|Other Abstract||C1q family proteins with C1q domain have been reported in vertebrates, but their biological roles are currently unknown. In this study, a C1q-like factor, designated Carassius auratus gibelio C1q-like factor (CagOC1q-like), was identified as a cortical granules component. Immunofluorescence localization revealed that the C1q family member was specifically expressed in follicular epithelial cells, and associated with cortical granules in fully grown oocytes. Moreover, it was discharged to the perivitelline space and egg envelope upon fertilization. As it is the first identified C1q family member that is expressed in follicular cells that surround oocyte, CagOC1q-like was applied to detection of follicular cell apoptosis and deletion. The entire cytological process of follicular cell apoptosis and deletion was clearly seen from double visualizations of follicular cells with CagOC1q-like immunofluorescence and apoptotic follicular cells labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) during oocyte maturation and ovulation.
The molecular characterizations of C1q-like factor were almost the same in gonochoristic color crucian carp (abbreviated CaOC1q) and gynogenetic gibel crucian carp(abbreviated CagOC1q), in previous studies. However, difference transcription level is found between CaOC1q and CagOC1q in mature eggs. The former was much higher than the latter. Interestingly, the transcription level of CagOC1q was almost under detection, compared to relatively high level of CaOC1q between ovulation and cardiopalmus stage. RNA in situ hybridization on cardiopalmus stage embryos revealed the specific expression of CaOC1q in the genital ridge, where little expression of CagOC1q was found. In order to study the role of C1q-like factor during embryo development, a morpholino antisense oligonucleotide (MO) was designed complementary to the putative translation initiation start site of both CaOC1q and CagOC1q mRNA. And the knockdown of C1q-like resulted in loss of PGCs in color crucian carp, whereas no obvious change of PGCs in gibel crucian carp. Perhaps our present studies will give new clue about how C1q family makes its function in reproduction and development.
We further study the functions of C1q-like in model organism, zebrafish. Through comprehensive searches of C1qDC (C1q-domain-containing) gene in zebrafish genome, cDNA and EST databases, we discovered zebrafish C1q-like, the the closest homologue of CaOC1q-like factor. The transcription and translation of C1q-like display a uniform distribution in early embryos, and are restricted to mid-hind brain and eye in later embryos. In vitro studies showed that C1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing caspase 3/9 activities. Moreover, its physiological roles were studied by morpholino-mediated knockdown in zebrafish embryogenesis. In comparison with control embryos, the C1q-like knockdown embryos display obvious defects in the head and craniofacial development mediated through p53-induced apoptosis, which was confirmed by the in vitro transcribed C1q-like mRNA or p53 MO co-injection. TUNEL assays revealed extensive cell death, and caspase 3/9 activity measurement also revealed about two folds increase in C1q-like morphant embryos, which was inhibited by p53 MO co-injection. Real-time quantitative PCR showed the upregulation expression of several apoptosis regulators such as p53, mdm2, p21, Bax and caspase 3, and down-regulation expression of hbae1 in the C1q-like morphant embryos. Knockdown of C1q-like in zebrafish embryos decreased hemoglobin production and impaired the organization of mesencephalic vein and other brain blood vessels. Interestingly, exposure of zebrafish embryos to UV resulted in an increase in mRNA expression of C1q-like, whereas over-expression of C1q-like was not enough resist to the damage. Furthermore, C1q-like transcription was up-regulated in response to pathogen Aeromonas hydrophila, and embryo survival significantly decreased in the C1q-like morphants after exposure to the bacteria. The data suggested that C1q-like might play an antiapoptotic and protective role in inhibiting p53-dependent and caspase 3/9-mediated apoptosis during embryogenesis, especially in the brain development, and C1q-like should be a novel regulator of cell survival during zebrafish embryogenesis.
It has been shown that mutations in at least four myotubularin family genes (MTM1, MTMR1, 2 and 13) are causative for human neuromuscular disorders. However, the pathway and regulative mechanism remain unknown. Here, we reported a new role for Mtmr8 in neuromuscular development of zebrafish. Firstly, we cloned and characterized zebrafish Mtmr8, and revealed the expression pattern predominantly in the eye field and somites during early somitogenesis. Using morpholino knockdown, then, we observed that loss-of-function of Mtmr8 led to defects in somitogenesis. Subsequently, the possible underlying mechanism and signal pathway were examined. We first checked the Akt phosphorylation, and observed an increase of Akt phosphorylation in the morphant embryos. Furthermore, we studied the PH/G domain function within Mtmr8. Although the PH/G domain deletion by itself did not result in embryonic defect, addition of PI3K inhibitor LY294002 did give a defective phenotype in the PH/G deletion morphants, indicating that the PH/G domain was essential for Mtmr8’s function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and found that both Mtmr8-MO1 and Mtmr8-MO2+LY294002 led to the disorganization of the actin cytoskeleton. In addition, we revealed a possible participation of Mtmr8 in the Hedgehog pathway, and cell transplantation experiments showed that Mtmr8 worked in a non-cell autonomous manner in actin modeling. The above data indicate that a conserved functional cooperation of Mtmr8 with PI3K regulates actin filament modeling and muscle development in zebrafish, and reveal a possible participation of Mtmr8 in the Hedgehog pathway. Therefore, this work provides a new clue to study the physiological function of MTM family members.
Embryonic morphogenesis of vascular and muscular systems is tightly coordinated, and recent studies revealed that some pathways, such as Hedgehog and PI3K, control both systems. In previous studies, we have revealed the function of Mtmr8 in regulating early muscle development through hedgehog and PI3K/Akt pathway. Later expression of Mtmr8 in the vasculature, suggests that zebrafish Mtmr8 may have more extensively functions during embryo development. Knockdown of Mtmr8 by morpholino resulted in severe defects in vascular development, including retardation in intersegmental vessel development and interruption of trunk dorsal aorta where hematopoietic stem cells arise, that might lead to a reduction in hematopoiesis. Moreover, Mtmr8 morphants showed loss of arterial endothelial cell identity in dorsal aorta, as indicated by the reduced or absent expression of the arterial marker EphrinB2a, which was effectively rescued by low concentration of PI3K inhibitor, and by over-expression of dnPKA mRNA or Vegf mRNA. Furthermore, the expression of Mtmr8 was especially migrated to the artery when treated with specific inhibitors of both PI3K and Hedgehog pathway, which abolished the expression of arterial marker. These data demonstrated that Mtmr8 might play a protect role in arterial differentiation through repressing the activity of PI3K, representing a novel element of the Hedgehog/PI3K/VEGF signaling cascade that controls arterial/venous fate.|