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. 2022 May 11;135(9):jcs259209. doi: 10.1242/jcs.259209

Fig. 7.

Fig. 7.

Suppression of rmnd5a function interferes with neural patterning. (A–C) Embryos injected with rmnd5a-mo were used for in situ hybridization with (A) sox2 (pan-neural marker), (B) shh (axial mesoderm and SHH target gene in the floor plate) and (C) nkx2.1 (marker of the ventral forebrain) at the indicated NF stages (st.; st. 14 in Aa, Ba; st. 15 in Bb; st. 17 in Ab, Bc, Bc′; st. 20 in Bd; st. 34 in Ac–Ac″, Be–Be″″ and C). Injected sides are shown on the right of the images (red color) and in Ac″, Ae″, Ae″″ and Ca‴, non-injected sides are shown on the left of the images and in Ac, Ae and Ca. Images show anterior views (Aa, Ab, Ac′, Ba-Bc, Be′, Be‴, Cb), lateral views (Ac, Ac″, Be, Be″, Be″″, Ca, Ca‴) or superior views (Bd). Expression of sox2 at NF stage 14 is shown in image Aa (anterior view; n=29/31); the yellow arrow indicates less patterned sox2 expression. The white arrows in images Ab, Bb and Bc indicate delayed development of the neural tube and smaller hemisphere. Yellow dashed lines in images Ab, Bb and Bd highlight sickle-shaped curvature towards the injected side. Green dashed line in image Ab indicates less patterned sox2 expression of the injected side within the presumptive brain region. Red arrow in image Ab indicates alleviated formation of the eye vesicle. Yellow arrow in image Ac′ marks the shifted olfactory anlage, and the oval indicates the remnant of the larval eye (visible in Ac–c″). (B) Suppression of rmnd5a function results in almost normal shh expression domains. Yellow arrows indicate the prechordal plate; image Bc′ shows an inner view of an opened larva at NF stage 17. Yellow dashed line in image Bd indicates bending of the larva. Reduction of forebrain and eye development can be seen upon Rmnd5a-knockdown (yellow dashed oval) compared to the non-injected side (green dashed oval) in image Be′. Green arrow in Be‴ shows primary expression of shh in the notochord; yellow arrow in Be‴ and Be″″ shows secondary domain of shh expression in the floor plate of the neural tube. (C) Expression of nkx2.1 in NF stage 34 embryos (images a,a′,a‴) and 20 µm vibratome sections (Ca1–a9, positions as indicated in image Ca; frontal plane shown in image Ca″). In image Ca″, the green arrow indicates the prospective lens on the non-injected side, the red arrow indicates a reduction of nkx2.1 expression on the injected side, the green oval indicates the prospective eye on the non-injected side, and the yellow oval indicates the prospective eye on the injected side. Asterisk indicates frontal section of the anterior head shown in Ca″ and Ca6. Ca and Ca‴ show lateral views of the non-injected side and injected side of the embryo, respectively. Scale bars: 300 μm. (D) Model of the primary cilium and a proposed function of the cilia-localized GID complex in the regulation of SHH protein homeostasis in the cilium. Left: primary cilium with a functional cilia-localized GID complex and functional SHH signaling response. Protein homeostasis of SHH components in the cilium is maintained (green). Right: primary cilium with aberrant protein homeostasis, reduction of SHH signaling components – namely GLI2 and PTCH1 – in the cilium and dysfunctional SHH signaling response without a functional GID complex (red).