Markus Kroeber

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Reduced expression of Pax6 in lens and cornea of mutant mice leads to failure of chamber angle development and juvenile glaucoma

Heterozygous mutations in PAX6 are causative for aniridia, a condition that is frequently associated with juvenile glaucoma. Defects in morphogenesis of the iridocorneal angle, such as lack of trabecular meshwork differentiation, absence of Schlemms canal and blockage of the angle by iris tissue, have been described as likely causes for glaucoma, and comparable defects have been observed in heterozygous Pax6-deficient mice. Here, we employed Cre/loxP-mediated inactivation of a single Pax6 allele in either the lens/cornea or the distal optic cup to dissect in which tissues both alleles of Pax6 need to be expressed to control the development of the tissues in the iridocorneal angle. Somatic inactivation of one allele of Pax6 exclusively from epithelial cells of lens and cornea resulted in the disruption of trabecular meshwork and Schlemms canal development as well as in an adhesion between iris periphery and cornea in juvenile eyes, which resulted in the complete closure of the iridocorneal angle in the adult eye. Structural changes in the iridocorneal angle presumably caused a continuous increase in intraocular pressure leading to degenerative changes in optic nerve axons and to glaucoma. In contrast, the inactivation of a single Pax6 allele in the distal optic cup did not cause obvious changes in iridocorneal angle formation. We conclude that the defects in iridocorneal angle formation are caused by non-autonomous mechanisms due to Pax6 haploinsufficiency in lens or corneal epithelial cells. Pax6 probably controls the expression of signaling molecules in lens cells that regulate the morphogenetic processes during iridocorneal angle formation.

Elevated amounts of myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression

Myocilin is a 55-57kDa secreted glycoprotein and member of the olfactomedin family, which is mutated in some forms of primary open-angle glaucoma. To assess the effects of elevated amounts of myocilin on aqueous humor outflow dynamics in an in vivo system, transgenic betaB1-crystallin-MYOC mice have been developed that strongly overexpress myocilin in their eyes. The transgenic overexpression of myocilin results in an almost five-fold increase of secreted normal myocilin in the aqueous humor of betaB1-crystallin-MYOC mice. In the present study, we wanted to use betaB1-crystallin-MYOC as a tool to identify the response of ocular tissues to the presence of higher than normal amounts of myocilin, and to identify changes in gene expression that could help to shed light on the functional in vivo properties of myocilin. RNA was isolated from ocular tissues of betaB1-crystallin-MYOC mice and wild-type littermates. Changes in gene expression were determined by hybridization of gene microarrays and confirmed by real time RT-PCR and Western blotting. The expression of genes that had been found to be differentially regulated in betaB1-crystallin-MYOC mice was further analyzed in cultured human trabecular meshwork (HTM) cells treated with recombinant myocilin. Although betaB1-crystallin-MYOC mice do not have an obvious phenotype, a statistically significant up- and downregulation of several distinct genes was found when compared to gene expression in wild-type littermates. Among the genes that were found to be differentially regulated were Wasl, Ceacam1, and Spon2, which are involved in cell adhesion and cell-matrix interactions. Differences in expression were also found for Six1 which encodes for a transcription factor, and for Pftk1 whose gene product is a cdc2-related protein kinase. The expression of these genes was also found to be regulated in vitro in HTM cells treated with recombinant myocilin. Substantially higher amounts in ocular tissues of betaB1-crystallin-MYOC mice were found for connexin 46 and alphaB-crystallin. In addition, several genes that encode for olfactomedin proteins showed distinct changes in expression. Olfml3 was significantly downregulated, while Lphn1, Lphn2, and Lphn3 were significantly upregulated. Our findings support a role for myocilin in modulating cellular adhesion, and suggest functional processes that involve other proteins of the olfactomedin family.

Heterozygote Wdr36-deficient mice do not develop glaucoma.

There is an ongoing controversy regarding the role of WDR36 sequence variants in the pathogenesis of primary open-angle glaucoma (POAG). WDR36 is a nucleolar protein involved in the maturation of 18S rRNA. The function of WDR36 is essential as homozygous Wdr36-deficient mouse embryos die before reaching the blastocyst stage. Here we provide a detailed analysis of the phenotype of heterozygous Wdr36-deficient mice. Loss of one Wdr36 allele causes a substantial reduction in the expression of Wdr36 mRNA. In the eyes of Wdr36(+/-) animals, the structure of the tissues involved in aqueous humor circulation and of the optic nerve head are not different from that of control littermates. In addition, one-year-old Wdr36(+/-) animals do not differ from wild-type animals with regards to intraocular pressure and number of optic nerve axons. The susceptibility of retinal ganglion cells to excitotoxic damage induced by NMDA is similar in Wdr36(+/-) and wild-type animals. Moreover, the amount of optic nerve axonal damage induced by high IOP is not different between Wdr36(+/-) and wild-type mice. Transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36(+/-) animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In addition, analysis of 18S rRNA maturation in Del605-607 Wdr36(+/-) or Wdr36(+/-) mice does not show obvious differences in rRNA processing or in the amounts of precursor forms when compared to wild-type animals. Our data obtained in Wdr36(+/-) mice do not support the assumption of a causative role for WDR36 in the pathogenesis of POAG.