Dataset Information

Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish.

ABSTRACT: Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3-Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3-Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3-Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis.

SUBMITTER: Perez-Camps M

PROVIDER: S-EPMC5042653 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish.

Perez-Camps Mireia M Tian Jing J Chng Serene C SC Sem Kai Pin KP Sudhaharan Thankiah T Teh Cathleen C Wachsmuth Malte M Korzh Vladimir V Ahmed Sohail S Reversade Bruno B

eLife 20160929

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, ...[more]

PMID: 27684073

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Project description:Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4-6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 µM TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological processes involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with these responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf-a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled (szl)-a gene encoding a secreted Frizzled-related protein that limits BMP signaling-was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin (chd) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may not be driven by decreased szl expression, and that TDCIPP-induced dorsalization may-similar to dorsomorphin-be due to interference with BMP signaling during early zebrafish development.

Dataset Information

Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish.

Publications

Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish.

Similar Datasets

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