The structural and functional organization of the adult mouse small intestinal epithelium lends itself to studying both the regulation and integration of cellular proliferation, differentiation, and death programs. The epithelium contains four principal cell types: absorptive enterocytes (comprising Ͼ80% of the total population), enteroendocrine cells, mucus-producing goblet cells, and Paneth cells. All four lineages are derived from a multipotent stem cell that is functionally anchored near the base of each of the small intestine's 1.1 million crypts of Lieberkü hn (1-4). Cell division is confined to these crypts (5). Enterocytes, enteroendocrine, and goblet cells migrate out of the crypt and up an adjacent villus. Migration is highly ordered and associated with terminal differentiation. Cell death occurs near the villus tip where cells are exfoliated into the lumen (6, 7). Proliferation, differentiation, and death take place in a spatially well-organized continuum that extends from the crypt to the apex of a villus. This sequence is completed rapidly (2-5 days in the case of enterocytes, enteroendocrine, and goblet cells; Refs. 1 and 8 -10) and is recapitulated throughout the lifespan of the mouse.The Paneth cell lineage differs from the others in a number of notable ways. It is the only lineage that executes its terminal differentiation program during a downward migration from the stem cell zone to the crypt base (11). It is the longest lived lineage, and the only one that exists entirely within the proliferative compartment. Each crypt contains 30 -50 mature Paneth cells that survive for 18 -23 days before degenerating and undergoing phagocytosis by their neighbors (11-13). Paneth cell age correlates with position in the crypt; the most mature cells are located at or near the crypt base (2). The size of the Paneth cell's apical secretory granules also correlates with age; larger granules are produced by older cells (2, 11).The function of the Paneth cell has not yet been clearly defined. Residency at the crypt base places this lineage in a position to release products from its apical granules that could affect establishment and/or maintenance of the stem cell's niche or influence the properties of the stem cell's descendants. A number of factors exported by Paneth cells could regulate epithelial proliferation and differentiation programs. They include tumor necrosis factor-␣ (14), guanylin (15), and epidermal growth factor (16). Two Paneth cell products have been implicated as modifiers of adenoma formation in mice heterozygous for a mutation in the adenomatous polyposis coli gene, Apc Min (17). Production of matrilysin, a matrix metalloprotein-

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Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position ؊5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steadystate level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT͞enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa.unfolded protein response ͉ choriocapillaris ͉ annexin V ͉ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining ͉ endoplasmic reticulum stress

Summary The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer- GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.