Twist is an essential regulator of the skeletogenic gene regulatory network in the sea urchin embryo

Abstract

Recent work on the sea urchin endomesoderm gene regulatory network (GRN) offers many opportunities to study the specification and differentiation of each cell type during early development at a mechanistic level. The mesoderm lineages consist of two cell populations, primary and secondary mesenchyme cells (PMCs and SMCs). The micromere-PMC GRN governs the development of the larval skeleton, which is the exclusive fate of PMCs, and SMCs diverge into four lineages, each with its own GRN state. Here we identify a sea urchin ortholog of the Twist transcription factor, and show that it plays an essential role in the PMC GRN and later is involved in SMC formation. Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. These data illustrate the significance of regulatory state maintenance and continuous progression during cell specification, and the dynamics of the sequential events that depend on those earlier regulatory states.

Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition

“…De-adhesion begins with adherens junctions disassembly and continues with cell membrane turnover; the membrane loses cadherins and catenins, and adds mesenchymal adhesion molecules with extracellular matrix affinity (D'Souza-Schorey, 2005;Yap et al, 2007;Thiery et al, 2009). In L. variegatus, alx1, twist and snail constitute a functional sub-circuit in the GRN that controls the repression and endocytosis of cadherin required for de-adhesion (Wu and McClay, 2007;Wu et al, 2008). Our analysis of both snail and twist knockdowns showed a failure of PMCs to complete EMT in spite of four other successful cell state changes: laminin hole creation (Fig.…”

“…This confirmed the previously described role for snail and twist in the deadhesion component in both this and other EMT models. Alx1, the known driver of both snail and twist expression (Wu and McClay, 2007;Wu et al, 2008), had the same knockdown phenotype as snail and twist, indicating alx1 is also required for de-adhesion. Therefore, alx1, twist and snail display control over adhesive state change and make a distinct sub-circuit of the GRN, a positivefeedback lockdown, that governs the de-adhesion process (Fig.…”

“…The biggest difference between the current S. purpuratus GRN model and that shown in Fig. 1 is the observation that snail and twist are part of the L. variegatus GRN (Wu et al, 2007;Wu et al, 2008). As specification of the skeletogenic GRN occurs prior to EMT, the network depicted in Fig.…”

“…See supplementary material Table S2 for MASO oligo sequences. The following concentrations were used for previously published Lv PMC transcription factor MASOs used in this study: 1.0 mM LvAlx1 (Ettensohn et al, 2003), 1.0 mM LvSnail2 (Wu and McClay, 2007), 1.5 mM LvTwist (Wu et al, 2008), 0.5 mM LvTbr (Croce et al, 2001) and 0.7 mM FoxN2/3-1 (Rho and McClay, 2011). mRNA for injection was transcribed in vitro using Ambion mMessage mMachine.…”

The control offoxN2/3expression in sea urchin embryos and its function in the skeletogenic gene regulatory network

“…Perturbation of these early-activated TFs at the top of the GRN thus causes catastrophic failures, whereas TFs activated at later stages of specification produce more specific responses if knocked down. For example, Snail and Twist, which are expressed in micromeres after the hatched blastula stage, play crucial roles during PMC ingression (Wu and McClay, 2007;Wu et al, 2008). Rather than regulating ingression, TFs like Hex or Tgif instead control skeletogenesis and biomineralization through activation of skeletogenic genes such as msp130, sm30 and sm50 (Oliveri et al, 2008).…”