RESUMO
Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1-5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6-11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.
Assuntos
Embrião de Mamíferos , Gastrulação , Modelos Biológicos , Neurulação , Organogênese , Animais , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Coração/embriologia , Mesencéfalo/embriologia , Camundongos , Tubo Neural/embriologia , Fator de Transcrição PAX6/deficiência , Fator de Transcrição PAX6/genética , Prosencéfalo/embriologia , Somitos/embriologiaRESUMO
Intra-retinal axon guidance involves a coordinated expression of transcription factors, axon guidance genes, and secretory molecules within the retina. Pax6, the master regulator gene, has a spatio-temporal expression typically restricted till neurogenesis and fate-specification. However, our observation of persistent expression of Pax6 in mature RGCs led us to hypothesize that Pax6 could play a major role in axon guidance after fate specification. Here, we found significant alteration in intra-retinal axon guidance and fasciculation upon knocking out of Pax6 in E15.5 retina. Through unbiased transcriptome profiling between Pax6fl/fl and Pax6-/- retinas, we revealed the mechanistic insight of its role in axon guidance. Our results showed a significant increase in the expression of extracellular matrix molecules and decreased expression of retinal fate specification and neuron projection guidance molecules. Additionally, we found that EphB1 and Sema5B are directly regulated by Pax6 owing to the guidance defects and improper fasciculation of axons. We conclude that Pax6 expression post fate specification of RGCs is necessary for regulating the expression of axon guidance genes and most importantly for maintaining a conducive ECM through which the nascent axons get guided and fasciculate to reach the optic disc.
Assuntos
Fasciculação Axônica/fisiologia , Orientação de Axônios/fisiologia , Fator de Transcrição PAX6/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Fasciculação Axônica/genética , Orientação de Axônios/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/genética , Neurogênese/fisiologia , Fator de Transcrição PAX6/deficiência , Fator de Transcrição PAX6/genética , Gravidez , RNA-Seq , Receptor EphB1/genética , Receptor EphB1/fisiologia , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Células Ganglionares da Retina/citologia , Semaforinas/genética , Semaforinas/fisiologiaRESUMO
Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.
Assuntos
Axônios/fisiologia , Diencéfalo/metabolismo , Tálamo/metabolismo , Animais , Diencéfalo/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Mutagênese , Netrina-1/metabolismo , Técnicas de Cultura de Órgãos , Fator de Transcrição PAX6/deficiência , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Semaforina-3A/metabolismo , Tálamo/patologiaRESUMO
OBJECTIVE: The corneas of heterozygous Pax6+/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6+/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types. RESULTS: In a preliminary study, ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/-;RCE:loxP reporter mice was also patchy. We attribute patchy Pax6 staining to mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.
Assuntos
Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Epitélio Corneano/patologia , Fator de Transcrição PAX6/deficiência , Fatores Etários , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos TransgênicosRESUMO
The evolution of unique organ structures is associated with changes in conserved developmental programs. However, characterizing the functional conservation and variation of homologous transcription factors (TFs) that dictate species-specific cellular dynamics has remained elusive. Here, we dissect shared and divergent functions of Pax6 during amniote brain development. Comparative functional analyses revealed that the neurogenic function of Pax6 is highly conserved in the developing mouse and chick pallium, whereas stage-specific binary functions of Pax6 in neurogenesis are unique to mouse neuronal progenitors, consistent with Pax6-dependent temporal regulation of Notch signaling. Furthermore, we identified that Pax6-dependent enhancer activity of Dbx1 is extensively conserved between mammals and chick, although Dbx1 expression in the developing pallium is highly divergent in these species. Our results suggest that spatiotemporal changes in Pax6-dependent regulatory programs contributed to species-specific neurogenic patterns in mammalian and avian lineages, which underlie the morphological divergence of the amniote pallial architectures.
Assuntos
Proteínas Aviárias/fisiologia , Encéfalo/embriologia , Encéfalo/fisiologia , Fator de Transcrição PAX6/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/genética , Embrião de Galinha , Elementos Facilitadores Genéticos , Evolução Molecular , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Neurogênese/genética , Neurogênese/fisiologia , Fator de Transcrição PAX6/deficiência , Fator de Transcrição PAX6/genética , Gravidez , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais , Especificidade da EspécieRESUMO
Purpose: Aniridia is a congenital disorder caused by variants in the PAX6 gene. In this study, we assessed the involvement of PAX6 in patients with aniridia from Australasia and Southeast Asia. Methods: Twenty-nine individuals with aniridia from 18 families originating from Australia, New Caledonia, Cambodia, Sri Lanka, and Bhutan were included. The PAX6 gene was investigated for sequence variants and analyzed for deletions with multiplex ligation-dependent probe amplification. Results: We identified 11 sequence variants and six chromosomal deletions, including one in mosaic. Four deleterious sequence variants were novel: p.(Pro81HisfsTer12), p.(Gln274Ter), p.(Ile29Thr), and p.(Met1?). Ocular complications were associated with a progressive loss of visual function as shown by a visual acuity ≤ 1.00 logMAR reported in 65% of eyes. The prevalence of keratopathy was statistically significantly higher in the Australasian cohort (78.6%) compared with the Southeast Asian cohort (9.1%, p=0.002). Variants resulting in protein truncating codons displayed limited genotype-phenotype correlations compared with other variants. Conclusions: PAX6 variants and deletions were identified in 94% of patients with aniridia from Australasia and Southeast Asia. This study is the first report of aniridia and variations in PAX6 in individuals from Cambodia, Sri Lanka, Bhutan, and New Caledonia, and the largest cohort from Australia.
Assuntos
Aniridia/genética , Deleção Cromossômica , Estudos de Associação Genética , Mosaicismo , Fator de Transcrição PAX6/genética , Adolescente , Adulto , Aniridia/diagnóstico , Aniridia/patologia , Sudeste Asiático , Australásia , Sequência de Bases , Criança , Estudos de Coortes , Éxons , Feminino , Expressão Gênica , Genótipo , Humanos , Padrões de Herança , Íntrons , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX6/deficiência , Linhagem , FenótipoRESUMO
Patterning of the telencephalic neuroepithelium is a tightly regulated process controlled by transcription factors and signalling molecules. The cortical primordium is flanked by two signalling centres, the hem medially, and the antihem laterally. The hem induces the formation of the hippocampus in adjacent neuroepithelium. Therefore, the position of the hem defines the position of the hippocampus in the brain. The antihem is positioned at the boundary between the dorsal and ventral telencephalon and proposed to provide patterning cues during development. LIM-homeodomain (LIM-HD) transcription factor LHX2 suppresses both hem and antihem fate in the cortical neuroepithelium. Upon loss of Lhx2, medial cortical neuroepithelium is transformed into hem, whereas lateral cortical neuroepithelium is transformed into antihem. Here, we show that transcription factor PAX6, known to regulate patterning of the lateral telencephalon, restricts this tissue from transforming into hem upon loss of Lhx2. When Lhx2 and Pax6 are both deleted, the cortical hem expands to occupy almost the complete extent of the cortical primordium, indicating that both factors act to suppress hem fate in the lateral telencephalon. Furthermore, the shift in the pallial-subpallial boundary and absence of the antihem, observed in the Pax6 mutant, are both restored in the Lhx2; Pax6 double mutant. Together, these results not only reveal a novel function for LHX2 in regulating dorsoventral patterning in the telencephalon, but also identify PAX6 as a fundamental regulator of where the hem can form, and therefore implicate this molecule as a determinant of hippocampal positioning.
Assuntos
Proteínas com Homeodomínio LIM/deficiência , Neurogênese/fisiologia , Fator de Transcrição PAX6/deficiência , Telencéfalo/embriologia , Fatores de Transcrição/deficiência , Animais , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: We studied whether regulation of Cdca7 (Cell division cycle associated 7) expression by transcription factor Pax6 contributes to Pax6's cellular actions during corticogenesis. The function of Cdca7 in mediating Pax6's effects during corticogenesis has not been explored. Pax6 is expressed by radial glial progenitors in the ventricular zone of the embryonic cortical neuroepithelium, where it is required for the development of a normal complement of Tbr2-expressing intermediate progenitor cells in the subventricular zone. Pax6's expression levels are graded across the ventricular zone, with highest levels laterally where Tbr2-expressing progenitors are generated in greatest numbers at early stages of corticogenesis. METHODS: We used in situ hybridization and immunohistochemistry to analyse patterns of Cdca7 and Pax6 expression in cortical tissue from wild-type and Pax6 -/- embryos. In each genotype we compared the graded expression of the two genes quantitatively at several ages. To test whether defects in Cdca7 expression in lateral cortical cells might contribute to the cellular defects in this region caused by Pax6 loss, we electroporated a Cdca7 expression vector into wild-type lateral cortex and examined the effect on the production of Tbr2-expressing cells. RESULTS: We found that Cdca7 is co-expressed with Pax6 in cortical progenitors, at levels opposite to those of Pax6. Lowest levels of Cdca7 are found in the radial glial progenitors of lateral cortex, where Pax6 levels are highest. Higher levels of Cdca7 are found in ventral telencephalon, where Pax6 levels are low. Loss of Pax6 causes Cdca7 expression to increase in the lateral cortex. Elevating Cdca7 in normal lateral cortical progenitors to levels close to those normally found in ventral telencephalon reduces their production of Tbr2-expressing cells early in lateral cortical formation. CONCLUSION: Our results suggest that Pax6 normally represses Cdca7 expression in the lateral cortex and that repression of Cdca7 in cells of this region is required for their production of a normal complement of Tbr2-expressing intermediate progenitors.