RESUMEN
How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2 and Isl1, including those dependent on Atoh7, and uncover the sequential and combinatorial interactions of these factors with the epigenetic landscape to control gene expression along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs).
Asunto(s)
Retina , Factores de Transcripción , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Retina/citología , Retina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Retinal ganglion cells (RGCs) are the only projection neurons in the neural retina. They receive and integrate visual signals from upstream retinal neurons in the visual circuitry and transmit them to the brain. The function of RGCs is performed by the approximately 40 RGC types projecting to various central brain targets. RGCs are the first cell type to form during retinogenesis. The specification and differentiation of the RGC lineage is a stepwise process; a hierarchical gene regulatory network controlling the RGC lineage has been identified and continues to be elaborated. Recent studies with single-cell transcriptomics have led to unprecedented new insights into their types and developmental trajectory. In this review, we summarize our current understanding of the functions and relationships of the many regulators of the specification and differentiation of the RGC lineage. We emphasize the roles of these key transcription factors and pathways in different developmental steps, including the transition from retinal progenitor cells (RPCs) to RGCs, RGC differentiation, generation of diverse RGC types, and central projection of the RGC axons. We discuss critical issues that remain to be addressed for a comprehensive understanding of these different aspects of RGC genesis and emerging technologies, including single-cell techniques, novel genetic tools and resources, and high-throughput genome editing and screening assays, which can be leveraged in future studies.
Asunto(s)
Diferenciación Celular , Células Ganglionares de la Retina/metabolismo , Animales , Linaje de la Célula , Regulación de la Expresión Génica , Humanos , Retina/metabolismo , Células Ganglionares de la Retina/citología , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: During development, all retinal cell types arise from retinal progenitor cells (RPCs) in a step-wise fashion. Atoh7 and Pou4f2 mark, and function in, two phases of retinal ganglion cell (RGC) genesis; Atoh7 functions in a subpopulation of RPCs to render them competent for the RGC fate, whereas Pou4f2 participates in RGC fate specification and RGC differentiation. Despite extensive research on their roles, the properties of the two phases represented by these two factors have not been well studied, likely due to the retinal cellular heterogeneity. RESULTS: In this report, we describe two novel knock-in mouse alleles, Atoh7zsGreenCreERT2 and Pou4f2FlagtdTomato , which labeled retinal cells in the two phases of RGC development by fluorescent proteins. Also, the Atoh7zsGreenCreERT2 allele allowed for indirect labeling of RGCs and other cell types upon tamoxifen induction in a dose-dependent manner. Further, these alleles could be used to purify retinal cells in the different phases by fluorescence assisted cell sorting (FACS). Single cell RNA-seq analysis of purified cells from Atoh7zsGreenCreERT2 retinas further validated that this allele labeled both transitional/competent RPCs and their progenies including RGCs. CONCLUSIONS: Thus, these two alleles are very useful tools for studying the molecular and genetic mechanisms underlying RGC formation.
Asunto(s)
Alelos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Retina/embriología , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.
Asunto(s)
Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/metabolismo , Vertebrados/metabolismo , Células Amacrinas/citología , Células Amacrinas/metabolismo , Animales , Apoptosis/genética , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Recuento de Células , División Celular/genética , Linaje de la Célula/genética , Proliferación Celular , Células Cultivadas , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Electroporación , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones Noqueados , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción Brn-3B/deficiencia , Factores de Transcripción/deficienciaRESUMEN
As with other retinal cell types, retinal ganglion cells (RGCs) arise from multipotent retinal progenitor cells (RPCs), and their formation is regulated by a hierarchical gene-regulatory network (GRN). Within this GRN, three transcription factors--atonal homolog 7 (Atoh7), POU domain, class 4, transcription factor 2 (Pou4f2), and insulin gene enhancer protein 1 (Isl1)--occupy key node positions at two different stages of RGC development. Atoh7 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Pou4f2 and Isl1 are downstream and regulate RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key step in RGC development, remains unclear. Here we report that ectopic expression of Pou4f2 and Isl1 in the Atoh7-null retina using a binary knockin-transgenic system is sufficient for the specification of the RGC fate. The RGCs thus formed are largely normal in gene expression, survive to postnatal stages, and are physiologically functional. Our results indicate that Pou4f2 and Isl1 compose a minimally sufficient regulatory core for the RGC fate. We further conclude that during development a core group of limited transcription factors, including Pou4f2 and Isl1, function downstream of Atoh7 to determine the RGC fate and initiate RGC differentiation.
Asunto(s)
Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclo Celular , Diferenciación Celular , Sistema Nervioso Central/metabolismo , Electrofisiología , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Retina/embriología , Retina/metabolismo , Células Madre/citologíaRESUMEN
Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general.
Asunto(s)
Factor Nuclear 6 del Hepatocito/metabolismo , Proteínas de Homeodominio/metabolismo , Retina/citología , Retina/embriología , Factores de Transcripción/metabolismo , Células Amacrinas/citología , Células Amacrinas/metabolismo , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 6 del Hepatocito/deficiencia , Factor Nuclear 6 del Hepatocito/genética , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Embarazo , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Células Horizontales de la Retina/citología , Células Horizontales de la Retina/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
The mammalian striatum controls the output of the basal ganglia via two distinct efferent pathways, the direct (i.e., striatonigral) and the indirect (i.e., striatopallidal) pathways. The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in a subpopulation of striatal progenitors; however, its specific role in striatal development remains unknown. Our genetic fate-mapping results show that Isl1-expressing progenitors give rise to striatal neurons belonging to the striatonigral pathway. Conditional inactivation of Isl1 in the telencephalon resulted in a smaller striatum with fewer striatonigral neurons and reduced projections to the substantia nigra. Additionally, conditional inactivation in the ventral forebrain (including both the telencephalon and diencephalon) revealed a unique role for Isl1 in diencephalic cells bordering the internal capsule for the normal development of the striatonigral pathway involving PlexinD1-Semaphorin 3e (Sema3e) signaling. Finally, Isl1 conditional mutants displayed a hyperlocomotion phenotype, and their locomotor response to psychostimulants was significantly blunted, indicating that the alterations in basal ganglia circuitry contribute to these mutant behaviors.
Asunto(s)
Cuerpo Estriado/embriología , Proteínas con Homeodominio LIM/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiología , Sustancia Negra/embriología , Factores de Transcripción/metabolismo , Animales , Conducta Animal/fisiología , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Cuerpo Estriado/citología , Proteínas del Citoesqueleto , Glicoproteínas/genética , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Homeodominio LIM/genética , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Semaforinas , Sustancia Negra/citología , Factores de Transcripción/genéticaRESUMEN
Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (â¼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Factor Nuclear 6 del Hepatocito/metabolismo , Neurogénesis/genética , Retina/citología , Células Horizontales de la Retina/metabolismo , Animales , Recuento de Células , Diferenciación Celular/genética , Supervivencia Celular , Embrión de Mamíferos , Proteínas del Ojo/genética , Proteínas Fluorescentes Verdes/genética , Factor Nuclear 6 del Hepatocito/genética , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/metabolismo , Vías Nerviosas/ultraestructura , Neuroglía/metabolismo , Neuroglía/fisiología , Neuronas/clasificación , Neuronas/metabolismo , Neuronas/ultraestructura , Proteína Quinasa C-alfa/metabolismo , Retina/embriología , Células Horizontales de la Retina/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Homeobox SIX3RESUMEN
p58IPK is a multifaceted endoplasmic reticulum (ER) chaperone and a regulator of eIF2α kinases involved in a wide range of cellular processes including protein synthesis, ER stress response, and macrophage-mediated inflammation. Systemic deletion of p58IPK leads to age-related loss of retinal ganglion cells (RGC) and exacerbates RGC damage induced by ischemia/reperfusion and increased intraocular pressure (IOP), suggesting a protective role of p58IPK in the retina. However, the mechanisms remain elusive. Herein, we investigated the cellular mechanisms underlying the neuroprotection action of p58IPK using conditional knockout (cKO) mouse lines where p58IPK is deleted in retinal neurons (Chx10-p58IPK cKO) or in myeloid cells (Lyz2-p58IPK cKO). In addition, we overexpressed p58IPK by adeno-associated virus (AAV) in the retina to examine the effect of p58IPK on RGC survival after ocular hypertension (OHT) in wild type (WT) mice. Our results show that overexpression of p58IPK by AAV significantly improved RGC survival after OHT in WT mice, suggesting a protective effect of p58IPK on reducing RGC injury. Conditional knockout of p58IPK in retinal neurons or in myeloid cells did not alter retinal structure or cellular composition. However, a significant reduction in the b wave of light-adapted electroretinogram (ERG) was observed in Chx10-p58IPK cKO mice. Deletion of p58IPK in retinal neurons exacerbates RGC loss at 14 days after OHT. In contrast, deficiency of p58IPK in myeloid cells increased the microglia/macrophage activation but had no effect on RGC loss. We conclude that deletion of p58IPK in macrophages increases their activation, but does not influence RGC survival. These results suggest that the neuroprotective action of p58IPK is mediated by its expression in retinal neurons, but not in macrophages. Therefore, targeting p58IPK specifically in retinal neurons is a promising approach for the treatment of neurodegenerative retinal diseases including glaucoma.
Asunto(s)
Glaucoma , Hipertensión Ocular , Animales , Ratones , Proteínas del Choque Térmico HSP40 , Activación de Macrófagos , Macrófagos/metabolismo , Microglía/metabolismo , Células Ganglionares de la Retina/metabolismoRESUMEN
Retinal vascular development is a complex process that is not yet fully understood. The majority of research in this area has focused on astrocytes and the template they form in the inner retina, which precedes endothelial cells in the mouse retina. In humans and dogs, however, astrocyte migration follows behind development of blood vessels, suggesting that other cell types may guide this process. One such cell type is the ganglion cell, which differentiates before blood vessel formation and lies adjacent to the primary retinal vascular plexus. The present study investigated the potential role played by ganglion cells in vascular development using Math5(-/-) mice. It has previously been reported that Math5 regulates the differentiation of ganglion cells and Math5(-/-) mice have a 95% reduction in these cells. The development of blood vessels and glia was investigated using Griffonia simplicifolia isolectin B4 labeling and GFAP immunohistochemistry, respectively. JB-4 analysis demonstrated that the hyaloid vessels arose from choriovitreal vessels adjacent to the optic nerve area. As previously reported, Math5(-/-) mice had a rudimentary optic nerve. The primary retinal vessels did not develop post-natally in the Math5(-/-) mice, however, branches of the hyaloid vasculature eventually dove into the retina and formed the inner retinal capillary networks. An astrocyte template only formed in some areas of the Math5(-/-) retina. In addition, GFAP(+) Müller cells were seen throughout the retina that had long processes wrapped around the hyaloid vessels. Transmission electron microscopy confirmed Müller cell abnormalities and revealed disruptions in the inner limiting membrane. The present data demonstrates that the loss of ganglion cells in the Math5(-/-) mice is associated with a lack of retinal vascular development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuroglía/patología , Células Ganglionares de la Retina/fisiología , Vasos Retinianos/patología , Animales , Animales Recién Nacidos , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Técnicas de Genotipaje , Proteína Ácida Fibrilar de la Glía , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , Vítreo Primario Hiperplásico Persistente/fisiopatología , Lectinas de Plantas/metabolismo , Neovascularización Retiniana/fisiopatología , Vasos Retinianos/metabolismoRESUMEN
Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.
Asunto(s)
Retina , Factores de Transcripción , Ratones , Animales , Factores de Transcripción/genética , Neuroglía , Neuronas , MamíferosRESUMEN
Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Genes del Desarrollo , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas con Homeodominio LIM , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Nervio Óptico/metabolismo , Nervio Óptico/ultraestructura , Retina/anomalías , Retina/embriología , Retina/ultraestructura , Células Ganglionares de la Retina/patología , Factor de Transcripción Brn-3B/genética , Factores de TranscripciónRESUMEN
Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Transcriptoma , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Mutación con Pérdida de Función , Ratones , ARN Citoplasmático Pequeño , Análisis de Secuencia , Células Madre , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The common deletion of the third exon of the growth hormone receptor gene (GHRd3) in humans is associated with birth weight, growth after birth, and time of puberty. However, its evolutionary history and the molecular mechanisms through which it affects phenotypes remain unresolved. We present evidence that this deletion was nearly fixed in the ancestral population of anatomically modern humans and Neanderthals but underwent a recent adaptive reduction in frequency in East Asia. We documented that GHRd3 is associated with protection from severe malnutrition. Using a novel mouse model, we found that, under calorie restriction, Ghrd3 leads to the female-like gene expression in male livers and the disappearance of sexual dimorphism in weight. The sex- and diet-dependent effects of GHRd3 in our mouse model are consistent with a model in which the allele frequency of GHRd3 varies throughout human evolution as a response to fluctuations in resource availability.
RESUMEN
Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5(HA) and Pou4f2(HA) alleles. We demonstrate that the tagged alleles recapitulated the wild-type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co-staining, we found that Math5 and Pou4f2 were transiently co-expressed in newly born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope-tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Epítopos/genética , Proteínas de Homeodominio/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Southern Blotting , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step in dolichol synthesis. Recessive mutations in DHDDS cause retinitis pigmentosa (RP59), resulting in blindness. We hypothesized that rod photoreceptor-specific ablation of Dhdds would cause retinal degeneration due to diminished dolichol-dependent protein N-glycosylation. Dhddsflx/flx mice were crossed with rod-specific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specific Dhdds knockout mice (Dhddsflx/flx iCre+). In vivo morphological and electrophysiological evaluation of Dhddsflx/flx iCre+ retinas revealed mild retinal dysfunction at postnatal (PN) 4 weeks, compared with age-matched controls; however, rapid photoreceptor degeneration ensued, resulting in almost complete loss of rods and cones by PN 6 weeks. Retina dolichol levels were markedly decreased by PN 4 weeks in Dhddsflx/flx iCre+ mice, relative to controls; despite this, N-glycosylation of retinal proteins, including opsin (the dominant rod-specific glycoprotein), persisted in Dhddsflx/flx iCre+ mice. These findings challenge the conventional mechanistic view of RP59 as a congenital disorder of glycosylation.
RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Neuronas Motoras/metabolismo , Factores de Transcripción Onecut/metabolismo , Médula Espinal/metabolismo , Factores de Transcripción/metabolismo , Animales , Expresión Génica , Proteínas de Homeodominio/genética , Locomoción/fisiología , Ratones , Ratones Transgénicos , Factores de Transcripción Onecut/genética , Factores de Transcripción/genéticaRESUMEN
Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.
Asunto(s)
Degeneración Nerviosa/patología , Enfermedades del Nervio Óptico/patología , Células Ganglionares de la Retina/patología , Animales , Muerte Celular/efectos de los fármacos , Toxina Diftérica/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Gliosis/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Nervio Óptico/patología , Nervio Óptico/ultraestructura , Enfermedades del Nervio Óptico/genética , Fragmentos de Péptidos/farmacología , Retina/efectos de los fármacos , Retina/embriología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/ultraestructura , Tamoxifeno/farmacología , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismo , Agudeza VisualRESUMEN
The vertebrate retina develops from an amorphous sheet of dividing retinal progenitor cells (RPCs) through a sequential process that culminates in an exquisitely patterned neural tissue. A current model for retinal development posits that sequential cell-type differentiation is the result of changes in the intrinsic competence state of multipotent RPCs as they advance in time and that the intrinsic changes are influenced by continuous changes in the extracellular environment. Although several studies support the proposition that newly differentiated cells alter the extrinsic state of the developing retina, it is still far from clear what role they play in modifying the extracellular environment and in influencing the properties of RPCs. Here, we specifically ablate retinal ganglion cells (RGCs) as they differentiate, and we determine the impact of RGC absence on retinal development. We find that RGCs are not essential for changing the competence of RPCs, but they are necessary for maintaining sufficient numbers of RPCs by regulating cell proliferation via growth factors. Intrinsic rather than extrinsic factors are likely to play the critical roles in determining retinal cell fate.