Colobomata, persistent optic fissures, frequently cause congenital blindness. Here, we focused on optic fissure fusion using in vivo time-lapse imaging in zebrafish. We identified the fusion initiating cells, which we termed “pioneer cells.” Based on morphology, localization, and downregulation of the neuroretinal (NR) precursor marker rx2, these cells could be considered as retinal pigment epithelial (RPE) progenitors. Notably, pioneer cells regain rx2 expression and integrate into the NR after fusion, indicating that they do not belong to the pool of RPE progenitors, supported by the lack of RPE marker expression in pioneer cells. They establish the first cellular contact between the margins in the proximal fissure region and separate the hyaloid artery and vein. After initiation, the fusion site is progressing distally, increasing the distance between the hyaloid artery and vein. A timed BMP (Bone Morphogenetic Protein) induction, resulting in coloboma, did not alter the morphology of the fissure margins, but it did affect the expression of NR and RPE markers within the margins. In addition, it resulted in a persisting basal lamina and persisting remnants of periocular mesenchyme and hyaloid vasculature within the fissure, supporting the necessity of BMP antagonism within the fissure margins. The hampered fissure fusion had severe effects on the vasculature of the eye.

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.

Decussation of axonal tracts is an important hallmark of vertebrate neuroanatomy resulting in one brain hemisphere controlling the contralateral side of the body and also computing the sensory information originating from that respective side. Here, we show that BMP interferes with optic chiasm formation and RGC pathfinding in zebrafish. Experimental induction of BMP4 at 15 hpf results in a complete ipsilateral projection of RGC axons and failure of commissural connections of the forebrain, in part as the result of an interaction with shh signaling, transcriptional regulation of midline guidance cues and an affected optic stalk morphogenesis. Experimental induction of BMP4 at 24 hpf, resulting in only a mild repression of forebrain shh ligand expression but in a broad expression of pax2a in the diencephalon, does not per se prevent RGC axons from crossing the midline. It nevertheless shows severe pathologies of RGC projections e.g., the fasciculation of RGC axons with the ipsilateral optic tract resulting in the innervation of one tectum by two eyes or the projection of RGC axons in the direction of the contralateral eye.

Motile cilia perform crucial functions during embryonic development and in adult tissues. They are anchored by an apical actin network that forms microridge-like structures on the surface of multiciliated cells. Using Xenopus as a model system to investigate the mechanisms underlying the formation of these specialized actin structures, we observed stochastic bursts of intracellular calcium concentration in developing multiciliated cells. Through optogenetic manipulation of calcium signaling, we found that individual calcium bursts triggered the fusion and extension of actin structures by activating non-muscle myosin. Repeated cycles of calcium activation promoted assembly and coherence of the maturing apical actin network. Inhibition of the endogenous inositol triphosphate-calcium pathway disrupted the formation of apical actin/microridge-like structures by reducing local centriolar RhoA signaling. This disruption was rescued by transient expression of constitutively active RhoA in multiciliated cells. Our findings identify repetitive calcium bursts as a driving force that promotes the self-organization of the highly specialized actin cytoskeleton of multiciliated cells.

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. Multiple factors are genetically linked to coloboma formation. However, especially the process of optic fissure fusion is still largely elusive on a cellular and molecular basis. We found a coloboma phenotype in mice with a targeted inactivation of the transforming growth factor 2 (TGFβ2). Here the optic fissure margins got in touch, however, failed to fuse. Transcriptomic analyses indicated TGFβ mediated ECM remodeling during optic fissure fusion. For functional analyses, we switched model systems and made use of zebrafish. We found TGFβ ligands expressed in the developing zebrafish eye, and the ligand binding receptor in the optic fissure. Using a new in vivo TGFβ signaling reporter, we also found active TGFβ signaling in the margins of the optic fissure. We addressed the function of Cadherin 6 ( cdh6 ), one of the TGFβ regulated genes, by knock down experiments in zebrafish and found a prominent coloboma phenotype. Cdh6 was often found involved in processes of epithelial to mesenchymal transition (EMT), strengthening our hypothesis that an EMT-like process is also necessary for optic fissure fusion. Furthermore, we found Gremlin 2b ( grem2b ) and Follistatin a ( fsta ), homologs of TGFβ regulated bone morphogenetic protein (BMP) antagonists, expressed in the optic fissure margins, indicating the necessity of a localized inhibition of BMP signaling. Finally, we show that induced BMP expression is sufficient to inhibit optic fissure fusion. Together with our previous findings this indicates a dual role of BMP signaling during optic fissure closure. Summary statement: TGFβ is crucial for optic fissure fusion, involving cdh6 . TGFβ mediated optic fissure fusion is potentially hampered by BMP signaling, which is blocked by TGFβ induced BMP antagonists within the optic fissure margins.

These have been added as references 66 and 67 at the ninth sentence of the 'DNA reagents' section of the Methods. The ninth sentence of the 'DNA reagents' section of the Methods incorrectly read 'Plasmids containing PIF6 and PHYB-mCherryCAAX (Plasmid #154913 and #51567, addgene, 490 Aresenal Way, Suite 100, Watertown, MA 02472) were subcloned into VF10 vectors with GFP, ezrin FERM domain (residues 1-310), or ezrin C-terminal domain (CTD) (residues 297-582) containing the phospho-mimetic T563E mutation.' The correct ninth sentence of the 'DNA reagents' section of the Methods is: 'Plasmids containing PIF6 and PHYB-mCherryCAAX (Plasmid #154913 created by Jonathan Clarke 66 and #51567 created by Chao Tang 67 , addgene, 490 Aresenal Way, Suite 100, Watertown, MA 02472) were subcloned into VF10 vectors with GFP, ezrin FERM domain (residues 1-310), or ezrin C-terminal domain (CTD) (residues 297-582) containing the phospho-mimetic T563E mutation.