Cdc42 and sec10 Are Required for Normal Retinal Development in Zebrafish.

Cilia are rod-like, microtubule-based organelles found on most mammalian cell types. Cilia can be classified as motile or nonmotile (primary) cilia. Motile cilia function mainly as motor organelles, whereas primary cilia are sensory organelles.1–3 Dysfunction of primary cilia results in human disorders, such as Bardet-Biedl (BBS), Joubert, and Senior-Loken syndromes, that affect multiple organs, resulting in central nervous system malformation, cystic kidney disease, and retinal dystrophy.4–5 Zebrafish knockdown models for several ciliopathies, including cep290, cc2d2a, inpp5e, ift57, ift88, and ift172, have kidney and retinal phenotypes that suggest insight into the mechanisms underlying these defects.6–9 The zebrafish eye is a well-laminated structure similar to the eyes of other vertebrates. Eye morphogenesis in the zebrafish starts at 11.5 hours post fertilization (hpf) and the eyecup is well-formed by 24 hpf. By 48 hpf, retinal lamination occurs in most of the retina.10 The vertebrate retina is organized into three laminae: the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). The photoreceptor cell in the ONL has a specialized morphology consisting of an inner segment (IS) and outer segment (OS) linked by connecting cilium, and is a highly polarized and light-sensitive cell. The IS, where protein synthesis occurs, is connected by the cilium to the OS, which consists of a microtubule-based axoneme and membrane disc stacks containing opsin required for phototransduction.11–13 The connecting cilia and basal body in the IS are observed at 50 hpf, and the OS is visible by 54 hpf. The first visual responses are present at approximately 70 hpf, and photoreceptor cells reach adult size by 576 hpf (24 days).10,14 The development and maintenance of photoreceptors requires intracellular trafficking in both photoreceptor and RPE cells. In photoreceptor cells, vesicles containing proteins destined for the OS traffic from the trans-Golgi network to the base of the connecting cilium via vesicular transport.15–17 In RPE cells, intracellular trafficking controls the phagocytosis of disk membranes shed from the tip of the OS.18 To replace lost membrane, the photoreceptor inner segments continuously provide material to the OSs.9 The connecting cilium, therefore, plays an important role as the bridge between the IS and OS. One of the proteins implicated in vesicular trafficking from the Golgi to the cilium is the small GTPase, Rab8.19 Disruption of Rab8 in Xenopus photoreceptor cells blocks rhodopsin-bearing post-Golgi vesicle trafficking, and results in the abnormal accumulation of rhodopsin carrier vesicles at the base of connecting cilium.15,16 Rab proteins perform functions through downstream effectors, such as the exocyst, a highly conserved eight-protein trafficking complex.20,21 We previously demonstrated that the exocyst is required for ciliogenesis in MDCK cells, due to its role in targeting and docking vesicles carrying ciliary proteins.22 We also showed that Cdc42, another small GTPase, localizes with the exocyst at the primary cilium, and biochemically and genetically interacts with exocyst Sec10.23 Although the roles of Cdc42 and Sec10 in epithelial cell biology are now better understood, their potential functions in eye development are still unknown. Interestingly, we recently found that knockdown of both cdc42 and sec10 in zebrafish resulted in small eyes, and knockdown of cdc42 led to loss of photoreceptor cilia.24,25 Here, we describe the role of cdc42 and sec10 in eye development using histological, functional, and embryonic manipulations in zebrafish. We find that cdc42 and sec10 knockdown results in increased retinal cell death, photoreceptor defects, and intracellular transport defects. We also demonstrate a synergistic genetic interaction between cdc42 and sec10, suggesting that cdc42 and sec10 act in the same pathway in retinal development. These findings indicate that sec10 and cdc42 play a central role in trafficking ciliary proteins to the photoreceptor cell for ciliogenesis.