TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

Abstract

The optic fissure is a transient gap in the developing vertebrate eye, which must be closed as development proceeds. A persisting optic fissure, coloboma, is a major cause for blindness in children. Although many genes have been linked to coloboma, the process of optic fissure fusion is still little appreciated, especially on a molecular level. We identified a coloboma in mice with a targeted inactivation of transforming growth factor β2 (TGFβ2). Notably, here the optic fissure margins must have touched, however failed to fuse. Transcriptomic analyses indicated an effect on remodelling of the extracellular matrix (ECM) as an underlying mechanism. TGFβ signalling is well known for its effect on ECM remodelling, but it is at the same time often inhibited by bone morphogenetic protein (BMP) signalling. Notably, we also identified two BMP antagonists among the downregulated genes. For further functional analyses we made use of zebrafish, in which we found TGFβ ligands expressed in the developing eye, and the ligand binding receptor in the optic fissure margins where we also found active TGFβ signalling and, notably, also gremlin 2b (grem2b) and follistatin a (fsta), homologues of the regulated BMP antagonists. We hypothesized that TGFβ is locally inducing expression of BMP antagonists within the margins to relieve the inhibition from its regulatory capacity regarding ECM remodelling. We tested our hypothesis and found that induced BMP expression is sufficient to inhibit optic fissure fusion, resulting in coloboma. Our findings can likely be applied also to other fusion processes, especially when TGFβ signalling or BMP antagonism is involved, as in fusion processes during orofacial development.

In Vivo Analysis of Optic Fissure Fusion in Zebrafish: Pioneer Cells, Basal Lamina, Hyaloid Vessels, and How Fissure Fusion is Affected by BMP

“…Next to some environmental and dietary factors, many genes have been linked to coloboma formation [7], resulting in a coloboma gene network [8,9] which recently was updated [6]. It consists of many signaling pathway components, e.g., of Wnt [10,11], fibroblast growth factor (FGF) [12,13], retinoic acid (RA) [14,15], Hippo [16], sonic hedgehog (Shh) [17], transforming growth factor β (TGFβ), and bone morphogenetic protein (BMP) [18,19].…”

“…Importantly, the morphology of coloboma phenotypes is highly variable, arguing at least for two main distinct coloboma classes [18,20]: morphogenetic coloboma, showing a vast cleft [1,11,16,21], and fusion defective coloboma, in which the margins were aligned but remained unfused [12,13,18]. The focus of our current analysis was the process of optic fissure fusion.…”

“…Notably, a loss, but also a gain of Pax2 results in coloboma, with a persistent basal lamina [34]. Furthermore, TGFβ was found important for basal lamina degradation [18]. Even though many factors were linked to the degradation of the basal lamina and recent transcriptional analyses include potential effector molecules [18,32,35], such factors need further analysis and validation.…”

“…Furthermore, TGFβ was found important for basal lamina degradation [18]. Even though many factors were linked to the degradation of the basal lamina and recent transcriptional analyses include potential effector molecules [18,32,35], such factors need further analysis and validation. Besides, it is also not clear whether the POM and the NR/RPE precursors cooperate during basal lamina degradation.…”

Pax2a regulates angiogenesis to facilitate mmp2-dependent basement membrane remodeling of the optic fissure

“…Work from zebrafish, mice and chick all point to an orderly progression involving: 1) retinal growth and cellular rearrangement leading to nasal and temporal retinal lobe apposition, 2) invasion of the fissure by endothelial and neural crest cells forming the hyaloid vasculature system, 3) cellular signaling, either between retinal rim cells or between rim cells and the migrating vasculature cells, 4) remodeling or removal of the basement membrane to enable physical connection of the rim cells and subsequent formation of a continuous retinal epithelial sheet via re-polarization and cell-cell adhesion. Step 1 has been nicely characterized in a few recent publications outlining the flow of retinal cells and morphological formation of the fissure (45–47). For step 2, several reports have carefully characterized the formation of the hyaloid vasculature system, including migration of hyaloid vasculature precursor cells into the fissure as soon as it forms (1–3).…”