Roles Of Retinoic Acid Signaling And The Cell Cycle Regulator Geminin In The Establishment Of Left-right Asymmetry

Establishment of body axis is a critical event in embryonic development. The anterior-posterior, dorsal-ventral, and left-right (LR) axes have been set up during gastrulation. Although mechanisms underlying determination of LR asymmetry have been extensively studied in the last two decades, many aspects such as formation of regulatory network, differences and coordinations of LR patterning of different organs remain largely unknown.In mice, involvements of RA signaling in regulating LR asymmetry have previously been reported. However, its regulatory mechanism, spatial and temperal specificities, as well as its role in coordinating LR patterning of different organs have never been studied. In this study in zebrafish, BMS453, a pan-retinoic acid receptor antagonist, and 4-diethylaminobenzaldehyde (DEAB), an inhibitor of RA synthase, were applied to block RA activity in different time frames. Then, effects on LR patterning were analyzed. Blocking RA activity from the 32-cell to the 2-somite (2S) stage resulted in randomized visceral laterality, with 41.0% of embryos displaying reversed liver and pancreas. But the heart laterality is only mildly affected with 12.1% of embryos displaying reversed heart looping, similar to that of the control (8.9%). In contrast, if RA activity was blocked after 2S, the majority (90.7%) displayed normal visceral laterality, whereas 24.3% and 36.4% of them showed reversed heart looping and midline heart, respectively. These results were confirmed by application of DEAB.When RA was blocked before 2S, further study indicated bilateral expressions of spaw and Iefty2, enhanced fgf8 transcription as well as its downstream targets erm and pea3, disturbed directional fluid flow and increases in cilia length in Kupffer's vesicle(KV), and decreased expression of cha. Although overexpression of fgf8 did not lead to increases in cilia length, kncok down of Fgf8 could efficiently rescue increased cilia length and decreased cha expression caused by BMS453 treatments, indicating the requirement of Fgf8 in mediating the regulatory role of RA in ciliogenesis and cha expression. Re-establishment of the asymmetric distribution of spaw in lateral plate mesoderm (LPM) in BMS453 treated embryos restored normal visceral laterality. These results indicated that RA signaling controls visceral laterality through the left-sided Nodal signal before 2S.Block of RA signaling after 2S resulted in perturbed heart laterality, but visceral laterality remained normal. Although block of RA signaling before 2S led to increased expression of bmp4 on both sides, its asymmetry remained unaffected. On the contrary, block of RA signaling after 2S led to disturbed bmp4 asymmetry. Re-establishment of the asymmetric distribution of bmp4 in the LPM in BMS453 treated embryos restored normal heart laterality. These results indicated that RA signaling controls heart laterality through asymmetric bmp4 in the LPM after 2S.To find out the source of RA that regulats visceral and heart laterality before or after 2S, expression patterns of raldh2, the key RA synthesis enzyme, were examined at different developmental stages. At the 80%-epiboly stage, expression of raldh2 was found in the dorsal region of the marginal zone except notochord, partially overlapping with that of fgf8. From 2S on, expression of raldh2 was found in the anterior LPM, with stronger expression on the left than the right side.Using zebrafish as the model system, we have studied the spatial-temporal functions of RA in regulating and coordinating LR patterning of different organs. Our results demonstrated that laterality of different organs could be controlled by different mechanisms and coordinated.Except for studying the role of RA in regulating cilia morphogenesis and visceral laterality, we also found another important factor geminin involved in regulating KV/cilia formation and organ laterality. Geminin plays an important role in coordinating the cell cycle with anterior-posterior patterning during embryonic development. However, whether it is involved in regulating LR patterning remains unknown. Here, we reported that geminin is required for setting up the LR asymmetry of heart and visceral organs during zebrafish development. When geminin was knocked down, heart and visceral laterality was disturbed. Further study demonstrated that the left-sided Nodal/spaw in the LPM as well as transcriptions of its downstream targets lefty2 and lefty1 were perturbed in geminin morphants. Upstream of the left-sided Nodal signal along the regulatory cascade of LR asymmetry, knock down of geminin resulted in defective KV formation and ciliogenesis rather than middle line defects. Predominant distribution of an antisense morpholino against geminin in dorsal forerunner cells (DFCs) led to defective KV morphogenesis and LR patterning, similar to those of geminin morphants, indicating a cell-autonomous role of geminin in regulating KV formation and ciliogenesis. Our results demonstrated that geminin is required for proper KV formation and ciliogenesis, thus playing an important part in setting up LR asymmetry.