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

REBIRTH Deutsche Forschungsgemeinschaft (German Research Foundation)

             nephrostome development
             motile cilium assembly

Xla Wt + fam183b MO (Fig.3.A.c)
Xla Wt + fam183b MO (Fig.3.A.d)
Xla Wt + fam183b MO (Fig.3.A.d)
Xla Wt + fam183b MO (Fig.3.B)
Xla Wt + fam183b MO (Fig.3.B)
Xla Wt + foxj1 (Fig.1.F)

	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.

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

Abdelkhalek, The mouse homeobox gene Not is required for caudal notochord development and affected by the truncate mutation. 2004, Pubmed , Xenbase
Afzelius, Male and female infertility problems in the immotile-cilia syndrome. 1983, Pubmed
Alten, Differential regulation of node formation, nodal ciliogenesis and cilia positioning by Noto and Foxj1. 2012, Pubmed
Banizs, Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus. 2005, Pubmed
Barriga, Animal models for studying neural crest development: is the mouse different? 2015, Pubmed , Xenbase
Beckers, The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left right patterning. 2007, Pubmed , Xenbase
Belo, Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. 1997, Pubmed , Xenbase
Berbari, The primary cilium as a complex signaling center. 2009, Pubmed
Blum, Morpholinos: Antisense and Sensibility. 2015, Pubmed , Xenbase
Blum, Symmetry breakage in the vertebrate embryo: when does it happen and how does it work? 2014, Pubmed , Xenbase
Boldt, An organelle-specific protein landscape identifies novel diseases and molecular mechanisms. 2016, Pubmed
Boon, MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia. 2014, Pubmed , Xenbase
Brody, Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. 2000, Pubmed
Chen, Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry. 1998, Pubmed
Choksi, Systematic discovery of novel ciliary genes through functional genomics in the zebrafish. 2014, Pubmed
Del Viso, Congenital Heart Disease Genetics Uncovers Context-Dependent Organization and Function of Nucleoporins at Cilia. 2016, Pubmed , Xenbase
de Vries, Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. 2000, Pubmed
Didon, RFX3 modulation of FOXJ1 regulation of cilia genes in the human airway epithelium. 2013, Pubmed
El-Brolosy, Genetic compensation: A phenomenon in search of mechanisms. 2017, Pubmed
Gerdes, The vertebrate primary cilium in development, homeostasis, and disease. 2009, Pubmed
Getwan, Toolbox in a tadpole: Xenopus for kidney research. 2017, Pubmed , Xenbase
Gloeckner, A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. 2007, Pubmed
Gomperts, Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells. 2004, Pubmed
Hall, Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes. 2013, Pubmed
Hirokawa, Cilia, KIF3 molecular motor and nodal flow. 2012, Pubmed
Jacquet, FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain. 2009, Pubmed
Jain, Temporal relationship between primary and motile ciliogenesis in airway epithelial cells. 2010, Pubmed
Kawai, DNA book. 2003, Pubmed
Kee, A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia. 2012, Pubmed
Lee, Riding the wave of ependymal cilia: genetic susceptibility to hydrocephalus in primary ciliary dyskinesia. 2013, Pubmed
Lyons, The reproductive significance of human Fallopian tube cilia. 2006, Pubmed
Mohr, High-throughput yeast two-hybrid screening of complex cDNA libraries. 2012, Pubmed
Moorman, Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol. 2001, Pubmed
Newton, Forkhead transcription factor Fd3F cooperates with Rfx to regulate a gene expression program for mechanosensory cilia specialization. 2012, Pubmed
Pennekamp, Situs inversus and ciliary abnormalities: 20 years later, what is the connection? 2015, Pubmed
Praveen, Unique among ciliopathies: primary ciliary dyskinesia, a motile cilia disorder. 2015, Pubmed
Rodríguez, High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. 2000, Pubmed
Rossi, Genetic compensation induced by deleterious mutations but not gene knockdowns. 2015, Pubmed
Schweickert, Cilia-driven leftward flow determines laterality in Xenopus. 2007, Pubmed , Xenbase
Spassky, Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis. 2005, Pubmed
Stannard, Ciliary function and the role of cilia in clearance. 2006, Pubmed
Stauber, Identification of FOXJ1 effectors during ciliogenesis in the foetal respiratory epithelium and embryonic left-right organiser of the mouse. 2017, Pubmed
Stubbs, The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. 2008, Pubmed , Xenbase
Takeda, Structure and function of vertebrate cilia, towards a new taxonomy. 2012, Pubmed
Talbot, A homeobox gene essential for zebrafish notochord development. 1995, Pubmed , Xenbase
Vick, Flow on the right side of the gastrocoel roof plate is dispensable for symmetry breakage in the frog Xenopus laevis. 2009, Pubmed , Xenbase
Vij, Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program. 2012, Pubmed
Vizcaíno, 2016 update of the PRIDE database and its related tools. 2016, Pubmed
Walentek, What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia. 2017, Pubmed , Xenbase
Yu, Foxj1 transcription factors are master regulators of the motile ciliogenic program. 2008, Pubmed