Beckers A et al. (2018), The evolutionary conserved FOXJ1 target gene Fa...

XB-ART-55341

Sci Rep 2018 Oct 02;81:14678. doi: 10.1038/s41598-018-33045-2.

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The evolutionary conserved FOXJ1 target gene Fam183b is essential for motile cilia in Xenopus but dispensable for ciliary function in mice.

Beckers A , Ott T , Schuster-Gossler K , Boldt K , Alten L , Ueffing M , Blum M , Gossler A .

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The transcription factor FOXJ1 is essential for the formation of motile cilia throughout the animal kingdom. Target genes therefore likely constitute an important part of the motile cilia program. Here, we report on the analysis of one of these targets, Fam183b, in Xenopus and mice. Fam183b encodes a protein with unknown function which is conserved from the green algae Chlamydomonas to humans. Fam183b is expressed in tissues harbouring motile cilia in both mouse and frog embryos. FAM183b protein localises to basal bodies of cilia in mIMCD3 cells and of multiciliated cells of the frog larval epidermis. In addition, FAM183b interacts with NUP93, which also localises to basal bodies. During frog embryogenesis, Fam183b was dispensable for laterality specification and brain development, but required for ciliogenesis and motility of epidermal multiciliated cells and nephrostomes, i.e. the embryonic kidney. Surprisingly, mice homozygous for a null allele did not display any defects indicative of disrupted motile ciliary function. The lack of a cilia phenotype in mouse and the limited requirements in frog contrast with high sequence conservation and the correlation of gene expression with the presence of motile cilia. This finding may be explained through compensatory mechanisms at sites where no defects were observed in our FAM183b-loss-of-function studies.

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Species referenced: Xenopus
Genes referenced: cep63 cetn4 fam183b foxj1 foxj1.2 hprt1 isyna1 mal mcc nup93 prl.2 slc22a18 tmem269 tubal3
GO keywords: epithelial cilium movement [+]
???displayArticle.morpholinos??? fam183b MO1 fam183b MO2

???displayArticle.disOnts??? ciliopathy
Phenotypes: Xla Wt + fam183b MO (Fig.3.A.b) [+]

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	Figure 1. Expression of Fam183b. (A) Phylogram of vertebrate Fam183 genes rooted on human FAM183A. Sequences used for alignment and phylogenetic analysis: human, HGNC-34347 and HGNC-34511; mouse, Q5NC57; chicken, F1P3Y5; Xen. tropicalis, XP_004914015; Xen. laevis, XP_018113781; zebrafsh, ZDBGENE-111103-1. (B) WISH of E7.75 wild type (wt) (a) and NotoGfp/Gfp (b) mutant embryos shows NOTOdependent expression of Fam183b in the LRO. (C) Analysis of Fam183b expression by RT-PCR of RNA from adult organs, as indicated. Full size gel is shown in Supplementary Figure S1. (D) SISH analysis of adult tissues, as indicated. Boxed areas in a–f outline regions shown at higher magnifcation in a’–f ’. Arrows point to sites of expression. FT: fallopian tube; CP: choroid plexus; PRL: photoreceptor layer; INL: inner nuclear layer; GCL: ganglion cell layer. (E) Expression of fam183a in Xenopus laevis. Fam183a mRNA was found at stage 20 in the foor plate (FP) and LRO (GRP; a,a’); at stage 34 in MCCs and nephrostomes (b); and at stage 45 (c) in the sub-commissural organ (SCO), the zona limitans intrathalamica (ZLI) and the foor plate of the brain (c’), in the dorsal lining of the branchial chamber (inset in c and c”), and the stomach (c”’). (F) Fam183a is a foxj1 target gene. Embryos were unilaterally injected on the lef side with a foxj1 mRNA and analysed for fam183a expression. foxj1, injected side. Scale bars: Da–f=500μm, a’–f ’=100μm.
	Figure 2. Subcellular localisation of FAM183B. (A) Co-localisation of mouse FAM183b-GFP with centrin4 in stage 33 Xenopus embryos. (B) Detection of C- and N-terminally Flag-tagged FAM183b in mIMCD3 cells by indirect immunofuorescence showing partial overlap with γ-tubulin. (C) Co-IP of tagged FAM183b and NUP93, indicating interaction. Red asterisks: co-IP; black asterisk: anti-Flag light chain detected by secondary antibody; black arrowhead: FAM183b at the expected apparent molecular weight; red arrowheads: background band detected in transfected CHO cells and IPs. Full size Western blots are shown in Supplementary Figure S2. (D) Co-localisation of endogenous NUP93 with CEP63 and γ-tubulin at centrosomes. Scale bars: Ba–d,a’–d’, Da,b,a’,b’=10μm.
	Figure 3. Functional analysis of fam183a in Xenopus laevis. (A) Embryos at the 4-cell stage were injected with fam183a-TBMO (b) or -SBMO (c), cultured to stage 33 and analysed for epidermal MCC ciliation (a–c) and ciliary beating (d). Note that cilia were reduced in length and number in MCCs of morphant specimens. Stippled boxes in (a–c) indicate the regions shown enlarged (a’–c”). Edemata (B), organ situs (C) and hydrocephalus (D) were analyzed at stage 45. Note that epidermal cilia defects and cardial edemata, were encountered in a statistically signifcant proportion of specimens, while rare LR defects and hydrocephalus were not signifcant. White arrowhead in (Bb) highlights cardial edema; arrangement of inner organs and gut coiling in (Ca,b) was illustrated by outlines and arrows, respectively; ventricular margins in (Da,b) were depicted by strippled outlines. FD: fuorescin dextran; g: gall bladder; h: heart; het., heterotaxia; i: intestine; mal: malformed; red. reduced; Sa: situs ambiguus; Si: situs inversus; Ss: situs solitus; +: dead.
	Figure 4. Generation and validation of a Fam183b-null allele. (A) Schematic drawing depicting the structure of the wild type locus, targeting vector and mutated allele. (B) RT-PCR with primers binding in Fam183b exon 1 and 4, Fam183b exon 2 and 4, and Hprt exon 7 and 9 on RNA from adult tissues. Full size gel is shown in Supplementary Figure S3. (C) Northern blot of total and polyA+ RNA from wt and Fam183b-mutant testes. Full size Northern blots are shown in Supplementary Figure S4. (D) WISH of E7.75 wt (a,b) and Fam183bΔex1/Δex1 (c,d) embryos. (b,d) higher magnifcation of ventral views of the LRO. (E) Western blot analysis of testis lysates from wt and Fam183bΔex1/Δex1 testes. Te full size Western blot is shown in Supplementary Figure S5.
	Figure 5. Histological analysis of Fam183b-mutant tissues. (A) Schematic drawing showing the planes of sections shown in (Ca–d). (B) Scheme depicting planes of sections shown in (Cg–j). (Ca–d) Representative sections of wild type (a,c) and Fam183bΔex1 mutant brains (b,d) at the two horizontal levels indicated in (A). Boxed areas indicate the regions shown at higher magnifcation in a’,b’,c’,c’’,d’ and d’’. (Ce,f) Representative sections of wild type (e) and Fam183bΔex1 mutant (f) lungs. Boxed areas indicate the regions shown at higher magnifcation in e’,f ’. (Cg–j) Representative sections of wild type (g,i) and Fam183bΔex1 mutant (h,j) nasal cavities at the two horizontal levels indicated in (B). Boxed areas indicate the regions shown at higher magnifcation in g’ and h’. Scale bars: a,b,c,d: 1mm; a’, b’,c’,c’’,d’,d’’: 500μm; e,f: 500μm; e’,f ’: 200μm; g–j: 500μm; g’,h’: 100μm.
	fam183b (family with sequence similarity 183, member B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.

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Abdelkhalek, The mouse homeobox gene Not is required for caudal notochord development and affected by the truncate mutation. 2004, Pubmed, Xenbase