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SOX1 Determines the Regional Identity of Neural Progenitors Differentiated from Human Embryonic Stem Cells

ABSTRACT: During human embryogenesis, primitive neural cells start to be generated at the time of gastrulation and gradually acquire regional identities, which is a process called neural patterning. But how intrinsic factors respond to exogenous patterning signals remains poorly understood. Human Embryonic Stem Cells (hESCs) provide a great model to recapitulate this process. Through exogenous manipulation of canonical WNT signaling activation during neural differentiation, dose-dependent specification of regionally defined neural progenitors ranging from the telencephalic forebrain to posterior hindbrain could be rapidly and efficiently induced. Unexpectedly, we find that SOX1, generally referred as a pan-neural gene, displays a regional specific distribution in the human neural patterning process. To investigate the expression and function of SOX1 efficiently, we have generated the SOX1-EGFP reporter and SOX1-knockout (KO) hESC lines using the CRISPR/Cas9 system. SOX1 is initially expressed across the mes–met border and peaked in the metencephalon region at the early regional specification stage. Its depletion leads to the posterior shift of the mes–met border. Therefore, SOX1 is required to define the border at a correct region. In-depth analysis of SOX1 ChIP-sequencing and transcriptome data will provide more insights into how SOX1 determines the mes-met border formation and identify the downstream targets of SOX1. This study identifies SOX1 as one of the intrinsic factors key for the prepattern establishment in the developing central nervous system, particularly for defining the isthmus position.

ORGANISM(S): Homo sapiens

PROVIDER: GSE138217 | GEO | 2020/08/26

REPOSITORIES: GEO

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Project description:The process that partitions the nascent vertebrate central nervous system into forebrain, midbrain, hindbrain, and spinal cord after neural induction is of fundamental interest in developmental biology, and is known to be dependent on Wnt/beta-catenin signaling at multiple steps. Neural induction specifies neural ectoderm with forebrain character that is subsequently posteriorized by graded Wnt signaling: embryological and mutant analyses have shown that progressively higher levels of Wnt signaling induce progressively more posterior fates. However, the mechanistic link between Wnt signaling and the molecular subdivision of the neural ectoderm into distinct domains in the anteroposterior (AP) axis is still not clear. To better understand how Wnt mediates neural AP patterning, we performed a temporal dissection of neural patterning in response to manipulations of Wnt signaling in zebrafish. We show that Wnt-mediated neural patterning in zebrafish can be divided into three phases: (I) a primary AP patterning phase, which occurs during gastrulation, (II) a mes/r1 (mesencephalon-rhombomere 1) specification and refinement phase, which occurs immediately after gastrulation, and (III) a midbrain-hindbrain boundary (MHB) morphogenesis phase, which occurs during segmentation stages. A major outcome of these Wnt signaling phases is the specification of the major compartment divisions of the developing brain: first the MHB, then the diencephalic-mesencephalic boundary (DMB). The specification of these lineage divisions depends upon the dynamic changes of gene transcription in response to Wnt signaling, which we show primarily involves transcriptional repression or indirect activation. We show that otx2b is directly repressed by Wnt signaling during primary AP patterning, but becomes resistant to Wnt-mediated repression during late gastrulation. Also during late gastrulation, Wnt signaling becomes both necessary and sufficient for expression of wnt8b, en2a, and her5 in mes/r1. We suggest that the change in otx2b response to Wnt regulation enables a transition to the mes/r1 phase of Wnt-mediated patterning, as it ensures that Wnts expressed in the midbrain and MHB do not suppress midbrain identity, and consequently reinforce formation of the DMB. These findings integrate important temporal elements into our spatial understanding of Wnt-mediated neural patterning and may serve as an important basis of a better understanding of neural patterning defects that have implications in human health.

Project description:The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis. However, their regulatory functions in stages from the blastula to early neurula remain largely unknown, primarily because loss-of-function studies have not been informative to date. In our present study, we systematically knocked down the B1 sox genes in zebrafish. Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b, which are active in the early embryo, resulted in very severe developmental abnormalities, confirming that the B1 sox genes are functionally redundant. We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through microarray analysis as well as in situ hybridization. Importantly, these phenotypic analyses of the quadruple knockdown embryos reveled that the B1 SOX proteins regulate the following distinct processes: (1) early dorsoventral patterning by controlling bmp2b/7; (2) gastrulation movements via the regulation of pcdh18a/18b and non-canonical wnt genes; (3) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 (positively) and ascl1a (negatively), and regional transcription factor genes, e.g. hesx1, zic1 and rx3; and (4) neural patterning by regulating signaling pathway genes, cyp26a1 in RA signaling, oep in Nodal signaling, shh, and mdkb. These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo. Microarray analysis was carried out to compare gene expression profiles at the 30% epiboly (30%E), 75% epiboly (75%E) and tailbud (TB) stages between wild-type embryos and the sox2/3/19a/19b quadruple knockdown embryos. RNAs from two independent samples were analyzed for each embryonic stage.

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SOX1 Determines the Regional Identity of Neural Progenitors Differentiated from Human Embryonic Stem Cells

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