Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing

Abstract

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation.

Multiscale analysis of architecture, cell size and the cell cortex reveals cortical F-actin density and composition are major contributors to mechanical properties during convergent extension

“…We chose α-actinin-1 because its expression increases sharply during neurulation (Session et al, 2016) (Fig. S6) and it is expressed at relatively high levels in the dorsal marginal zone (Popov et al, 2017). Moderate to high levels of α-actinin-1 overexpression strongly disrupted normal development by inhibiting blastopore closure (Fig.…”

Notch1 is asymmetrically distributed from the beginning of embryogenesis and controls the ventral center

“…S6A-E). In addition, Notch1 Mo downregulated other genes important for the ventral program, such as trim29 (Hayes et al, 2007;Cibois et al, 2015;Popov et al, 2017) (Fig. S6H), xk81a1 (Yan et al, 2009;Scerbo et al, 2017) (Fig.…”

A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs

“…The ‘inputs’ in this GRN represent the TFs and signaling molecules (transduced via intracellular TFs) important for mesendoderm formation and early endoderm patterning, many of which have been elucidated [8,9]. Recently, genome-wide approaches have identified additional localized maternal and zygotic transcripts encoding transcription factors [10-16]. Based on a comprehensive catalogue of the X. tropicalis TFs [14], 130 TFs are found to be enriched vegetally (in comparison to the animal pole), and we have focused on the ∼50 TFs expressed at relatively high abundance in the vegetal tissue (Transcripts Per Million values ≥ 50).…”

“…These data have allowed us to incorporate, to a greater extent, temporal information into a graphical organization of our network. Additionally, HTS data revealed vegetally enriched transcripts [14-16], which can be difficult to visualize by whole mount in situ hybridization.…”

“…These loops, along with negative and double negative feedback loops, appear to be particularly useful in regionalizing the dorsal and ventral sides of the mesendoderm. It is likely that more of these types of loops will be identified in the future, as we better define the expression domains of more transcription factors during Xenopus gastrulation [14-16]. Also, as this review focused primary on mesendodermal genes, the molecular mechanism for the regionalization of the embryo between mesendoderm and prospective ectoderm is not reflected on this network.…”