Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin

Abstract

Most bilaterians exhibit a left-right asymmetric distribution of their internal organs. The sea urchin larva is notable in this regard since most adult structures are generated from left sided embryonic structures. The gene regulatory network governing this larval asymmetry is still a work in progress but involves several conserved signaling pathways including Nodal, and BMP. Here we provide a comprehensive analysis of Hedgehog signaling and it's contribution to left-right asymmetry. We report that Hh signaling plays a conserved role to regulate late asymmetric expression of Nodal and that this regulation occurs after Nodal breaks left-right symmetry in the mesoderm. Thus, while Hh functions to maintain late Nodal expression, the molecular asymmetry of the future coelomic pouches is locked in. Furthermore we report that cilia play a role only insofar as to transduce Hh signaling and do not have an independent effect on the asymmetry of the mesoderm. From this, we are able to construct a more complete regulatory network governing the establishment of left-right asymmetry in the sea urchin.

Keywords: Cilia; Hedgehog; Nodal; Right–left asymmetry; Sea urchin.

Figure 1

Asymmetric gene expression in Lytechinus variegatus. A–B: Nodal and Lefty expression throughout development visualized by in situ mRNA hybridization. Timepoints sampled are in hours post fertilization (12–26). The embryos at 12hpf are oriented with the oral ectoderm to the left (asterix) while all other embryos are rotated along the vertical axis 90° with the oral ectoderm facing into the image. C: Double fluorescent in situ mRNA hybridization shows Pitx2 (red) expressed in the right coelomic pouch. SoxE (green) is expressed in the left coelomic pouch, the future site of the adult rudiment.

Figure 2

Hh signaling regulates late Nodal expression. (A) Three treatments knock down Hh signaling. Patched (Ptc) expression in the coelomic pouches (arrowheads) requires Hh signaling. With three different approaches, a hedgehog morpholino, cyclopamine, or inclusion of a kinesin-II antibody in the cytoplasm, Ptc expression is eliminated. Each of these approaches were used to knock down Hh signaling in the experiments in this paper with similar results. (B) Expression of Nodal from the prism stage to pluteus stage shows normal right-sided nodal in control embryos, while this expression is absent or reduced in cyclopamine-treated embryos. (C) The knockdown is quantified using QPCR of cyclopamine and control DMSO treated embryos. Error bars represent standard error.

Figure 3

Hh signaling does not affect early asymmetric nodal in the NSM. In situ hybridization of Nodal showing it’s three expression domains, oral (asterix), gut (red arrowhead) and right-side ectoderm (white arrowhead), in control morpholino injected and Hh morpholino injected, from mesenchyme blastula stage (14hpf) to prism stage (22hpf) (A). Pitx2 expression is shown from it’s earliest expression (22hpf) through the early pluteus stage (28hpa) in control morpholino and Hh morpholino injected embryos (B). Percentages indicate the fraction of cases that had identical expression patterns. In the remaining fraction, staining was either absent or indeterminate because of background staining. In no cases did we observe left-right inverted staining patterns.

Figure 4

SoxE expression is maintained in Hh knockdown embryos. In situ hybridization for expression of the transcription factors FoxF, Pitx2, and SoxE in control, Hh knockdown, Nodal knockdown, and Hh plus Nodal knockdown embryos. Nodal signaling was inhibited from early gastrula onwards using the drug SB431542 (second column). Hh was knocked down by injecting a morpholino to Hh at 1 cell stage (third column). Both Hh and Nodal inhibition was achieved by Hh MASO injection at one cell stage followed by SB431542 treatment from early gastrula onwards (fourth column). Percentages indicate the fraction of embryos exhibiting identical staining; the remaining fractions exhibited some level of staining resembling control embryos. In no cases did we observe left-right inverted staining patterns

Figure 5

Cilia do not establish left-right asymmetry. In normal embryos at the pluteus stage robust cilia staining is seen using an antibody to acetylated tubulin (green). Injection of a monoclonal kinesin-II antibody disrupts cilia assembly resulting in short, non-motile cilia stubs. When assayed by in situ hybridization these embryos exhibit reduced Nodal staining, consistent with Hh pathway inhibition, but normal SoxE expression when compared to controls suggesting that cilia do not influence the left-right symmetry-breaking event. Percentages indicate the fraction of embryos exhibiting identical staining.

Figure 6

A regulatory network driving symmetry breaking in the sea urchin. A gene regulatory network summarizing the interactions that establish left-right asymmetry in the sea urchin. The network is informed by work from the Lepage lab, the Su lab and the Smith lab and drawn using the biotapestry software (Bessodes et al., 2012; Duboc et al., 2005; Longabaugh et al., 2009; Materna et al., 2013).