Transforming growth factor‐β signal regulates gut bending in the sea urchin embryo

Abstract

During gastrulation, one of the most important morphogenetic events in sea urchin embryogenesis, the gut bends toward the ventral side to form an open mouth. Although the involvement of transforming growth factor-β (TGF-β) signals in the cell-fate specification of the ectoderm and endoderm along the dorsal-ventral axis has been well reported, it remains unclear what controls the morphogenetic behavior of gut bending. Here, using two sea urchin species, Hemicentrotus pulcherrimus and Temnopleurus reevesii, we show that TGF-β signals are required for gut bending toward the ventral side. To search for the common morphogenetic cue in these two species, we initially confirmed the expression patterns of the dorsal-ventral regulatory TGF-β members, nodal, lefty, bmp2/4, and chordin, in T. reevesii because these factors are appropriate candidates to investigate the cue that starts gut bending, although genetic information about the body axes is entirely lacking in this species. Based on their expression patterns and a functional analysis of Nodal, the dorsal-ventral axis formation of T. reevesii is likely regulated by these TGF-β members, as in other sea urchins. When the Alk4/5/7 signal was inhibited by its specific inhibitor, SB431542, before the late gastrula stage of T. reevesii, the gut was extended straight toward the anterior tip region, although the ectodermal dorsal-ventral polarity was normal. By contrast, H. pulcherrimus gut bending was sensitive to SB431542 until the prism stage. These data clearly indicate that gut bending is commonly dependent on a TGF-β signal in sea urchins, but the timing of the response varies in different species.

Direct TGF‐ß signaling via alk4/5/7 pathway is involved in gut bending in sea urchin embryos

“…The morphant morphology observed in this treatment was almost identical to alk4/5/7 morphants as well as SB431542‐treated embryos in P. lividus 16 . Therefore, we conclude that, in H. pulcherrimus , Alk4/5/7‐MO is effective in blocking TGF‐ß signals, such as SB431542, which we previously used for blocking the signals in a whole embryo 4 …”

“…Because alk4/5/7 is expressed everywhere (Figure 2) and SB431542 treatment blocks TGF‐ß signaling in all cells, we could not conclude that it is essential for the elongating gut to directly receive TGF‐ß signaling to bend itself toward the ventral side during gastrulation, which we have reported in a previous paper 4 . To investigate this, we generated chimera embryos in which TGF‐ß signaling was inhibited only in the vegetal half by combining normal animal halves and Alk4/5/7‐MO‐injected vegetal halves and measured the angle of gut bending (Figure 3A, G).…”

“…In our previous study, we reported that Smad2/3 are phosphorylated, that is, activated, and enter the nuclei on the ventral side of the tip of the gut during its bending 4 . Although we could not conclude that the activation of Smad2/3 is directly regulated through gut Alk4/5/7 with SB431542 treatment, it is suggested that direct TGF‐ß reception in the gut activates downstream Smad2/3 based on the chimeric experiments shown in this study.…”

“…To investigate this, we generated chimera embryos in which TGF‐ß signaling was inhibited only in the vegetal half by combining normal animal halves and Alk4/5/7‐MO‐injected vegetal halves and measured the angle of gut bending (Figure 3A, G). The method for measuring gut bending has been described previously 4 . Alk4/5/7‐MO‐ or zebrafish‐UROD‐MO (negative control)‐injected vegetal halves and tagRFP mRNA‐injected animal halves were combined.…”

“…We previously showed that the transforming growth factor‐beta (TGF‐ß) signaling pathway, which includes the key regulatory factors for the specification of the dorsal‐ventral axis, is necessary for correct gut bending by blocking the pathway with its receptor inhibitor SB431542 (Figure 1A‐D). 4 …”