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Understanding the mechanisms that regulate the transition between the proliferative and a post-mitotic state of retinal progenitor cells (RPCs) is key to advancing our knowledge of retinal growth and maturation. In the present study we determined that during zebrafish embryonic retinal neurogenesis, two paired-type homeobox genes - vsx2 and dmbx1 - function in a mutually antagonistic manner. We demonstrate that vsx2 gene expression requires active Fgf signaling and that this in turn suppresses dmbx1 expression and maintains cells in an undifferentiated, proliferative RPC state. This vsx2-dependent RPC state can be prolonged cell-autonomously by knockdown of dmbx1, or it can be suppressed prematurely by the over-expression of dmbx1, which we show can inhibit vsx2 expression and lead to precocious neuronal differentiation. dmbx1 loss of function also results in altered expression of canonical cell cycle genes, and in particular up-regulation of ccnd1, which correlates with our previous finding of a prolonged RPC cell cycle. By knocking down ccnd1 and dmbx1 simultaneously, we show that RPCs can overcome this phenotype to exit the cell cycle on time and differentiate normally into retinal neurons. Collectively, our data provide novel insight into the mechanism that enables RPCs to exit the cell cycle through a previously unrecognized antagonistic interaction of two paired-type homeobox genes that are central regulators of an Fgf-vsx2-dmbx1-ccnd1 signaling axis.

Intestinal epithelial cell tight junctions (TJs) contribute to the integrity of the intestinal barrier allowing for control of the physical barrier between external antigens or bacterial products and the internal environment. Zonula occludens-1 (ZO-1) is a protein that modulates intestinal TJs, and serum levels of ZO-1 has been suggested as a biomarker of disrupted barrier function in humans. Previous studies suggested that increased intestinal permeability was associated with evidence of TJ abnormalities. However, there is limited information on the serological measurement of ZO-1 and its relation to other tests of barrier function in healthy subjects. We investigated the correlation of serum ZO-1, with physiologic measures of intestinal permeability (as the ratio of the fractional excretion of lactulose-mannitol or LMR) in a cohort of 39 healthy FDRs of Crohn's disease (CD) patients. No significant correlation was found between LMR and ZO-1 levels (r2 = 0.004, P < 0.71), or intestinal fatty acid binding proteins (I-FABP) (r2 = 0.004, P < 0.71). In conclusion, our data show that ZO-1 and I-FABP are not a marker of gut permeability as defined by LMR.

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Background Crohn’s disease (CD) is thought to be due to an interaction between environmental factors and the gut microbiome that activates an immune response in genetically susceptible hosts. Epidemiologic studies suggest diet is an important variable in CD development; however, little is known about the mechanism by which diet contributes to pathogenesis. It has recently been shown that diet plays a substantial role in shaping microbiome composition (MC). We hypothesize that specific diet patterns are associated with differences in MC that may be related to CD risk. Aims To characterize associations between diet patterns with MC and fecal calprotectin (FC) in healthy first-degree relatives (FDR’s) of CD patients in the Genetic, Environmental, Microbial (GEM) Project. Methods A validated food frequency questionnaire (FFQ) was used to assess diet for North American FDR’s at recruitment. Each question was summarized as a score based on weekly consumption frequency. The Dirichlet method of unsupervised clustering was used to generate dominant diet clusters. Diet-microbiome associations were assessed using the two-part microbiome model. Stool microbiota at recruitment was characterized by 16s RNA sequencing of V4 region using MiSeq platform. Baseline FC was measured by BUHLMANN ELISA test. Results 2766 FDR’s had FFQ’s at recruitment; mean age 18.52 years, 53% female. The Dirichlet method identified 4 clusters, some of which resembled known diet patterns: Superbowl (mainly organ meats, non-red meat, beer, spirits), High Carbohydrate (HC), Mediterranean (MD) and Western (WD) diets. HC was associated with increased relative abundance of V. veillonella (P=2.68E-4). Both HC and MD were associated with decreased abundance of E. klebsiella (P=2.64E-5 and P=1.02E-5 respectively). WD was associated with decreased abundance of L. dorea (P=5.74E-5), a genus considered high risk for CD. A per question analysis demonstrated significant associations between several taxa and individual foods. Diet clusters were then correlated with FC, and a decrease in FC was observed with MD (estimate -16.96, P=0.012). There were no associations with FC in a per question analysis. Conclusions Dominant dietary patterns and certain individual foods are associated with specific gut MC. As well, MD is inversely associated with FC and therefore has potential use as an intervention for lowering inflammation. Understanding relationships between diet, MC and FC in individuals at high risk for CD would be beneficial in defining new dietary strategies in predictably modulating future risk of CD. Funding Agencies CCCHelmsley Charitable Trust

Background Fecal calprotectin concentration (FC), a measure of gut inflammation is reported to be significantly higher in healthy first-degree relatives (FDR) of Crohn’s disease (CD) patients compared to healthy controls. In contrast, FC in spouses of CD patients was not significantly different from controls, suggesting that a genetic predisposition rather than a shared environmental factor affects FC. Aims We investigated the genetic association with FC in healthy FDRs of CD patients. Notably, these subjects are known to be enriched with CD risk alleles. Methods We investigated 1455 healthy Caucasian FDRs of CD patients from the GEM Project. Subjects were genotyped by HumanCoreEXOME chip and ImmunoChip platforms and then imputed by the Haplotype Reference Consortium v1.1 panel (Michigan Imputation Server). SNPs with a minor allele frequency<5% were removed. FC was measured using BUHLMANN ELISA kit. Heritability was estimated using a pedigree based SOLAR program and a SNP-based GCTA software. Genome wide association of FC was tested using the GEE framework that accounts for family clusters, age, sex, first 3 genetic principal components and multiplex family status (≥2 FDRs diagnosed with CD). In addition, CD-polygenic risk scores were derived based on summary statistics and imputed SNPs from a recent GWAS by pruning and thresholding (P+T) and LDPred algorithm (PMID:31002795). Results Among 1455 subjects, 45.2% were male, median age was 19 years (IQR 13–26), 8.8% were from multiplex families, and median FC was 52 mg/kg (IQR 31–87; 20.8% had FC>100). We estimated the heritability of FC to be 27% (27.1%, standard error=9%, p<0.001 by pedigree approach; 27.9%, SE=12%, p<0.001 by SNP approach). An untargeted GWAS failed to show any significant association with FC (i.e. p<5x10-8). The lowest p value was obtained for rs224631 (p=5x10-7). Strikingly, an increase in CD polygenic risk scores was significantly associated with an increase of FC (p=5.2x10-5 with P+T method). Conclusions We demonstrate that FC concentration is a heritable trait in unaffected FDRs of CD patients. Although the association between genetic variants with FC did not reach GWAS significance, CD-polygenic risk score, which incorporates small effect size CD-associated SNPs, was significantly associated with FC concentrationin this cohort. Our results suggest that FC concentration is influenced genetically with contributions from CD-associated SNPs in unaffected FDRs of CD probands. It remains to be determined if the genetic influence to FC concentration is dependent/independent with the future development of CD. Submitted on behalf of The CCC-GEM Project research team Funding Agencies CCCHelmsley Charitable Trust/ Mount Sinai Hospital Fellowship Award

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