TGF-β sensu stricto signaling regulates skeletal morphogenesis in the sea urchin embryo

Abstract

Cell-cell signaling plays a prominent role in the formation of the embryonic skeleton of sea urchins, but the mechanisms are poorly understood. In the present study, we uncover an essential role for TGF-β sensu stricto signaling in this process. We show that TgfbrtII, a type II receptor dedicated to signaling through TGF-β sensu stricto, is expressed selectively in skeletogenic primary mesenchyme cells (PMCs) during skeleton formation. Morpholino (MO) knockdowns and studies with a specific TgfbrtII inhibitor (ITD-1) in both S. purpuratus and Lytechinus variegatus embryos show that this receptor is required for biomineral deposition. We provide pharmacological evidence that Alk4/5/7 is the cognate TGF-β type I receptor that pairs with TgfbrtII and show by inhibitor treatments of isolated micromeres cultured in vitro that both Alk4/5/7 and TgfbrtII function cell-autonomously in PMCs. Gene expression and gene knockdown studies suggest that TGF-β sensu stricto may be the ligand that interacts with TgfbrtII and support the view that this TGF-β superfamily ligand provides an essential, permissive cue for skeletogenesis, although it is unlikely to provide spatial patterning information. Taken together, our findings reveal that this model morphogenetic process involves an even more diverse suite of cell signaling pathways than previously appreciated and show that PMCs integrate a complex set of both generalized and spatially localized cues in assembling the endoskeleton.

Inhibition of microRNA suppression of Dishevelled results in Wnt pathway-associated developmental defects in sea urchin

“…To identify the underlying molecular mechanism that led to Dvl miRNATP-and Dvl mRNA-induced skeletal defects, we examined expressions of the following gene categories: (1) Alx1, which is essential for PMC specification (Ettensohn et al, 2003); (2) biomineralization genes SM29, SM49, SM50 (Adomako-Ankomah and Ettensohn, 2013); (3) Hnf6, which regulates SM50 (Otim et al, 2004;Otim, 2017); (4) factors that have been shown to be important for PMC positioning, including Nodal, BMP2/4 (Yaguchi et al, 2008;Duboc et al, 2010), Pax2/ 5/8 (Rottinger et al, 2008), Vegf3 (Duloquin et al, 2007;Adomako-Ankomah and Ettensohn, 2013), Alk2/4/7, Slc26a5 and TGF-βrtII (Piacentino et al, 2015(Piacentino et al, , 2016Sun and Ettensohn, 2017); and (5) Kirrel, which is involved in PMC fusion (Ettensohn and Dey, 2017) (Fig. 8A).…”

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis

“…Remarkably, we found that Alx1 targets also include important, PMC-specific signaling receptors that play crucial roles in PMC guidance, migration and patterning. For example, we found that Alx1 provides direct transcriptional inputs into a VEGF receptor (Sp-Vefgr-Ig10) (Adomako-Ankomah and Ettensohn, 2013;Duloquin et al, 2007), an FGF receptor (Sp-Fgfr2_1) (Lapraz et al, 2006) and a TGFβ receptor (Sp-Tgfbr2) (Sun and Ettensohn, 2017). Taken together, direct Alx1 targets define a genetic subcircuit that impinges on almost all aspects of PMC behavior, including migration, fusion and biomineralization.…”

From genome to anatomy: The architecture and evolution of the skeletogenic gene regulatory network of sea urchins and other echinoderms

“…PMCs also express a suite of matrix metalloprotease genes, arranged in tandem on a single chromosome, which encode the metalloprotease activities required for spiculogenesis in vivo and in vitro (Ingersoll & Wilt, 1998;Roe, Park, Strittmatter, & Lennarz, 1989). Last, a PMC-specific TgfbrtII receptor was recently shown to be required for biomineral deposition (Sun & Ettensohn, 2017).…”

“…The possible regulation of PMC effector gene expression by these pathways has not been explored in detail, and it should be noted that both pathways (and even VEGF signaling) could modulate skeletal growth by transcription-independent mechanisms. A PMC-specific, Type II TGF-β receptor, encoded by tgfbrtII (a target of both Ets1 and Alx1), is required for biomineral deposition and likely acts by binding TGF-β sensu stricto (Sun and Ettensohn, 2016). Piacentino et al, (2015) also found that TGF-β signaling regulates skeletal development and showed that the formation of anterior skeletal elements is particularly sensitive to signaling through this pathway.…”