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Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.
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Although the small GTPase RAB37 acts as an organizer of autophagosome biogenesis, the upstream regulatory mechanism of autophagy via guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange in maintaining retinal function has not been determined. We found that retinitis pigmentosa GTPase regulator (RPGR) is a guanine nucleotide exchange factor that activates RAB37 by accelerating GDP-to-GTP exchange. RPGR directly interacts with RAB37 via the RPGR-RCC1-like domain to promote autophagy through stimulating exchange. Rpgr knockout (KO) in mice leads to photoreceptor degeneration owing to autophagy impairment in the retina. Notably, the retinopathy phenotypes of Rpgr KO retinas are rescued by the adeno-associated virus-mediated transfer of pre-trans-splicing molecules, which produce normal Rpgr mRNAs via trans-splicing in the Rpgr KO retinas. This rescue upregulates autophagy through the re-expression of RPGR in KO retinas to accelerate GDP-to-GTP exchange; thus, retinal homeostasis reverts to normal. Taken together, these findings provide an important missing link for coordinating RAB37 GDP-GTP exchange via the RPGR and retinal homeostasis by autophagy regulation.
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Autofagia , Proteínas de Transporte , Proteínas do Olho , Fatores de Troca do Nucleotídeo Guanina , Camundongos Knockout , Retina , Proteínas rab de Ligação ao GTP , Animais , Retina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Camundongos , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Células HEK293 , Camundongos Endogâmicos C57BL , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Spermatogenesis depends on the supporting of the Sertoli cells and their communications with germ cells. However, the regulation of crosstalk between the Sertoli cells and germ cells remains unclear. RESULTS: In this report, we used conditional knockout technology to generate the Sertoli cells-specific knockout of Rnf20 in mice. The Amh-Rnf20-/- male mice were infertile owing to spermatogenic failure that mimic the Sertoli cell-only syndrome (SCOS) in humans. Knockout of Rnf20 resulted in the H2BK120ub loss in the Sertoli cells and impaired the transcription elongation of the Cldn11, a gene encoding a component of tight junction. Notably, RNF20 deficiency disrupted the cell adhesion, caused disorganization of the seminiferous tubules, and led to the apoptotic cell death of both spermatogonia and spermatocytes in the seminiferous tubules. CONCLUSIONS: This study describes a Rnf20 knockout mouse model that recapitulates the Sertoli cell-only syndrome in humans and demonstrates that RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells.
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BACKGROUND: Neurocognitive disorders and psychosocial difficulties are common in patients with Turner syndrome and multiple neurodegenerative diseases, yet there is no effective cure. Human primordial germ cells (hPGCs) are pluripotent germline stem cells in early embryo, which pass genetic information from one generation to the next, whereas all somatic cells will die along with the end of life. However, it is not known whether patient hPGCs with Turner syndrome contain information of neurocognitive and psychosocial illness. RESULTS: In this report, we used a high-density of culture system of embryoids derived from iPSCs of a patient with Turner syndrome to ask how pathogenetic pathways are associated with onset of neurocognitive and psychosocial disorders. The hPGC-Like Cells (hPGCLCs) were in vitro specified from iPSCs of 45,XO, 46,XX and 46,XY by the high-density induction of embryoids. Amazingly, we found that the specification process of the hPGCLCs in 45,XO, compared to those in 46,XX and 46,XY, enriched several common pathogenetic pathways regulating neurocognitive and psychosocial disorders, that shared among multiple neurodegenerative diseases and Turner syndrome. The downregulated chemical synaptic transmission pathways, including glutamatergic, GABAergic, and nicotine cholinergic synapses, indicated synaptic dysfunctions, while upregulated pathways that were associated with imbalance of mitochondrial respiratory chain complexes and apoptosis, may contribute to neuronal dysfunctions. Notably, downregulation of three types of ubiquitin ligases E1-E2-E3 and lysosome-associated sulfatases and RAB9A, owing to haploinsufficiency and parental preference of the X chromosome expression, indicated that two pathways of cellular degradation, lysosome and ubiquitin-proteasome, were impaired in the specification process of 45,XO hPGCLCs. This would lead to accumulation of undesired proteins and aggregates, which is a typically pathological hallmark in neurodegenerative diseases. CONCLUSIONS: Our data suggest that the specification process of the hPGCLCs in 45,XO, compared to those in 46,XX and 46,XY, enriched pathogenetic pathways that are associated with the onset of neurocognitive and psychosocial disorders.
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The germline cells are essential for the propagation of human beings, thus essential for the survival of mankind. The germline stem cells, as a unique cell type, generate various states of germ stem cells and then differentiate into specialized cells, spermatozoa and ova, for producing offspring, while self-renew to generate more stem cells. Abnormal development of germline stem cells often causes severe diseases in humans, including infertility and cancer. Primordial germ cells (PGCs) first emerge during early embryonic development, migrate into the gentile ridge, and then join in the formation of gonads. In males, they differentiate into spermatogonial stem cells, which give rise to spermatozoa via meiosis from the onset of puberty, while in females, the female germline stem cells (FGSCs) retain stemness in the ovary and initiate meiosis to generate oocytes. Primordial germ cell-like cells (PGCLCs) can be induced in vitro from embryonic stem cells or induced pluripotent stem cells. In this review, we focus on current advances in these embryonic and adult germline stem cells, and the induced PGCLCs in humans, provide an overview of molecular mechanisms underlying the development and differentiation of the germline stem cells and outline their physiological functions, pathological implications, and clinical applications.
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Células-Tronco Embrionárias , Células Germinativas , Adulto , Diferenciação Celular/genética , Feminino , Células Germinativas/metabolismo , Humanos , Masculino , Meiose/genética , Espermatozoides/fisiologiaRESUMO
Over the past 440 years since the discovery of the medicinal value of swamp eels, much progress has been made in the study of their biology. The fish is emerging as an important model animal in sexual development, in addition to economic and pharmaceutical implications. Tracing genomic history that shapes speciation of the fish has led to discovery of the whole genome-wide chromosome fission/fusion events. Natural intersex differentiation is a compelling feature for sexual development research. Notably, identification of progenitors of germline stem cells that have bipotential to differentiate into either male or female germline stem cells provides new insight into sex reversal. Here, we review these advances that have propelled the field forwards and present unsolved issues that will guide future investigations to finally elucidate vertebrate sexual development using the new model.
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Recombinação Genética , Vertebrados , Animais , Humanos , MasculinoRESUMO
Gonadal trans-differentiation from ovary to testis occurs in a same individual, suggesting a role of epigenetic regulation. However, histone modifications concerning the sex reversal process remain elusive. We analyzed histone modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology was used to test chromatin immunoprecipitation of gonads. Western blot analysis was performed to analyze protein expression. Immunofluorescence analysis was conducted to localize proteins in gonadal tissues. Here, we report a developmental atlas of histone modifications in the gonadal differentiation, including acetylation, methylation, and ubiquitination. We provided a detail distribution map of these modification sites including novel histone modifications along histones H2a, H2b, H3, and H4, and revealed their relationship with types of gonadal differentiation. We then determined a testis-enriched histone modification site, H2b monoubiquitination at K120, and its association with spermatogenesis. ChIP-seq demonstrated that the modification was highly enriched in the male sex-determining gene dmrt1 (doublesex and mab-3 related transcription factor 1), in particular, in its exon regions, suggesting its role in transcriptional regulation of dmrt1 in testis. Together, these data not only provide a new resource for epigenetic study in gonadal development, but also define an association of histone modifications with gonadal differentiation from ovary to testis.
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Transdiferenciação Celular/genética , Código das Histonas , Histonas/genética , Fatores de Transcrição , Animais , Proteínas de Ligação a DNA , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Gônadas/crescimento & desenvolvimento , Masculino , Modelos Animais , Processamento de Proteína Pós-Traducional , Diferenciação Sexual/genética , Smegmamorpha , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação , Sequenciamento Completo do GenomaRESUMO
Infertile ovotestis (mixture of ovary and testis) often occurs in intersex individuals under certain pathological and physiological conditions. However, how ovotestis is formed remains largely unknown. Here, we report the first comprehensive single-cell developmental atlas of the model ovotestis. We provide an overview of cell identities and a roadmap of germline, niche, and stem cell development in ovotestis by cell lineage reconstruction and a uniform manifold approximation and projection. We identify common progenitors of germline stem cells with two states, which reveal their bipotential nature to differentiate into both spermatogonial stem cells and female germline stem cells. Moreover, we found that ovotestis infertility was caused by degradation of female germline cells via liquid-liquid phase separation of the proteasomes in the nucleus, and impaired histone-to-protamine replacement in spermatid differentiation. Notably, signaling pathways in gonadal niche cells and their interaction with germlines synergistically determined distinct cell fate of both male and female germlines. Overall, we reveal a cellular fate map of germline and niche cell development that shapes cell differentiation direction of ovotestis, and provide novel insights into ovotestis development.
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Células Germinativas/crescimento & desenvolvimento , Seleção Sexual/genética , Animais , Diferenciação Celular , Feminino , MasculinoRESUMO
Mitophagy is an essential mechanism in maintaining cellular homeostasis, in which damaged and superfluous mitochondria are selectively degraded by the autophagy-lysosome pathway. Our recent study revealed that SPATA33 functions as a novel receptor for mitophagy in the priming of mitochondria for degradation in male germline cells. SPATA33 directly mediates the interaction of the outer mitochondrial membrane protein VDAC2 with the autophagy machinery component ATG16L1 during mitophagy. Upon starvation induction, SPATA33 can promote mitophagy as an autophagy receptor. Thus, SPATA33 confers cargo selectivity during mitophagy in germline cells. These findings provide new insights into selective autophagy and mitochondrial homeostasis.
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Autofagia , Mitofagia , Animais , Células Germinativas , Mitocôndrias , Proteínas MitocondriaisRESUMO
Nearly all diseases in humans, to a certain extent, exhibit sex differences, including differences in the onset, progression, prevention, therapy, and prognosis of diseases. Accumulating evidence shows that macroautophagy/autophagy, as a mechanism for development, differentiation, survival, and homeostasis, is involved in numerous aspects of sex differences in diseases such as cancer, neurodegeneration, and cardiovascular diseases. Advances in our knowledge regarding sex differences in autophagy-mediated diseases have enabled an understanding of their roles in human diseases, although the underlying molecular mechanisms of sex differences in autophagy remain largely unexplored. In this review, we discuss current advances in our insight into the biology of sex differences in autophagy and disease, information that will facilitate precision medicine.Abbreviations: AD: Azheimer disease; AMBRA1: autophagy and beclin 1 regulator 1; APP: amyloid beta precursor protein; AR: androgen receptor; AMPK: AMP-activated protein kinase; ATG: autophagy related; ATP6AP2: ATPase H+ transporting accessory protein 2; BCL2L1: BCL2 like 1; BECN1: beclin 1; CTSD: cathepsin D; CYP19A1: cytochrome P450 family 19 subfamily A member 1; DSD: disorders of sex development; eALDI: enhancer alternate long-distance initiator; ESR1: estrogen receptor 1; ESR2: estrogen receptor 2; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GABARAP: GABA type A receptor-associated protein; GLA: galactosidase alpha; GTEx: genotype-tissue expression; HDAC6: histone deacetylase 6; I-R: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; m6A: N6-methyladenosine; MYBL2: MYB proto-oncogene like 2; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB9A, RAB9A: member RAS oncogene family; RAB9B, RAB9B: member RAS oncogene family; RAB40AL: RAB40A like; SF1: splicing factor 1; SOX9: SRY-box transcription factor 9; SRY: sex determining region Y; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; VDAC2: voltage dependent anion channel 2; WDR45: WD repeat domain 45; XPDS: X-linked parkinsonism and spasticity; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.
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Autofagossomos/metabolismo , Autofagia/fisiologia , Doença/etiologia , Medicina de Precisão , Caracteres Sexuais , Proteínas Reguladoras de Apoptose/metabolismo , HumanosRESUMO
Selective autophagic degradation of mitochondria (mitophagy) is important in maintaining proper cellular homeostasis. Here, we found that SPATA33 is a novel autophagy mediator for mitophagy in testis. The SPATA33 protein localizes on mitochondria via its binding of the carboxyl terminal with the outer mitochondrial membrane protein VDAC2. Upon starvation induction, SPATA33 is recruited to autophagosome by binding the autophagy machinery ATG16L1 via its N-terminal along with mitochondria. Notably, Spata33 knockout inhibited autophagy and overexpression can promote autophagosome formation for mitochondrial sequestration. Therefore, SPATA33 confers selectivity for mitochondrial degradation and promotes mitophagy in male germline cells.
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Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular , Células Germinativas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/patologia , Testículo/fisiologia , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
Spermatogenesis is an essential process for producing sperm cells. Reproductive strategy is successfully evolved for a species to adapt to a certain ecological system. However, roles of newly evolved genes in testis autophagy remain unclear. In this study, we found that a newly evolved gene srag (Sox9-regulated autophagy gene) plays an important role in promoting autophagy in testis in the lineage of the teleost Monopterus albus. The gene integrated into an interaction network through a two-way strategy of evolution, via Sox9-binding in its promoter and interaction with Becn1 in the coding region. Its promoter region evolved a cis element for binding of Sox9, a transcription factor for male sex determination. Both in vitro and in vivo analyses demonstrated that transcription factor Sox9 could bind to and activate the srag promoter. Its coding region acquired ability to interact with key autophagy initiation factor Becn1 via the conserved C-terminal, indicating that srag integrated into preexisting autophagy network. Moreover, we determined that Srag enhanced autophagy by interacting with Becn1. Notably, srag transgenic zebrafish revealed that Srag exerted the same function by enhancing autophagy through the Srag-Becn1 pathway. Thus, the new gene srag regulated autophagy in testis by integrated into preexisting autophagy network.
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Autofagia/genética , Evolução Biológica , Enguias/fisiologia , Fatores de Transcrição SOX9/metabolismo , Testículo/fisiologia , Animais , Animais Geneticamente Modificados , Autofagossomos/fisiologia , Masculino , Peixe-ZebraRESUMO
BACKGROUND: Both testis and ovary can be produced sequentially in an individual with the same genome when sex reversal occurs in the teleost Monopterus albus, and epigenetic modification is supposed to be involved in gonadal differentiation. However, DNA methylation regulation mechanism underlying the gonadal differentiation remains unclear. RESULTS: Here, we used liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to simultaneously determine endogenous levels of both 5-methyl-2'-deoxycytidine (m5dC) and 5-hydroxymethyl-2'-deoxycytidine (hm5dC) during gonadal differentiation. Overall DNA methylation level was upregulated from ovary to testis via ovotestis. As a de novo methylase, dnmt3aa expression was also upregulated in the process. Notably, we determined transcription factor Foxa1 for dnmt3aa gene expression. Site-specific mutations and chromatin immunoprecipitation showed that Foxa1 can bind to and activate the dnmt3aa promoter. Furthermore, DNA methylation levels of key genes foxl2 (forkhead box L2) and cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) in regulation of female hormone synthesis were consistently upregulated during gonadal differentiation. CONCLUSIONS: These data suggested that dynamic change of DNA methylation modification is associated with gonadal differentiation.
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BACKGROUND: Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. RESULTS: Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). CONCLUSIONS: Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.
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Edição de Genes/instrumentação , Mutação , Peixe-Zebra/genética , Anormalidades Múltiplas/genética , Animais , Sequência de Bases , Anormalidades do Olho/genética , Humanos , Macrostomia/genéticaRESUMO
Detecting selection signatures in genomes that relates to transcription regulation has been challenges in genetic analysis. Here, we report a set of transcription factors EBF1, E2F1 and EGR2 for transcription activation of RAB37 promoter by a comparative analysis of promoter activities of RAB37 in humans, mice, and pigs. Two of the transcription factors bound to and co-regulated RAB37 promoter in each species. SNPs were further screened in pig RAB37 gene by population genomics in pig populations from both China and Europe. Three SNPs were identified in second CpG island upstream of core promoter of RAB37. These SNP variations led to at least 5 haplotypes, representing 5 multiple alleles of RAB37 in pig population. Distribution of these alleles in different genetic background of breeds showed a role of artificial selection for the variations of these multiple alleles. Of them, RAB37-c acquired the highest ability to activate gene expression in comparison with the other promoters, thus enhanced autophagy efficiently. These findings provide better understanding of transcription activation of RAB37 and artificial selection via RAB37 for autophagy regulation.
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Autofagia , Proteínas rab de Ligação ao GTP , Alelos , Animais , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único/genética , Suínos , Ativação Transcricional/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Teleost fishes account for over half of extant vertebrate species. A core question in biology is how genomic changes drive phenotypic diversity that relates to the origin of teleost fishes. RESULTS: Here, we used comparative genomic analyses with chromosome assemblies of diverse lineages of vertebrates and reconstructed an ancestral vertebrate genome, which revealed phylogenomic trajectories in vertebrates. We found that the whole-genome-wide chromosome fission/fusions took place in the Monopterus albus lineage after the 3-round whole-genome duplication. Four times of genomic fission/fusions events resulted in the whole genome-wide chromosome fusions in the genomic history of the lineage. In addition, abundant recently evolved new genes for reproduction emerged in the Monopterus albus after separated from medaka. Notably, we described evolutionary trajectories of conserved blocks related to sex determination genes in teleosts. CONCLUSIONS: These data pave the way for a better understanding of genomic evolution in extant teleosts.
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General education is an important part in higher education, which emphasizes the educational idea of integration of generality with specialty, and practices people-oriented education concept. However, there are some difficulties and puzzles in general education. Now the general education system with Chinese characteristics is needed to be established through practice and development. In this paper, we enumerate how to integrate knowledge of human genetics in practice of general education, teaching cases, and relevant analysis with concepts of general education. Using questions as "what are human beings?" as a leverage, we introduce teaching contents closely related to daily life. For example, we explain the past, present and future of human beings through contemporary evolutionary genomics teaching. In addition, we introduce problem-based deep thinking for students, thus integrating classical attributes of human beings into general education.
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Currículo , Genética Humana/educação , Ensino , Humanos , Conhecimento , EstudantesRESUMO
Retinopathy, owing to damage to the retina, often causes vision impairment, and the underlying molecular mechanisms are largely unknown. Using a gene targeting strategy, we generated mice with the essential gene Tubgcp4 knocked out. Homozygous mutation of Tubgcp4 resulted in early embryonic lethality due to abnormal spindle assembly caused by GCP4 (gamma-tubulin complex protein 4, encoded by Tubgcp4) depletion. Heterozygotes were viable through dosage compensation of one wild-type allele. However, haploinsufficiency of GCP4 affected the assembly of γ-TuRCs (γ-tubulin ring complexes) and disrupted autophagy homeostasis in retina, thus leading to photoreceptor degeneration and retinopathy. Notably, GCP4 exerted autophagy inhibition by competing with ATG3 for interaction with ATG7, thus interfering with lipidation of LC3B. Our findings justify dosage effects of essential genes that compensate for null alleles in viability of mutant mice and uncover dosage-dependent roles of GCP4 in embryo development and retinal homeostasis. These data have also clinical implications in genetic counseling on embryonic lethality and in development of potential therapeutic targets associated with retinopathy.