Ramakrishna P. Alur

Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. Here, we profile global gene expression during optic fissure closure using laser capture microdissected (LCM) tissue from the margins of the fissure. From these data, we identify a unique role for the C2H2 zinc finger proteins Nlz1 and Nlz2 in normal fissure closure. Gene knockdown of nlz1 and/or nlz2 in zebrafish leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. We also identify misregulation of pax2 in the developing eye of morphant fish, suggesting that Nlz1 and Nlz2 act upstream of the Pax2 pathway in directing proper closure of the optic fissure.

Nitisinone improves eye and skin pigmentation defects in a mouse model of oculocutaneous albinism

Mutation of the tyrosinase gene (TYR) causes oculocutaneous albinism, type 1 (OCA1), a condition characterized by reduced skin and eye melanin pigmentation and by vision loss. The retinal pigment epithelium influences postnatal visual development. Therefore, increasing ocular pigmentation in patients with OCA1 might enhance visual function. There are 2 forms of OCA1, OCA-1A and OCA-1B. Individuals with the former lack functional tyrosinase and therefore lack melanin, while individuals with the latter produce some melanin. We hypothesized that increasing plasma tyrosine concentrations using nitisinone, an FDA-approved inhibitor of tyrosine degradation, could stabilize tyrosinase and improve pigmentation in individuals with OCA1. Here, we tested this hypothesis in mice homozygous for either the Tyrc-2J null allele or the Tyrc-h allele, which model OCA-1A and OCA-1B, respectively. Only nitisinone-treated Tyrc-h/c-h mice manifested increased pigmentation in their fur and irides and had more pigmented melanosomes. High levels of tyrosine improved the stability and enzymatic function of the Tyrc-h protein and also increased overall melanin levels in melanocytes from a human with OCA-1B. These results suggest that the use of nitisinone in OCA-1B patients could improve their pigmentation and potentially ameliorate vision loss.

Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human

Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one alleles function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo, likely by reducing protein stability. These results suggest that hypomorphic alleles of PAX2/Pax2 can lead to significant disease in humans and mice.

A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma

Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

aldh7a1 Regulates Eye and Limb Development in Zebrafish

Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of aldh7a1 as a gene necessary for normal eye development. We show that morpholino knockdown of aldh7a1 in zebrafish causes uveal coloboma and misregulation of nlz1, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of aldh7a1 leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The aldh7a1 morphant phenotype is partially rescued by co-injection of nlz1 mRNA suggesting that nlz1 is functionally downstream of aldh7a1 in regulating cell proliferation in the optic cup. These results support a role of aldh7a1 in ocular development and skeletal abnormalities in zebrafish.

nlz1 is required for cilia formation in zebrafish embryogenesis.

The formation of cilia is a fundamental developmental process affecting diverse functions such as cellular signaling, tissue morphogenesis and body patterning. However, the mechanisms of ciliogenesis during vertebrate development are not fully understood. In this report we describe a novel role of the Nlz1 protein in ciliogenesis. We demonstrate morpholino-mediated knockdown of nlz1 in zebrafish causes abnormal specification of the cells of Kupffers vesicle (KV); a severe reduction of the number of cilia in KV, the pronephros, and the neural floorplate; and a spectrum of later phenotypes reminiscent of human ciliopathies. In vitro and in vivo data indicate that Nlz1 acts downstream of Foxj1a and Wnt8a/presumed canonical Wnt signaling. Furthermore, Nlz1 contributes to motile cilia formation by positively regulating Wnt11/presumed non-canonical Wnt signaling. Together, our data suggest a novel role of nlz1 in ciliogenesis and the morphogenesis of multiple tissues.

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3

Purpose Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.