SUN1/2Regulate Convergence And Extension Movements During Zebrafish Embryogenesis

SUN domain-containing proteins are an evolutionarily conserved family of proteins that share a conserved C-terminal SUN (Sadlp/Unc84homology) domain. The SUN domain was first defined as a domain of shared homology between Sad1in Schizosaccharomyces pombe and UNC-84in Caenorhabditis elegans. In mammalian, there are multiple SUN domain-containing proteins, among these proteins SUN1and SUN2are widely expressed in a variety of tissues. Structurally, SUN1and SUN2contain transmembrane domains spanning the inner nuclear membrane and a conserved C-terminal SUN domain localizing to the lumen of the nuclear envelope. The N-terminal of SUN proteins have been shown to be in the nucleoplasm and to interact with nuclear lamins. These are in favor of the formation of LINC (linker of nucleoskeleton and cytoskeleton) complex, which provides a direct connection between the nucleoskeleton and cytoskeleton, and is believed to be important for nuclear positioning and nuclear migration.Kai Lei has reported that Sun1; Sun2double knockout mice (Sun1-/-; Sun2-/-) caused neonatal death, indicating that SUN1and SUN2may have essential functions in embryonic development. While the functional importance of SUN1and SUN2in nuclear positioning and nuclear migration at cellular levels has been reported, their physiological role in early embryonic development is not well documented. Gastrulation is an important process during early stage of the zebrafish embryonic development that gives rise to the three germ layers. Convergence and extension (C&E) movement is the major morphogenetic process, which shapes the body axis, narrows all the germ layers in the mediolateral direction and extends them along the anterior-posterior axis at the dorsal midline. In vertebrates, the Wnt/Planar Cell Polarity (Wnt/PCP) pathway is a key regulator of C&E movements, essential for several polarized cell behaviors, including directed cell migration, and mediolateral and radial cell intercalation. To investigate the in vivo function of SUN1and SUN2in early embryonic development, we carried out loss-of-function analyses by using antisense morpholino oligonucleotides (MO). Part I Expression pattern of zebrafish Sun1and Sun2.To understand the potential roles of SUN1and SUN2during zebrafish development, we examined their spatial and temporal expression pattern. The expression of Sun1and Sun2at different stages of zebrafish development was detected by whole-mount in situ hybridization and RT-PCR. Whole-mount in situ hybridization analyses revealed that the expression of Sun1and Sun2were maternally deposited (8-cell stage), and later ubiquitous through whole embryo cells. The RT-PCR analysis showed that Sun1was intially upregulated expression from30%epiboly stage, and Sun2maintained high exprssion from30%epiboly stage to tailbud stage. These results indicate that Sun1and Sun2play an important role in zebrafish embryogenesis. Part II Knockdown of Sun1and Sun2disrupts C&E movements during gastrulation.To further analyze the function of SUN1and SUN2during zebrafish embryogenesis, we first performed knockdown experiments by injecting translation blocking MO at the one-to four-cell stage, which induced specific knockdown of SUN1and SUN2protein expression. Sun1or Sun2morphants (morpholino-injected embryos) were found to have slightly reduced elongation of the body axis at24hour post fertilization (hpf), but still do not have significant defect even with large doses of MO. But while embryos were coinjected with Sun1and Sun2MOs, we found that knockdown of Sun1and Sun2caused severe gastrulation defects. And these gastrulation defects showed a dose dependent manner. These results suggest that SUN1and SUN2are functionally redundant. To identity the cause of this gastrulation defects, we analyzed the expression patterns of several marker genes for the axial mesoderm (ntl), paraxial mesoderm{myod1and tbx6) and neurectoderm (dlx3b and krox20). The results suggest that, knockdown of Sun1and Sun2disrupts cell migration of these germ layers, and eventually impacts C&E movements during gastrulation.Part Ⅲ The mechanism of SUN1/2involved in the C&E movements. Previous studies have shown that loss of Sun1was always associated with an altered distribution of nuclear pore complexes (NPC) as well as altered nuclear shape. In vertebrates, β-catenin, a key fator of Wnt signaling pathway, functions as a component of the cadherin complex, which controls cell-cell adhesion and influences cell migration. To determine whether SUN1and SUN2contribute to NPCs functionality and nuclear transport of β-catenin, we examined the distribution of β-catenin in embryo cells. No obvious changes were observed about the distribution of β-catenin protein in nuclear and cytoplasm after injection of Sun1and Sun2MOs. These results indicate that, knockdown of Sun] and Sun2does not affect nuclear transport of protein β-catenin, and loss-of-function of SUN1and SUN2does not interfere with function of NPCs in nuclear transportation. Notably, Wnt/PCP pathway plays a pivotal role in C&E movemets in vertebrates. To detect whether knockdown of Sun1and Sun2impacted Wnt/PCP pathway, we performed western blot experiments using P-JNK antibody. In our study, we found that JNK phosphorylation (P-JNK) was decreased in Sun1and Sun2morphants. To further identify relevant transcriptional targets downstream of the JNK signaling cascade associated with zebrafish C&E. we used RT-PCR to survey mRNA levels of candidate genes liv1and slat3. We found that their mRNA levels were decreased in morphants. Taken together, these observations provide solid evidence demonstrating that SUN1/2positively regulates C&E movemets of zebrailsh embryos via Wnt/PCP pathway.In conclusion. SUN1and SUN2play a crucial role in the regulation of C&E movemcts by Wnt/PCP pathway. The results described in this research indicate that SUN1and SUN2is essential for zebrafish embryogenesis.