Functional Study Of Cell Apoptosis In The Development Of Zebrafish Cloaca

Anorectal malformations(ARMs) comprise a wide spectrum of diseases, which include malformations of the distal anus and rectum as well as the urinary and genital tracts. They occur in approximately 1 in 5000 live births. The occurrence of ARM is the result of abnormal embryo development. The etiology of ARM is unknown. Genetic factors are important contributing factors in the pathogenesis of ARM. Anorectal and urogenital malformations resulted from defective cloaca development account for a large proportion in ARMs.The cloaca is a transient embryonic cavity. The urogenital sinus and the rectum are formed as a result of cloacal separation. Several mechanisms have been proposed to explain the processes of cloacal division based on animal studies. At the same time, the cell behavior during the process of cloacal development remains unclear. Researchs on the relationship between cell behavior and ARM disease also exist many gaps. The molecular mechanism of vertebrate embryogenesis is conservative. Zebrafish has many advantages compared with mice. The embryo of zebrafish is transparent, external fertilization and development. We observed the developmental process of zebrafish cloaca and found that it was similar to the process in human and mice. They all need structure remodeling of the cloaca cells. So zebrafish is an ideal model to study cloacal development during embryogenesis.Zebrafish cloaca develops from proctodeum. At 24hpf(hours post-fertilization), basic structure of cloaca has been formed, which can be divided into the left wall(LW), the right wall(RW)and the ventral cloacal membrane(VCM). Cloacal remodeling processes involve the separation of LW and RW, formation of the intermediate cavity, disappearance of cloacal membrane, and formation of the urogenital sinus and anus. At 36 hpf, it can be clearly observed that cloacal walls become thinner and pronephric duct connects with the cloaca. At 48 hpf, the posterior gut developing along the anterior to the posterior axis has reached LW, and the cavity becomes bigger. At 60 hpf, LW connects with RW only by the ventral cloacal membrane. From 60 hpf to 72 hpf, cloacal morphology changes greatly, including disappearance of the ventral cloacal membrane and morphologic change of the rhabdoid cloaca walls. Following disappearance of VCM and separation of cloaca, the pronephric duct opens to the outside at about 72 hpf. At 84 hpf, the posterior gut has fused with cells from LW and inverted U-shape has formed at the distal end of two openings. From 96 hpf to 120 hpf, the cloacal cells connected with the posterior gut become thickened, which will form anal canal and anus. The hindgut opens to the outside via them. During the process, we found that the posterior gut would reach the cloaca and fuse with it. So we support that cloaca division is achieved by autonomous behavior of cloacal cells or response to surrounding signal.We detected cloacal cell apoptosis at different stages during the cloacal development by TUNEL and Caspase3 antibody staining. We found that apoptotic peaks appeared at about 1.5dpf and 4.5dpf. Overexpression of bcl2 l could inhibit early cloacal cell apoptosis and result in the existence of redundant cloaca membrane. The phenotype was similar to cloacal malformation in human congenital anomalies. Knock-down of baxa gene could also inhibit early cloacal cell apoptosis and result in cloacal malformation. Z-VAD-FMK, pan-caspase inhibiotor, could block early cell apoptosis of cloacal membrane, cause that cloacal cells gathered together and could not form inverted U-shape. We concluded that cell apoptosis plays an important role in normal cloaca development.