The serologically defined colon cancer antigen-3 (SDCCAG3) is involved in the regulation of ciliogenesis

A primary cilium is present on most eukaryotic cells and represents a specialized organelle dedicated to signal transduction and mechanosensing. Defects in cilia function are the cause for several human diseases called ciliopathies. The serologically defined colon cancer antigen-3 (SDCCAG3) is a recently described novel endosomal protein mainly localized at early and recycling endosomes and interacting with several components of membrane trafficking pathways. Here we describe localization of SDCCAG3 to the basal body of primary cilia. Furthermore, we demonstrate that decreased expression levels of SDCCAG3 correlate with decreased ciliary length and a reduced percentage of ciliated cells. We show that SDCCAG3 interacts with the intraflagellar transport protein 88 (IFT88), a crucial component of ciliogenesis and intraciliary transport. Mapping experiments revealed that the N-terminus of SDCCAG3 mediates this interaction by binding to a region within IFT88 comprising several tetratricopeptide (TRP) repeats. Finally, we demonstrate that SDCCAG3 is important for ciliary localization of the membrane protein Polycystin-2, a protein playing an important role in the formation of polycystic kidney disease, but not for Rab8 another ciliary protein. Together these data suggest a novel role for SDCCAG3 in ciliogenesis and in localization of cargo to primary cilia

Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia

A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure

Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse

Urothelial Defects from Targeted Inactivation of Exocyst Sec10 in Mice Cause Ureteropelvic Junction Obstructions.

Most cases of congenital obstructive nephropathy are the result of ureteropelvic junction obstructions, and despite their high prevalence, we have a poor understanding of their etiology and scarcity of genetic models. The eight-protein exocyst complex regulates polarized exocytosis of intracellular vesicles in a large variety of cell types. Here we report generation of a conditional knockout mouse for Sec10, a central component of the exocyst, which is the first conditional allele for any exocyst gene. Inactivation of Sec10 in ureteric bud-derived cells using Ksp1.3-Cre mice resulted in severe bilateral hydronephrosis and complete anuria in newborns, with death occurring 6-14 hours after birth. Sec10 FL/FL;Ksp-Cre embryos developed ureteropelvic junction obstructions between E17.5 and E18.5 as a result of degeneration of the urothelium and subsequent overgrowth by surrounding mesenchymal cells. The urothelial cell layer that lines the urinary tract must maintain a hydrophobic luminal barrier again urine while remaining highly stretchable. This barrier is largely established by production of uroplakin proteins that are transported to the apical surface to establish large plaques. By E16.5, Sec10 FL/FL;Ksp-Cre ureter and pelvic urothelium showed decreased uroplakin-3 protein at the luminal surface, and complete absence of uroplakin-3 by E17.5. Affected urothelium at the UPJ showed irregular barriers that exposed the smooth muscle layer to urine, suggesting this may trigger the surrounding mesenchymal cells to overgrow the lumen. Findings from this novel mouse model show Sec10 is critical for the development of the urothelium in ureters, and provides experimental evidence that failure of this urothelial barrier may contribute to human congenital urinary tract obstructions

Sec10 is necessary for maintenance of the uroplakin barrier in the developing ureter urothelium.

<p>Embryos were collected after timed-matings to evaluate ureters at the UPJ prior to formation of the obstruction, which occurs between E17.5 –E18.5. (A, B) Immunostaining for E-cadherin (green) and SMA (red) at the UPJ in <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> and control E16.5 ureters confirmed initial patency of the mutant UPJs. (C, D) Immunostaining for uroplakin-3 (red) and vimentin to mark the mesenchymal cells (green) revealed that at E16.5, <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> ureters already demonstrated patchy uroplakin-3 localization. Areas of urothelium with noticeably absent uroplakin-3 are marked with arrows (D), while in control ureters, uroplakin-3 was distributed all around the luminal surface (C). (E, F) By E17.5, staining for E-cadherin (green) and SMA (red) revealed that the urothelial layer was visibly abnormal in morphology and consisted of mostly a single layer. (G, H) At E17.5, uroplakin-3 was strongly detectable in all control ureters, but completely absent in <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> ureters around the UPJ. (I, J) To measure proliferation in the smooth muscle layers of developing ureters at the UPJ region, we immunostained for Ki67, which is expressed only in mitotic cells. (K) Proliferation rates were calculated from counting all Ki67⁺;SMA⁺ cells and Ki67^-;SMA⁺ smooth muscle cells in <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> and control ureters at E16.5 and E17.5. Proliferation rates were compared between mutant and control ureters by student t-tests, with means ± SD and n’s for each group shown. In all images, nuclei were counterstained with DAPI.</p

Hydronephrosis in <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> kidneys is due to physical obstructions in ureters at the UPJ, resulting in anuria and heart failure.

<p>(A, B) Urinary tracts, including both kidneys, intact ureters, and bladder were removed from mutant <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> mice and littermate controls (<i>Sec10</i>^<i>FL/FL</i> and <i>Sec10</i>^<i>Fl/+</i>;<i>Ksp-Cre</i>) at E18.5 and P0. Blue dye was injected into the renal pelvis, and in control kidneys the dye migrated down the ureters and accumulated in the bladder as expected (representative E18.5 sample shown in A). In every <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> kidney with hydronephrosis tested at E18.5 and P0, the dye stopped at the UPJ (arrow, representative E18.5 sample shown in B). (C) Occasionally in the dissected mutant newborn kidneys with hydronephrosis, a microscopic examination revealed a deposit of white debris within the ureter above the UPJ region, also suggesting a physical blockage of the ureter lumen (arrow, C). (D, E) In one of the few newborn <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> kidneys that did not show hydronephrosis, dye injections traveled to the bladder, but revealed a visibly abnormal ureter lumen with rough irregular edges (E) not observed in controls (D). (F) Aspirations from bladders of newborn pups confirmed that no urine was present in the bladders of <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> pups with bilateral hydronephrosis, compared with a normal distribution found in littermate controls (shown are means ± SD). (G) All newborn <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> pups with bilateral obstructions and hydronephrosis died 6–14 hours after birth, with necropsies revealing heart wall distension and cardiac hemorrhaging. Shown are two representative <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> hearts (right) dissected immediately after death (12-hours post-birth), compared to an age-matched littermate control heart (left).</p

Generation of the <i>floxed-Sec10</i> mouse strain and Sec10 conditional knockout mice.

<p>(A, B) Shown is a schematic of the murine <i>Sec10</i> gene before (A) and after (B) recombination with the <i>Sec10</i> conditional targeting vector. Digestion with <i>XbaI</i> restriction enzyme yields a 12.5 kb DNA fragment containing exons 5–12 in wild type animals, but the targeting vector introduced new <i>XbaI</i> sites to yield smaller fragments. (C) Southern blotting of genomic DNA digested with <i>XbaI</i> from wild type C57Bl/6J mice, the injected Sec10 ES clone, and chimeric pups demonstrated homologous recombination with the targeting vector. (D) The final <i>floxed-Sec10</i> strain was created by mating mice with germline transmission of the <i>Sec10</i> targeting vector with <i>FLPe</i> mice to remove the large Neomycin cassette. (E) Upon exposure to Cre recombinase, exons 7–10 were deleted. (F) <i>Ksp-Cre</i> mice were crossed with a <i>tdTomato</i> reporter mouse strain, and Cre activity was confirmed to be specific to epithelium of ureters, Wolffian ducts (WD), and the collecting system of the kidney as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129346#pone.0129346.ref035" target="_blank">35</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129346#pone.0129346.ref037" target="_blank">37</a>]. Shown is the genitourinary system of E13.5 <i>Ksp-Cre/+;tdTomato/+</i> embryos, with red fluorescence confirming strong activation of Cre recombinase even at this early stage. (G) PCRs from genomic DNA of various tissues were able to genotype <i>floxed-Sec10</i> alleles (upper gel), confirm <i>Cre</i> transgenes in our strains (middle gel), and detect deletion of exons 7–10 specifically in <i>Cre</i>-expressing tissues (lower gel). Positions of primers used in F are shown in D and E. (H) In <i>Sec10</i>^<i>FL/FL</i>;<i>Ksp-Cre</i> mice, Western blotting showed reduced Sec10 protein in whole kidney lysates and isolated ureters, compared with <i>Sec10</i>^<i>FL/FL</i> littermate controls. (I, J) Western band intensities were measured and Sec10 protein levels were normalized against β-actin and compared via student t-tests (n = 5 for each group, shown are means ± SD).</p