Intraocular calcidiol: Uncovering a role for vitamin D in the eye.

Vitamin D has emerged as a potentially important molecule in ophthalmology. To date, all ophthalmic data pertaining to vitamin D has been restricted primarily to tear and serum analysis in human patients. Considering the isolated nature of the eye, we sought to determine the presence of intraocular vitamin D in ocular disease. METHODS: 25-Hydroxyvitamin D3 (25(OH)D3) concentrations were measured in the eye and blood of 120 participants undergoing ophthalmic procedures. Ocular localization of the 1,25-dihydroxyvitamin D3-generating (CYP27B1) and deactivating (CYP24A1) hydroxylases was performed by immunohistochemistry. Gene expression of CYP27B1, CYP24A1 and VEGF-A was measured in eyes from patients with and without disease. RESULTS: 25(OH)D3 was quantified in 112 ocular samples. In 40 cataract patient samples, the average 25(OH)D3 concentration was 0.057 ng/mL, compared to 72 retinal disease patient samples, average of 0.502 ng/mL (p < 0.001). Intraocular 25(OH)D3 did not correlate with serum levels of 25(OH)D3. There was no difference between the level of 25(OH)D3 measured in the aqueous and vitreous humour. The vitamin D-specific CYPs 27B1 and 24A1, strongly localized to complementary regions of the ciliary body, retinal pigment epithelium and neural retina. Gene expression analysis confirmed retinal CYP27B1 correlated strongly with VEGF-A in eyes from diabetic patients (r = 0.92, p < 0.001). CONCLUSIONS: Our data confirms that vitamin D is present in the humours of the human eye and that local synthesis/degradation is possible via the ocular CYP27B1 and CYP24A1. This argues for a functional role for local vitamin D production and signaling in the eye and suggests that vitamin D may be an important intraocular mediator in disease pathogenesis.

BRACHYURY directs histone acetylation to target loci during mesoderm development.

T-box transcription factors play essential roles in multiple aspects of vertebrate development. Here, we show that cooperative function of BRACHYURY (T) with histone-modifying enzymes is essential for mouse embryogenesis. A single point mutation (TY88A) results in decreased histone 3 lysine 27 acetylation (H3K27ac) at T target sites, including the T locus, suggesting that T autoregulates the maintenance of its expression and functions by recruiting permissive chromatin modifications to putative enhancers during mesoderm specification. Our data indicate that T mediates H3K27ac recruitment through a physical interaction with p300. In addition, we determine that T plays a prominent role in the specification of hematopoietic and endothelial cell types. Hematopoietic and endothelial gene expression programs are disrupted in TY88A mutant embryos, leading to a defect in the differentiation of hematopoietic progenitors. We show that this role of T is mediated, at least in part, through activation of a distal Lmo2 enhancer.

Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40-50% of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12% of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2-ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

Tulp3 is a critical repressor of mouse hedgehog signaling.

Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby-like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3(-/-) embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3(-/-)/Shh(-/-) compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh(-/-) embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway.