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In vitro construction of an embryonic caudal organizer

ABSTRACT: in vitro construction of an embryonic caudal organizer

PROVIDER: PRJNA1129168 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29632266_1.fastq.gz	Fastqsanger.gz
	SRR29632266_2.fastq.gz	Fastqsanger.gz
	SRR29632267_1.fastq.gz	Fastqsanger.gz
	SRR29632267_2.fastq.gz	Fastqsanger.gz
	SRR29632268_1.fastq.gz	Fastqsanger.gz

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in vitro construction of an embryonic caudal organizer

Project description:Purpose: Characterization of cell types in bmp4 explants and BMP4 induced human embryoids. Methods: Single cell transcriptomes of bmp4 explants were harvested at 6 hpf, 8 hpf,10 hpf, 12 hpf, 18 hpf and 24 hpf. Single cell transcriptomes of BMP4 induced human embryoids were harvested at Day 5 and Day 6. Libraries were prepared using Single Cell 3′Library & gel Bead kit v3.1 (10x Genomics, Cat# PN-1000121) according to the manufacturer’s protocol for 10000 cell recovery. Results: paraxial mesoderm, lateral plate mesoderm, and their derivatives were detected in bmp4 explants, and NMP, somite and neural cell fates were detected in bmp4 induced human embryoids. Conclusions: A well-organized caudal-like structure was induced in bmp4 explants and caudal cell fates were specified in BMP4 induced human embryoids.

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Project description:Studies in animal models have been fundamental for understanding brain development but only a fraction of these findings have been validated in a human context. Here, we tissue-engineered a model (MiSTR) of human neural tube regionalization based on human embryonic stem cells (hESCs) and microfluidic cell culturing. By exposing differentiating hESCs to a WNT signalling gradient we mimicked early rostro-caudal neural patterning, and with >80% reproducibility generated a coherent tissue with progressive caudalization from forebrain over midbrain to hindbrain, including formation of isthmic organiser characteristics. Single-cell transcriptomics revealed that rostro-caudal organization was established already at 24 hours of differentiation, before expression of neural markers. Moreover, the transcriptomic hallmarks of rostro-caudal MiSTR organization accurately recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. MiSTR thereby represents a novel in vitro model of human neurodevelopment to deconstruct and systematically analyze factors responsible for rostro-caudal neural tube patterning.

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Project description:Generating properly organized and differentiated embryonic structures in vitro from aggregates of pluripotent cells remains a major challenge. In a living embryo, the interplay in between signaling molecules known as morphogens and their antagonists lead to gradients of activity that instruct cells about their fate, leading to patterning and morphogenesis. Here we show that experimentally engineering a morphogen signaling center within an aggregate of mouse pluripotent stem cells is sufficient to mimic the function of an embryo organizer and to trigger embryonic development. The resulting embryoids are remarkably well patterned along anterior-posterior and dorsal-ventral axes, generate three germ layers through a process of gastrulation and differentiate germ layer derivatives (including notochord, vasculature, neural tube, neural crest, endoderm) similar to an authentic embryo at E9-E9.5.

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Project description:Spemann-Mangold organizer-specific transcription factors (TFs), Otx2, Lim1/Lhx1, and Gsc, are essential for embryonic head specification. However, the molecular mechanisms by which those TFs interact with the genome to direct head formation are largely unknown. Here we employed ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and found that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we defined two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and Lim1/Lhx1 (activator) or Goosecoid (repressor) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

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