical movement of the nuclei, and the depth of nuclear migration correlates with the probability that the next cell division will be neurogenic. This study focuses on the mechanisms underlying the relationships between IKNM, cell signaling, and neurogenesis. In particular, we have explored the role IKNM has on endosome biology within RPCs. Through genetic manipulation and live imaging in zebrafish, we find that early (Rab5-positive) and recycling (Rab11a-positive) endosomes polarize in a dynamic fashion within RPCs and with reference to nuclear position. Functional analyses suggest that dynamic polarization of recycling endosomes and their activity within the neuroepithelia modulates the subcellular localization of Crb2a, consequently affecting multiple signaling pathways that impact neurogenesis including Notch, Hippo, and Wnt activities. As nuclear migration is heterogenous and asynchronous among RPCs, Rab11a-affected signaling within the neuroepithelia is modulated in a differential manner, providing mechanistic insight to the correlation of IKNM and selection of RPCs to undergo neurogenesis.
RESUMO
This chapter provides an overview of the early developmental origins of six ocular tissues: the cornea, lens, ciliary body, iris, neural retina, and retina pigment epithelium. Many of these tissue types are concurrently specified and undergo a complex set of morphogenetic movements that facilitate their structural interconnection. Within the context of vertebrate eye organogenesis, we also discuss the genetic hierarchies of transcription factors and signaling pathways that regulate growth, patterning, cell type specification and differentiation.
Assuntos
Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Fatores de Transcrição/genética , Animais , Corpo Ciliar/embriologia , Corpo Ciliar/crescimento & desenvolvimento , Corpo Ciliar/metabolismo , Córnea/embriologia , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , Olho/embriologia , Olho/crescimento & desenvolvimento , Humanos , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Cristalino/metabolismo , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap-/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap-/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.
Assuntos
Glucose/metabolismo , Fígado/embriologia , Nucleotídeos/biossíntese , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Glucose/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Camundongos , Nucleotídeos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinase 3 , Transativadores/genética , Proteínas de Sinalização YAP , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Retina/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores Notch/metabolismo , Elementos Reguladores de Transcrição , Retina/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Although many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find that changes to cell geometry associated with dense tissue packing play a significant role in regulating differentiation rate in the zebrafish neural tube. Specifically, progenitors that are displaced away from the apical surface due to crowding, tend to differentiate in a Notch-dependent manner. Using simulations we show that interplay between progenitor density, cell shape and changes in differentiation rate could naturally result in negative-feedback control on progenitor cell number. Given these results, we suggest a model whereby differentiation rate is regulated by density dependent effects on cell geometry to: (1) correct variability in cell number; and (2) balance the rates of proliferation and differentiation over development to 'fill' the available space.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células-Tronco Neurais/metabolismo , Tubo Neural/embriologia , Neurogênese/fisiologia , Peixe-Zebra/embriologia , Animais , Células-Tronco Neurais/citologia , Tubo Neural/citologia , Receptores Notch/genética , Receptores Notch/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The Atoh7 transcription factor catalyzes the rate-limiting step in the specification of retinal ganglion cells (RGCs). As a tool to study vertebrate retinal development, we validate an antibody that recognizes human and mouse Atoh7 polypeptide, using informative knockout and transgenic mouse tissues and overexpression experiments. The transient features of Atoh7 protein expression during retinal neurogenesis match the expected pattern at the tissue and cellular level. Further, we compare endogenous Atoh7 to established RGC markers, reporter mouse lines and cell cycle markers, demonstrating the utility of the antibody to investigate molecular mechanisms of retinal histogenesis.
Assuntos
Anticorpos Monoclonais/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Células Cultivadas , Embrião de Mamíferos/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Retina/citologia , Células Ganglionares da Retina/citologia , Homologia de SequênciaRESUMO
The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma.
Assuntos
Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Epitélio Pigmentado da Retina/citologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Alelos , Animais , Apoptose/genética , Núcleo Celular/metabolismo , Proliferação de Células , Coloboma/patologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células HEK293 , Humanos , Morfogênese/genética , Mutação , Fenótipo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/transplante , Transdução de Sinais/genética , Fatores de Transcrição de Domínio TEA , Transativadores/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Transgenes , Regulação para Cima , Proteínas de Sinalização YAP , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.
Assuntos
Tamanho Corporal/genética , Proteínas de Peixes/metabolismo , Morfogênese/genética , Oryzias/anatomia & histologia , Oryzias/embriologia , Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas de Peixes/genética , Proteínas Ativadoras de GTPase/metabolismo , Genes Essenciais/genética , Gravitação , Humanos , Mutação/genética , Tamanho do Órgão/genética , Oryzias/genética , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Esferoides Celulares/citologia , Esferoides Celulares/metabolismoRESUMO
To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Movimento Celular , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Morfogênese , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Retina/citologia , Retina/embriologia , Retina/metabolismo , Serina-Treonina Quinase 3 , Transativadores/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.