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Dr Shiv Kumar

Dr Shiv Kumar

Research Fellow in Neurobiology

Researcher profile

Email
sk288@st-andrews.ac.uk

 

Research areas

Transcriptional Regulation Mechanism Study of PV.1 during Neuro-ectoderm and Mesoderm Formation in Early Embryos of Xenopus laevis

Bone morphogenetic protein4 (BMP4) and FGF signaling actively participates in dorso-ventral axis formation, ventral mesodermal induction (VMZ), germ-layer specification and positioning differentiation, cancer and metastasis during early development of vertebrate. Our study demonstrated that signaling crosstalk of BMP4/Smad-1 and FGF/Xbra induces the expression of ventx1.1 (posterior-ventral.1, PV.1) in Xenopus embryos, separately. Ventx1.1 is a well-known homeobox transcription factor and neural repressor in Xenopus. Ventx1.1 leads the ectoderm and ventral mesoderm formation in Xenopus embryos. Moreover, ectopic expression of Ventx1.1 reduces the expression of dorsal, early and late neural specific genes, including zic3, foxd4l1.1 chordin, noggin, Xngnr and n-cam in animal cap explants of Xenopus embryos, causing the inhibition of neurogenesis in Xenopus. Our study demonstrates that the physical interaction of Smad-1 and Xbra physically interacts and cooperate synergistically to induce the expression of ventx1.1 in VMZ, leading to ectoderm and neural inhibition during gastrula of Xenopus embryos. Moreover, ChIP-Seq and ChIP-PCR shows that Smad-1 and Xbra directly binds within the 5?-flanking upstream region of ventx1.1 and enhances the each other?s direct binding within the proximal promoter of ventx1.1 during gastrula. Meanwhile, we observe that genome-wide ChIP-Seq of earliest neural specific fork-head winged transcription factor, namely FoxD4l1.1 directly binds to close vicinity of putative transcription initiation site within the promoter region of ventx1.1 in Xenopus gastrulae. Our study demonstrates that FoxD14l.1 inhibits ventx1.1 expression in neuroectoderm (Future brain) region, directly and indirectly. Ectopic expression of FoxD4l1.1 inhibits the BMP4-mediated activation as well as chromatin binding of Smad-1 through inhibiting the C-terminal phosphorylation during gastrulae. Additionally, FoxD4l.1 physically interacts and sequesters Xbra to inhibit Smad-1 and Xbra binding in Xenopus embryos. ChIP-PCR assay exhibits that ectopic expression of FoxD4l1.1 reduces the binding of Smad-1 and Xbra within the promoter region of ventx1.1. FoxD4l1.1-mediated inhibition of ventx1.1transcription triggers the neural induction and leads the ectoderm-neuroectoderm transition in Xenopus gastrulae. Another finding indicates that Ventx1.1 directly inhibits the neural specific gene expression of Zic3 in ectoderm and VMZ of Xenopus embryos, inhibiting the neural induction in Xenopus. Using chromatin immunoprecipitation (ChIP)-Seq, genome-wide binding pattern of Ventx1.1 in Xenopus gastrulae, we report that Ventx1.1 associates with its own 5?-flanking sequence. We present evidence that Ventx1.1 binds a cis-acting Ventx1.1 response element (VRE) in its own promoter, leading to repression of its own transcription. Site-directed mutagenesis of the VRE in the Ventx1.1 promoter significantly abrogated this inhibitory autoregulation of Ventx1.1 transcription. Notably, Ventx1.1 and Xcad2, an activator of Ventx1.1 transcription, competitively co-occupied the VRE in the Ventx1.1 promoter. In support of this, mutation of the VRE down-regulated basal and Xcad2-induced levels of Ventx1.1 promoter activity. In addition, overexpression of Ventx1.1 prevents Xcad2 from binding to the Ventx1.1 promoter, and vice versa. This finding suggests that Ventx1.1 negatively regulates its own transcription in competition with Xcad2, thereby fine-tuning its own expression levels during dorsoventral patterning of Xenopus early embryo.

 

My overall research collectively proposes a putative theory that preoccupied territory of germ layer-specific transcription factors, including Ventx1.1 and Ventx2.1 (ectoderm and VMZ), FoxD4l1.1 and Zic3(neuroectoderm), Goosecoid (dorsal mesoderm), Xbra (Mesoderm) and vegT, Sox17, Sia, eomesdermin (dorsal mesoderm and endoderm) determine their territory and inhibits the expression of other specifier during germ layer specification, cell polarity, axis patterning, differentiation, gastrulation movement and convergent extension during embryonic development of vertebrate. The tempo-spatial expression of preoccupied determinants is tightly précised and fine-tuned by maternal-zygotic signalling molecules, leading to controlled and disciplined germ layer specification and placode development, axis patterning (dorso-ventral and anterior-posterior axis formation) and gastrulation movement and convergent extension during embryonic development of vertebrate.  

 

Currently, I am working on neuron physiology at the University of St. Andrews, Fife Scotland UK

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