During embryogenesis genes are expressed in temporal and spatial manner to perform functions. Based on our previous work, two differentially expressed genes, CagMdkb and CagApoA-IV, were identified from the suppression subtractive hybridization (SSH) cDNA plasmid libraries between 10-somite embryos and gastrula embryos in gibel carp. Their expression patterns and functions during embryogenesis were investigated in present study.
Mdk is a secreted protein that plays an important role in neurogenesis and involves in human tumors. In this study, RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during the following embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein is localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal is detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein is also extended to numerous small neurons in forebrain, midbrain and hindbrain, as well as nerve fibers in spinal cord. Co-localization with 3A10 antibody reveals CagMdkb immunoreactivity on the developing Mauthner neuron, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppresses forebrain structures formation, and CagMdkb function depends on the secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish brain formation.
ApoA-IV is a member of apolipoprotein family. Whole-mount in situ hybridization reveals CagApoA-IV is predominantly transcribed at YSL in the early embryos, and later the transcripts are restricted in the liver in 5-day larva. In adult the transcripts are mainly in liver and intestine. The newly synthesized CagApoA-IV protein was also firstly detected in the YSL in early stage. At larvae stage CagApoA-IV protein is abundantly localized in the brain ventricles, in pharynx and gill lamellae, in the endothelium layers of swim bladder, in the epithelial complex of pronephric tubules, in the atria and ventricle of the heart, in the sinusoids of the liver, in the dorsal aorta and intersegmental vessels and in the pancreas islet. When anti-sense morpholinos were injected into 1-cell stage embryos, the expression of the CagApoA-IV was reduced from gastrula stage, which leads to severe deficiencies in embryos and larvae at later stages. The dominant optic deficiencies include the shrink brain, abnormal cardiovascular system and decreased yolk absorbability. The anatomic sections also show severe deficiencies in other mesodermal and endodermal organs such as liver, intestine, kidney, and swim bladder. The data indicate that CagApoA-IV not only plays important roles in digestive system development but also affects brain and cardiovascular system formation which implicates deduced CagApoA-IV expression may related with the diseases in brain and cardiovascular system.