Full Transcriptome Analysis of Early Dorsoventral Patterning in Zebrafish

Abstract

Understanding the molecular interactions that lead to the establishment of the major body axes during embryogenesis is one of the main goals of developmental biology. Although the past two decades have revolutionized our knowledge about the genetic basis of these patterning processes, the list of genes involved in axis formation is unlikely to be complete. In order to identify new genes involved in the establishment of the dorsoventral (DV) axis during early stages of zebrafish embryonic development, we employed next generation sequencing for full transcriptome analysis of normal embryos and embryos lacking overt DV pattern. A combination of different statistical approaches yielded 41 differentially expressed candidate genes and we confirmed by in situ hybridization the early dorsal expression of 32 genes that are transcribed shortly after the onset of zygotic transcription. Although promoter analysis of the validated genes suggests no general enrichment for the binding sites of early acting transcription factors, most of these genes carry “bivalent” epigenetic histone modifications at the time when zygotic transcription is initiated, suggesting a “poised” transcriptional status. Our results reveal some new candidates of the dorsal gene regulatory network and suggest that a plurality of the earliest upregulated genes on the dorsal side have a role in the modulation of the canonical Wnt pathway.

Spatial reconstruction of single-cell gene expression data

“…5g , purple). Notably, these cells were not an artifact of the isolation process: they were identified in 10 separate embryos, in each experimental batch, and previous in situ analysis for foxo3b , aplnrb , and isg15 revealed their individual scattered expression 38 , 39 .…”

Response to Nodal morphogen gradient is determined by the kinetics of target gene induction

“…Nodal signaling induces and patterns mesendoderm and establishes left-right asymmetry ( Conlon et al, 1994 ; Shen, 2007 ; Grande and Patel, 2009 ; Schier, 2009 ; Duboc et al, 2010 ; Shiratori and Hamada, 2014 ). The Nodal signaling pathway regulates dozens of genes, ranging from transcription factors to cytoskeletal components, in order to pattern embryonic tissues ( Bennett et al, 2007 ; Liu et al, 2011 ; Fodor et al, 2013 ). In embryonic stem cells, Nodal signaling is required for self-renewal as well as specification of endoderm and mesoderm ( James et al, 2005 ; Vallier et al, 2005 ; Schier, 2009 ; Oshimori and Fuchs, 2012 ; Chen et al, 2013 ).…”

Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing

“…Gene expression profiling is an excellent starting point to study a large variety of biological mechanisms associated with specific developmental stages, mutations, and/or disease conditions (Fodor et al, 2013; Jiang et al, 2014; Stockhammer et al, 2010; Vesterlund et al, 2011; Wells et al, 2013). RNA sequencing (RNA-seq) has become an established method for transcriptome profiling, especially with recent reduction of the associated costs.…”

“…The zebrafish pineal organ, which is made up of projection neurons and photoreceptors, is an important neuroendocrine organ that secretes melatonin in response to circadian stimuli (Cahill, 1996; Fodor et al, 2013; Gamse et al, 2002; Jiang et al, 2014; Stockhammer et al, 2010; Wells et al, 2013). Cell fate decisions between projection neurons and photoreceptors in the pineal organ is determined by the coordinated actions of BMP and Notch signaling (Cau et al, 2008; Quillien et al, 2011).…”