Identification of BMP-regulated genes in early gastrulation stage zebrafish embryos
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE135100 | GEO | 2020/07/30
REPOSITORIES: GEO
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Project description:The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning, with high BMP signal activating ventral-lateral mesoderm markers directly, and low BMP signal inducing neural tissues. The Zinc finger SWIM domain-containing protein 4 (zswim4) is expressed in the dorsal blastopore lip at the onset of Xenopus gastrula and then enriched at the forming neuroectoderm at mid-gastrula stages. Overexpression of zswim4 in Xenopus embryos causes inhibition of the anterior axis and shortened, curved body, and knockdown or knockout of zswim4 disturbed embryonic body axis formation and head development. The expression of ventral-lateral mesoderm marker genes was reduced after zswim4 overexpression and increased in embryos with zswim4 knockdown. Neural marker genes were repressed in zswim4 morphant. Mechanistically Zswim4 attenuates BMP signal through reducing protein stability of Smad1 in both Xenopus embryos and HEK293T cells. Zswim4 interacts with Smad1 and promotes ubiquitination of Smad1 in HEK293T cells. To identify the interaction partner of Zswim4 in regulating Smad1 stability, we performed SILAC based IP in HEK293T cells, and the precipitates were analyzed by Mass Spectrometry.
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Project description:Optogenetics is a rapidly advancing technology that combines photochemical, optical and synthetic biology techniques to control cellular behaviour and physiology. Combining sensitive light responsive optogenetic intervention tools with human pluripotent stem cell differentiation models has the potential to refine differentiation and unpick the processes by which morphogenetic signals direct tissue patterning, organisation and cell specification. Here, we utilised an optogenetic bone morphogenetic protein (BMP) signalling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We initially engineered light-sensitive hESCs through CRISPR/Cas9-mediated integration of the optoBMP system into the AAVS1 locus. Activation of optoBMP with blue light in lieu of BMP family growth factors during differentiation resulted in activation of BMP signalling pathway mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells left in the dark. Furthermore, cells differentiated with light were capable of forming chondrogenic pellets consisting of a hyaline-like cartilaginous matrix.
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Project description:Identification of BMP-regulated genes in early gastrulation stage zebrafish embryos
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Project description:Spemann and Mangold Organizer (SMO) is of fundamental importance for the dorsal ventral body axis formation during vertebrate embryogenesis. Maternal Huluwa (Hwa) has been identified as the dorsal determinant that is both necessary and sufficient for the SMO formation. However, it remains unclear how Hwa is regulated. Here we report that the E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) is essential for restricting the spatial activity of Hwa, and therefore the proper SMO formation in Xenopus leavis. ZNRF3 interacts with and ubiquitinates Hwa thereby regulating its lysosomal trafficking and protein stability. Perturbation of ZNRF3 leads to the accumulation of Hwa and induction of ectopic axis in embryos. Ectopic expression of ZNRF3 promotes Hwa degradation and damps the axis-inducing activity of Hwa. Thus, our findings identify a substrate of ZNRF3, but also highlight the importance of the regulation of Hwa temporospatial activity in body axis formation in vertebrate embryos.
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Project description:FBS and BMP influence the somatic reprogramming induced by Oct4/Sox2/Klf4/c-Myc similarly. Bone morphogenetic proteins (BMPs) are abundant in serum and activate Smad1/5/8 to regulated target genes. BMPs play a important role in ES self-renewal, neurogenesis, osteogenesis, angiogenesis. We performed this experiment for identify what targets are regulated by BMPs and FBS.
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Project description:A control vs. genetic knockout experiment aimed at determining what RNAs are upregulated or downregulated in E13.5 mouse limb tissue lacking the Lmx1b gene. Because LMX1B is required for dorsal-ventral patterning of the limb, this screen gives insight into what putative downstream targets of Lmx1b contribute to dorsal-ventral patterning.
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Project description:Studies of the Xenopus organizer have laid the foundation for our understanding of the conserved signaling pathways that pattern vertebrate embryos during gastrulation. Here, we use this wealth of knowledge as leverage in the design and analysis of a genomic visualization of organizer-related gene transcription. Using ectopic expression of the two major activities of the organizer, BMP and Wnt inhibition, as well as endogenous tissues, we generate a focused set of samples that represent different aspects of organizer signaling. The genomic expression values of each sample are then measured with oligonucleotide arrays. From this data, genes regulated by organizer signaling are selected and then clustered by their patterns of regulation. A new GO biological process annotation of the Xenopus genome allows us to rapidly identify clusters that are highly enriched for known gastrula patterning genes. Within these clusters, we can predict the expression patterns of unknown genes with remarkable accuracy, leading to the discovery of new organizer-related gastrula stage expression patterns for 19 genes. Moreover, the patterns of gene response observed within these clusters allow us to parse apart the contributions of BMP and Wnt inhibition in organizer function. We find that the majority of gastrula patterning genes respond transcriptionally to these activities according to only a few stereotyped patterns, allowing us to describe suites of genes that are likely to share similar regulatory mechanisms. These suites of genes demonstrate a mechanism where BMP inhibition initiates the organizer program before gastrulation, and Wnt inhibition maintains and drives the organizer program during gastrulation. Experiment Overall Design: In order to describe and separate the genomic expression changes induced by the two main organizing activities, BMP inhibition and Wnt inhibition, we created a panel of gastrula stage ventral tissues that ectopically overexpressed one or both of these activities, and compared these samples to endogenous dorsal and ventral gastrula stage tissue. Two well-studied organizer secreted factors, Noggin and Dickkopf-1 (Dkk-1), were used to ectopically inhibit BMP and Wnt signaling, respectively. Four different overexpression mixtures were injected ventrally into 4-cell embryos: noggin and eGFP (anti-BMP); noggin, dkk-1, and eGFP (anti-BMP and anti-Wnt); dkk-1 and eGFP (anti-Wnt); and eGFP alone. eGFP mRNA was used to trace targeting. Plasmid DNA was used for noggin and dkk-1 overexpression, instead of mRNA, in order to limit ectopic expression of these genes to post-MBT stages, more closely mimicking the endogenous regulation of these genes. The ventrally injected embryos were grown to early stage 10, sorted for appropriately targeted eGFP florescence, and then bisected between the dorsal and ventral halves at either stage 10 (early gastrula) or stage 11.5 (late gastrula). For the noggin and/or dkk-1 injected embryos, only the ventral halves of the embryos were saved, eliminating endogenous organizer tissues. For the embryos injected with only eGFP, both the ventral and dorsal halves were saved, creating separate ventral and dorsal control conditions. These five conditions were each generated twice at both stage 10 and 11.5, with each set of five samples coming from a single clutch of embryos, creating twenty total tissue samples from 4 different clutches. For each batch of injections some sorted embryos were allowed to develop through tailbud stages in order to validate the phenotypes induced by our constructs. Total RNA was then isolated from the twenty tissue samples and applied to Affymetrix oligonucleotide arrays.
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