Volume 25, Issue 24, Pages 3267-3273 (December 2015) Gmnc Is a Master Regulator of the Multiciliated Cell Differentiation Program  Feng Zhou, Vijay Narasimhan, Mohammad Shboul, Yan Ling Chong, Bruno Reversade, Sudipto Roy  Current Biology  Volume 25, Issue 24, Pages 3267-3273 (December 2015) DOI: 10.1016/j.cub.2015.10.062 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Gmnc Is Required for MCC Formation in the Kidney Tubules of the Zebrafish Embryo (A and B) The kidney tubules of 48 hr post-fertilization (hpf) wild-type embryos are populated with clusters of MCCs (A). Boxes delineate the regions imaged at a higher magnification in (A′) and (A″). Multiple basal bodies are indicated by brackets (A′ and A″). In contrast, 100% (n = 47, from two independent experiments) of the gmnc splice-blocking MO-injected embryos were devoid of MCCs (B and B′). Examples of single basal bodies are marked by asterisks (B′ and B″). Acetylated-tubulin (Ac; green), DAPI (DNA; blue; rendered in black and white in A″ and B″), and γ-tubulin (marks basal bodies; red; for A′, A″, B′, and B″ only). (C and D) In contrast to wild-type embryos (C), kidney cysts (black arrow) form in 3-day-old gmnc morphants (93%, n = 40) (D). (E and F) The F1 generation from intercrossed F0 gmnc CRISPR mutants was a mixture of wild-type animals (E) and animals carrying the gmnc deletion (F). The kidney tubules of the latter did not harbor MCCs at 36 hpf. (G and H) Approximately one-quarter (two different pairs of F1 heterozygotes were analyzed; pair 1: 26%, n = 31; pair 2: 27%, n = 44) of the F2 generation from intercrossed heterozygous F1 parents lacked MCCs in the kidney tubules at 36 hpf (H). The other three-quarters of the embryos appeared wild-type (G). Scale bars, 10 μm (C and D); 5 μm (all others). See also Figure S1. Current Biology 2015 25, 3267-3273DOI: (10.1016/j.cub.2015.10.062) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Gmnc Functions Downstream of Notch Activation and Upstream of mcidas (A–C) At 48 hpf, the kidney tubules of mib mutant embryos display supernumerary MCCs, which appear as a contiguous bundle (B), in contrast to wild-type embryos, whose kidney tubules harbor distinct clusters of MCCs (A). A majority of the mib embryos injected with the gmnc MO were devoid of MCCs (75%; n = 10) (C), replicating the phenotype of the gmnc morphants (Figure 1B). Acetylated-tubulin, green; DAPI, blue. (D and E) Similar to the mib mutants (B), jagged2a morphants at 48 hpf harbor supernumerary MCCs lining the kidney tubules (D). Embryos co-injected with jagged2a and gmnc MOs exhibited the gmnc morphant phenotype and lacked MCCs (100%; n = 10) (E). (F–H) At 24 hpf, the zebrafish mcidas gene is expressed in the kidney tubules in a spotted pattern (asterisks) that corresponds to the MCC precursors (F). mcidas expression domain is expanded in mib mutant embryos (G) and is completely lost when gmnc is knocked down in the mib background (100%; n = 20) (H). (I and J) The Tg(foxj1b::GFP) reporter fish strain (T2BGSZ10) reveals that, at 24 hpf, foxj1b is expressed in the MCC precursor cells of the kidney ducts (I). foxj1b::GFP expression was lost in the gmnc MO-injected T2BGSZ10 embryos (100%; n = 16) (J). GFP (foxj1b), green; DAPI, blue. Scale bars, 50 μm (F–H); 10 μm (all others). See also Figure S2. Current Biology 2015 25, 3267-3273DOI: (10.1016/j.cub.2015.10.062) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 gmnc Is Expressed in the Kidney Tubules and Is Modulated by Notch Signaling and Foxj1a Activation (A) End-point PCRs on cDNA extracted from 1.5, 5, 14, 18, and 24 hpf embryos, respectively, revealed that whereas the maternal contribution of the foxj1a transcript is minimal (1.5 hr), the gmnc transcript is maternally deposited. actin-b1 (actb1) was used as the loading control. (B and C) Representative staining from in situ hybridization using a probe against gmnc showed that the gene is expressed in the developing nasal placodes (arrows) at 18 hpf (B) and is also expressed in a spotted pattern in the mid-section of the kidney ducts (asterisks) at 24 hpf (C). Scale bars, 50 μm. (D) qPCRs were performed on uninjected or jagged2a MO-injected embryos at 24 hpf. Similar to the ciliogenic genes like foxj1a, foxj1b, and mcidas (mci), whose levels increased in the N-deficient background, gmnc transcript levels also increased moderately (by ∼3-fold). Expression levels in the uninjected condition were arbitrarily assigned a value of 1. rplpo was used as an internal (loading) control. Error bars represent the SEM from two independent experiments. (E) qPCRs were performed on non-heat-shocked or heat-shocked Tg(hsp70::foxj1a) embryos. The heat-shock treatment induced the foxj1a transgene level by ∼15-fold, and the foxj1a overexpression induced the gmnc level by ∼10-fold. Expression levels in the non-heat-shocked condition were arbitrarily assigned a value of 1. actin-b1 (actb1) was used as an internal (loading) control. Error bars represent the SEM from three independent experiments. Current Biology 2015 25, 3267-3273DOI: (10.1016/j.cub.2015.10.062) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 gmnc Is Necessary and Sufficient for MCC Differentiation in Xenopus (A) End-point PCRs on stage-30 control and gmnc morphants showed mis-splicing of the Xenopus gmnc pre-mRNA. (B) Western-blot analysis using an antibody against human GMNC showed that endogenous Gmnc was depleted in the gmnc morphant embryos at stage 35 and that the injected human GMNC RNA translated to a large amount of the GMNC protein. Actin was used as a loading control. (C and D) The development of gmnc morphant embryos was delayed (observed in 93% of the 43 animals tested). Despite this, in situ hybridization revealed no overt defects in the mucus-secreting goblet cell differentiation (81%; n = 21) (C) or in the differentiation of scattered cells, which are involved in ionic homeostasis regulation (78%; n = 18) (D). (E and F) In contrast to the lack of differentiation defects in goblet cells and scattered cells, the differentiation of MCCs, the third main cell type present on the Xenopus embryonic skin, and marked by ccdc19 (encoding a ciliary protein), was significantly reduced in the gmnc morphants (87%; n = 52) (F) compared to control embryos (E). (G and H) Immuno-staining of acetylated-tubulin revealed that multiple cilia were markedly reduced in the gmnc morphants (94%; n = 34) (H) compared to control embryos (G). (I–K) High-magnification images of the surface ectoderm at stage 30 in control (I), gmnc morphants (J), or emrbyos co-injected with gmnc MO and 80 pg of human GMNC RNA (K) showed that overexpression of the hGMNC RNA not only rescued the MCC defects in the gmnc morphants but was sufficient to produce ectopic MCCs (79%; n = 14). See also Figure S4. Current Biology 2015 25, 3267-3273DOI: (10.1016/j.cub.2015.10.062) Copyright © 2015 Elsevier Ltd Terms and Conditions