Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-β (TGF-β) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

Background: During visual system development, multiple signalling pathways cooperate to specify axial polarity within the retina and optic tectum. This information is required for the topographic mapping of retinal ganglion cell axons on the tectum. Meis1 is a TALE-class homeodomain transcription factor known to specify anterior-posterior identity in the hindbrain, but its role in visual system patterning has not been investigated. Results: meis1 is expressed in both the presumptive retina and tectum. An analysis of retinal patterning reveals that Meis1 is required to correctly specify both dorsal-ventral and nasal-temporal identity in the zebrafish retina. Meis1-knockdown results in a loss of smad1 expression and an upregulation in follistatin expression, thereby causing lower levels of Bmp signalling and a partial ventralization of the retina. Additionally, Meis1-deficient embryos exhibit ectopic Fgf signalling in the developing retina and a corresponding loss of temporal identity. Meis1 also positively regulates ephrin gene expression in the tectum. Consistent with these patterning phenotypes, a knockdown of Meis1 ultimately results in retinotectal mapping defects. Conclusions: In this work we describe a novel role for Meis1 in regulating Bmp signalling and in specifying temporal identity in the retina. By patterning both the retina and tectum, Meis1 plays an important role in establishing the retinotectal map and organizing the visual system.

Holoprosencephaly (HPE) is the most common human congenital forebrain defect, affecting specification of forebrain tissue and subsequent division of the cerebral hemispheres. The causes of HPE are multivariate and heterogeneous, and include exposure to teratogens, such as retinoic acid (RA), and mutations in forebrain patterning genes. Many of the defects in HPE patients resemble animal models with aberrant RA levels, which also show severe forebrain abnormalities. RA plays an important role in early neural patterning of the vertebrate embryo: expression of RA-synthesizing enzymes initiates high RA levels in the trunk, which are required for proper anterior–posterior patterning of the hindbrain and spinal cord. In the forebrain and midbrain, RA-degrading enzymes are expressed, protecting these regions from the effects of RA. However, the mechanisms that regulate RA-synthesizing and RA-degrading enzymes are poorly understood. Mutations in the gene TGIF are associated with incidence of HPE. We demonstrate in zebrafish that Tgif plays a key role in regulating RA signaling, and is essential to properly pattern the forebrain. Tgif is necessary for normal initiation of genes that control RA synthesis and degradation, resulting in defects in RA-dependent central nervous system patterning in Tgif-depleted embryos. The loss of the forebrain-specific RA-degrading enzyme cyp26a1 causes a forebrain phenotype that mimics tgif morphants. We propose a model in which Tgif controls forebrain patterning by regulating RA degradation. The consequences of abnormal RA levels for forebrain patterning are profound, and imply that in human patients with TGIF deficiencies, increased forebrain RA levels contribute to the development of HPE.

The eye primordium arises as a lateral outgrowth of the forebrain, with a transient fissure on the inferior side of the optic cup providing an entry point for developing blood vessels. Incomplete closure of the inferior ocular fissure results in coloboma, a disease characterized by gaps in the inferior eye and recognized as a significant cause of pediatric blindness. Here, we identify eight patients with defects in tissues of the superior eye, a congenital disorder that we term superior coloboma. The embryonic origin of superior coloboma could not be explained by conventional models of eye development, leading us to reanalyze morphogenesis of the dorsal eye. Our studies revealed the presence of the superior ocular sulcus (SOS), a transient division of the dorsal eye conserved across fish, chick, and mouse. Exome sequencing of superior coloboma patients identified rare variants in a Bone Morphogenetic Protein (Bmp) receptor (BMPR1A) and T-box transcription factor (TBX2). Consistent with this, we find sulcus closure defects in zebrafish lacking Bmp signaling or Tbx2b. In addition, loss of dorsal ocular Bmp is rescued by concomitant suppression of the ventral-specific Hedgehog pathway, arguing that sulcus closure is dependent on dorsal-ventral eye patterning cues. The superior ocular sulcus acts as a conduit for blood vessels, with altered sulcus closure resulting in inappropriate connections between the hyaloid and superficial vascular systems. Together, our findings explain the existence of superior coloboma, a congenital ocular anomaly resulting from aberrant morphogenesis of a developmental structure. Ocular coloboma is a disease characterized by gaps in the lower portion of the eye and can affect the iris, lens, or retina, and cause loss of vision. Coloboma arises from incomplete closure of a transient fissure on the underside of the developing eye. Therefore, our identification of patients with similar tissue defects, but restricted to the superior half of eye, was surprising. Here, we describe an ocular developmental structure, the superior ocular sulcus, as a potential origin for the congenital disorder superior coloboma. Formation and closure of the sulcus are directed by dorsal-ventral eye patterning, and altered patterning interferes with the role of the sulcus as a pathway for blood vessel growth onto the eye.

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.