Figure 4. MHC protein detected by immunostaining after perturbation of putative myogenic regulators.

MHC protein localization was tested by immunostaining in fluo-control MO-injected pluteus larvae (72 hr) (A) and in embryos of the same age injected with MOs against FoxY (B), FoxC (C), FoxF (D), FoxL1 (E), MyoD2 (F), Six1/2N (G), and Tbx6 (H) (for MO injection controls see also Materials and methods and Figure 2—figure supplement 2). The ciliary band and gut internal cilia were stained by immunohistochemistry with an anti-acetylated tubulin antibody. Each picture is a stack of merged confocal Z sections with MHC in red and acetylated tubulin in green. Nuclei were labeled blue with DAPI. All embryos are seen in lateral view with the oral side on the right. White arrows indicate the position of cardiac sphincters. White lines indicate muscle fibers (mf). Below each panel, statistics of muscle fiber phenotype observed are reported as normal (6–7 mf), mild (4–5 mf), or strong (0–2 mf). A co-expression analysis of Six1/2 and FoxC is reported in Figure 4—figure supplement 1. Analysis of the temporal expression profile of two distinct Six1/2 isoforms and visualization of pigmentation after perturbing Six1/2N isoform are reported in Figure 4—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.07343.013

Figure 4—figure supplement 1. Control experiments for MOs.

Circumesophagael muscles were tested by phalloidin staining in fluo-control MO-injected pluteus larvae (72 hr) (A, A′) and in embryos of the same age injected with MOs against FoxC (B, B′), FoxF (C, C′), and FoxL1 (D, D′) at different concentrations. Each picture is a stack of merged confocal Z sections. Phalloidin is seen in green and nuclei are labeled blue with Hoechst. All embryos are seen in lateral view with the oral side on the left. Below each panel, statistics of muscle fiber phenotype observed are reported.

DOI: http://dx.doi.org/10.7554/eLife.07343.014

Figure 4—figure supplement 2. Co-expression analysis of Six1/2 and FoxC by double FISH.

Relative spatial expression domains of Six1/2 and FoxC at the mid gastrula stage (42 hr). Image is a stack of merged confocal Z sections in all channels. Inset shows representative single confocal section of the tip of the archenteron. Color code of channel association to each gene is shown in each panel. Nuclei are stained blue with DAPI. Embryo is seen in a frontal view.

DOI: http://dx.doi.org/10.7554/eLife.07343.015

Figure 4—figure supplement 3. The two Six1/2 isoforms.

(A) Upstream sequence of the of the Six1/2 gene. Two ATGs are shown in red. The upstream one, highlighted in bold, corresponds to the first ATG in the long isoform (Six1/2N) that is probably generated by an alternative transcription start. The downstream ATG indeed corresponds to the first ATG of the short isoform in which transcription starts a few nucleotides upstream of it (Andrew Ransick, personal communication). Highlighted in different colors show the regions where the different set of qPCR primers were designed: the ones used to amplify the upstream sequence belonging to Six1/2N isoform only, are in yellow, while the ones used to amplify part of the homeobox domain (in bold), common to both isoforms, are highlighted in olive green. The target sequence used to design the MO against the long isoform Six1/2N is highlighted in cyan. (B) Temporal expression profiles of Six1/2 distinct isoforms during sea urchin embryogenesis. Graph shows the number of transcripts per embryo during embryogenesis revealed by qPCR. Six1/2HD represents the sum of the number of transcripts of the two isoforms, while Six1/2N shows only the number of transcripts for Six1/2N. The columns represent average of various measurements, and the error bars are standard deviations. (C, D) Bright-field images were taken with DIC of (C) control uninjected larva and (D) Six1/2N morphant (72 hr) for visualizing the effect on pigmentation.

DOI: http://dx.doi.org/10.7554/eLife.07343.016

Figure 4—figure supplement 4. Control experiments for MOs.

Circumesophagael muscles were tested by phalloidin staining in fluo-control MO-injected pluteus larvae (72 hr) (A, A′ in Figure 4—figure supplement 1) and in embryos of the same age injected with MOs against Six1/2 (A, A′) and Tbx6 (B, B′) at a concentration of 100 μM. Each picture is a stack of merged confocal Z sections. Phalloidin is seen in green and nuclei are labeled blue with Hoechst. All embryos are seen in lateral view with the oral side on the left. Below each panel, statistics of muscle fiber phenotype observed are reported.

DOI: http://dx.doi.org/10.7554/eLife.07343.017