Evolutionary emergence of the rac3b / rfng / sgca regulatory cluster refined mechanisms for hindbrain boundaries formation

Abstract

Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel // regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new -regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

Yap/Taz-TEAD ACTIVITY LINKS MECHANICAL CUES TO CELL PROGENITOR BEHAVIOR DURING HINDBRAIN SEGMENTATION

“…At this stage, the hindbrain boundary cells appear at the interface between rhombomeres ( Figure 1A-D) and expand all along the dorsoventral domain of the hindbrain (Cheng et al, 2004). They express a specific set of genes (Letelier et al, 2018), and remarkably, no proneural genes. The latter are expressed in zones that flank rhombomere boundaries in which they drive neurogenesis (Nikolaou et al, 2009).…”

“…Functionally, it was shown that these boundary cells act as an elastic mesh to prevent cell mixing between adjacent rhombomeres. This is due to the assembly of actomyosin cable-like structures in the apical side of these cells that generate tension within this cell population (Calzolari et al, 2014;Letelier et al, 2018). Given the compelling evidence for the relevance of YAP and TAZ as downstream mediators of mechanical cues, we investigated the role of Yap/Taz-TEAD activity within boundary cells as putative sensors of changes in cell and tissue architecture.…”

“…Our previous work demonstrated the role of contractile forces exerted by F-actin cables and their associated myosin motors to generate mechanical tension in boundary cells, specially through regulation of small GTPases of the RHO family (Calzolari et al, 2014;Letelier et al, 2018). Thus, our aim was to address whether integrity of the actomyosin cytoskeleton was necessary for Yap/Taz-activity within the All rights reserved.…”

“…10, 2018; boundary cells. To do so, Yap/Taz-activity was determined after blunting endogenous tensile forces in the Tg[4xGTIIC:d2GFP] embryos by several means (Figure 3): i) inhibition of myosin II using pharmacological treatments such as para-Nitroblebbistatin or Rockout (Calzolari et al, 2014), ii) disruption of the actomyosin cable assembly by downregulating the function of the boundary specific small GTPase Rac3b, with spliceblocking morpholino (Letelier et al, 2018), and iii) conditional inhibition of Rac3b by clonal expression of a dominant negative form of Rac3b (Myc:hsp:Rac3bDN) (Letelier et al, 2018). Upon inhibition of myosin II before the onset of Yap/Taz-activity within the boundaries, the activity was lost ( Figure 3B Figure 3A, n = 3/20; and 3F, n = 8/36).…”

“…This is due to the enrichment of actomyosin cable-like structures at the apical side of boundary cells that generate tension allowing these cells to behave as an elastic mesh (Calzolari et al, 2014;Letelier et al, 2018). During neurogenesis, hindbrain boundaries constitute a node for signaling pathways instructing the differentiation and organization of neurons in the neighboring rhombomeres (Cheng et al, 2004;Riley et al, 2004;Cooke et al, 2005;Terriente et al, 2012).…”