| Including fish and amphibians, aquatic vertebrates have lateral line which is a very important sensory organ. During the evolutionary process of the system, in spite of the teleost electroreceptors has lost, the lateral line system of cartilaginous fish and none neopterygii is composed of mechanicoreceptors(neuromasts) by feelings of mechanical vibration and electroreceptors(ampullary organs) by feelings of weak electric field. Lateral line receptors of the two kinds of different functions are composed by sensory cells, support cells and nerve cells, and they have their origins in a series of lateral line placodes. Cephalic lateral line placodes form the sensory crista by extension, and then in the central area of the sensory crista the neuromast primordium to be formed and the primordia of the ampullary organ are formed from the flanking region of it. So far, the molecular basis of the differentiation of the two types of lateral line receptors is not clear. This is the key question, however, of understanding that how the electroreceptors lost along the evolution process of the fish phylogeny, then, gain again in certain lineage of fish groups.To approach the question, we chose an electroreceptive fish, Siberian sturgeon, to study. There has been a comprehensive morphological understanding of the lateral line system in sturgeon. Recentely, some of the genes and signaling pathways associated with the lateral line system are reported. Those studies find that the development of the mechanicoreceptor is controled by Shh, Wnt, RA and other signal. Since the mechanicoreceptor and the electroreceptor are both originated from the common ancestoral lateral line placodes, then how the same lateral line placodes differentiate to give rise of two different receptors, the mechno-neuromasts and the electro-ampullary organs? What roles do these gene/signals play in the process of differentiation? We used transcriptome sequencing, whole mount in situ hybridization to study the differentiation mechanism of mechanoreceptors and electroreceptors of the lateral line system in Siberian sturgeon at the molecular level. Siberian sturgeon(Acipenser baerii) belongs to Osteichthyes, Actinopterygii, Acipenseriformes, which is one of the oldest fish existing on the earth. In addition to have the mechanoreceptors, Siberian sturgeon is one of the few teleost fish with electroreceptors, which occupies extremely important position in the evolution of the fish phylogeny. Therefore, study of the differentiation mechanism of mechanoreceptors and electroreceptors of the lateral line system in sturgeon that provides significant value in understanging of the evolution the lateral line system in fish. 1 Effect of small molecules on development of the lateral line receptors in Siberia sturgeonHedgehog signal, RA signal and canonical Wnt/β-catenin pathway act vital role on the early embryonic development, organ formation, tissue regeneration and other physiological processes. In order to find out that if these signaling pathways playing an important role in the differentiation of neuromasts and ampullary organs of the lateral line receptors in Siberian sturgeon, we used small molecules of 20μM cyclopamine, 0.1μM retinoic acid, 1μM retinoic acid, 10μM DEAB, 20μM DEAB and 50 mM lithium chloride to treat stage 29 of Siberian sturgeon embryo for 4 days, then, to observe the morphological changes and changes in numbers of the neuromasts and ampullary organs by using the optical microscope with or without FM1-43 staining and the scanning electron microscope(SEM). Results showed that cyclopamine not only affected the development of eyes, vibrissas, lips, dorsal fin and caudal fin, but also inhibited the development of the neuromasts and ampullary organs in the Siberian sturgeon. Even more, cyclopamine especially affected the development of ampullary organs. The preliminary results indicated that Hedgehog signaling pathway connected with cyclopamine might be involved in the development of the neuromasts and ampullary organs. In low concentration of retinoic acid group the neuromasts and ampullary organs was not significantly inhibited, that was strongly contrasted with the results in the high concentration retinoic acid group. In the later treatment group, the receptors were significantly inhibited, especially the ampullary organs, which was almost not generated at all. In two different concentration groups of DEAB treatment, the sturgeon larvae did not show any obvious difference in formation rate and the morphological effects while comparing to the control group. Also, there were no formation or morphological effects on the sturgeon larvae in lithium chloride treatment group. The preliminary results indicated that Hedgehog signaling pathway connected with cyclopamine and RA signaling pathway connected with retinoic acid that might be involved in the development of the neuromasts and ampullary organs. 2 Hedgehog signaling is involved in the development of lateral line receptors in Siberia sturgeonBased on the results of small molecules, we further analysed the role of Hedgehog signaling pathway in the development of the lateral line receptors in terms of the gene expression. First, we comparatively analysed the transcriptome of regeneration process of the neuromasts and ampullary organs. We found the differences in expression and dynamic changes of Shh and Patched1 genes through the transcriptome analysis in neuromasts and ampullary organ regeneration. Furthermore, we inhibited Hedgehog signaling by using cyclopamine from stage 29 to stage 37 that resulted the reduction of the numbers of ampullary organs significantely. By using the whole mount in situ hybridization to analyze the expression of Shh, Patched1, Smoothened, Gli2 genes at the molecular level, which resulted that the Shh, Patched1, Smoothened, Gli2 genes were widely expressed in the stage 32 and stage 36, but there were no significant difference of the expression of genes between the treatment and the control groups. From the beginning of stage 41, the expression scope of these genes became narrow, and mainly concentrated in the suborbital and the lips area. In cyclopamine treatment of the Siberian sturgeon, Shh gene was weakly expressed in the ventral suborbital region and strongly expressed in the lips area, and Smoothened gene was weakly expressed in both the ventral suborbital region and the lips area. While comparing the results with the control group, the expression of Shh and Smoothened in treatment groups were slightly decreased, but not significantly. However, Patched1, Gli2 genes in the ventral suborbital region and the lips area of cyclopamine treatment group were almost no expression and the difference was obvious by comparing with the control group. These results indicated that Hedgehog signaling pathway might be related to the development of neuromasts and ampullary organs, which suggested that the Hedgehog signaling might play an important role in the differentiation of neuromasts and ampullary organs.In conclution, in order to find out the target genes and their signaling pathways in developmental differentiation of the two types of lateral line receptors, this study elected the Siberian sturgeon as the model animal due to its evolutionary importance. We first, observed and described the morphology of neuromasts and ampullary organs of the sturgeon larvae at stage 43-44 by using scanning electron microscopy and FM1-43 fluorescence staining, then, we examined and found that cyclopamine, retinoic acid inhibited the development of neuromasts and ampullary organ. Furthermore, we comparatively analysed the transcriptome during the regeneration process of neuromasts and ampullary organs, and found out the key genes of Shh, Patched1, Smoothened, Gli2 on the Hedgehog signaling expression were differentely in two types of receptors. Finally, the results from the whole mount in situ hybridization indicated that the expression of Patched1 and Gli2 were inhibited significantly in the ventral lateral region of the larval by the hedgehog inhibitor cyclopamine. We speculated that Hedgehog was possiblly involved in the differentiation of the neuromasts and ampullary organs in the lateral line placodes. |
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