Male germ cell-associated kinase is required for axoneme formation during ciliogenesis in zebrafish photoreceptors

ABSTRACT Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.


Fig. S1. Amino acid comparison between zebrafish, mouse, and human MAK.
Amino acid sequences of zebrafish, mouse, and human MAK proteins are aligned using Clustal Omega.Grey shading indicates sequence differences.The kinase domain is indicated by an underline.The bottom table shows the percentage of amino acid identity in the full-length protein, the kinase domain, and the C-terminal domain.
Amino acid sequence identity percentage

Figure S1
Full  ) is used to draw the outline of the total retinal area.Next, using the "threshold" command of Image J (Fiji), we converted zpr1-positive and -negative areas to a binary scale, 1 and 0, respectively.The number of pixels corresponding to 1 or 0 within the neural retina was determined.Using the "measurement" command of Image J (Fiji), the percentage of zpr1-positive areas relative to the total retinal area was calculated as the percentage of the number of 1 pixels relative to the number of 0+1 pixels.Means and standard deviations were calculated from data obtained for 2-5 sections of more than two embryos.

Fig. S2 .
Fig. S2.Measurement of ONL thickness, rod cell number, and the fraction of zpr1-positive area with regard to total retinal area.(A) Images with retinas were straightened with Image J (Fiji).Straightened images were divided into 10 equal areas (A) with 9 deciles (D) between adjacent areas.The thickness of the ONL was measured at the deciles and cell numbers were counted in each area.(B) Cryosections labeled with zpr1 antibody were scanned under a laser-scanning microscope (LSM510; Carl Zeiss, FV3000; Olympus).First, a one-section image containing the central retina per eye was selected from each individual sample.Using the "split channel" command of Image J (Fiji), confocal scanning RGB color images were split into either R, G or B channel.Only the zpr1 channel (green channel in this figure) is used to draw the outline of the total retinal area.Next, using the "threshold" command of Image J (Fiji), we converted zpr1-positive and -negative areas to a binary scale, 1 and 0, respectively.The number of pixels corresponding to 1 or 0 within the neural retina was determined.Using the "measurement" command of Image J (Fiji), the percentage of zpr1-positive areas relative to the total retinal area was calculated as the percentage of the number of 1 pixels relative to the number of 0+1 pixels.Means and standard deviations were calculated from data obtained for 2-5 sections of more than two embryos.

Fig. S5 .
Fig. S5.mak mRNA expression is reduced in pday mutants and MAK protein is localized in ciliary axonemes.(A) In situ hybridization of zebrafish embryos at 72 hpf with mak RNA probe.Upper panels indicate in situ hybridization with sense (left) and anti-sense RNA probes (right).Bottom panels indicate in situ hybridization of wild-type and pday mutant embryos with mak anti-sense probe.In pday mutant embryos, mak mRNA expression is reduced.Scale bars: 1 mm.(B) Projection view of 3D confocal image of wild-type transgenic Tg[crx: eGFP-MAK] ONL with anti-GFP and anti-acetylated a-tubulin antibodies.Nuclei were counterstained with Hoechst 33342.Left panel indicates a radially sectioned projection view of the retina which is shown in the right panel of Fig. 5C.The eGFP-MAK signals overlap with anti-acetylated a-tubulin signals.Right panel indicates tangentially sectioned projection view, which was digitally produced by rotating the radially sectioned view using Imaris software (Bitplane).Scale bars: 1 µm.