XB-ART-52237Sci Rep. September 28, 2016; 6 28297.
Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by hypoxia-inducible factor 2α.
Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons.
PubMed ID: 27323909
PMC ID: PMC4915019
Article link: Sci Rep.
Genes referenced: ccnd1 ccnd2 ccndx dio3 isl1 mut nkx6-1 olig2 vsx1
Article Images: [+] show captions
|Figure 1. Zebrafish cyclin Dx is a member of the cyclin D family, and zccndx is expressed during early development.(A) Phylogenetic tree analysis of zccndx with other cyclin D proteins. Four groups of the cyclin D family, ccnD1, D2, D3, and Dx, exhibited perfect separation. The zccndx protein was grouped with Xenous ccndx. Analysis was performed using the parsimony method with 1000 bootstraps. (B) The zccndx protein binds to Cdk4. COS-1 cells were cotransfected with the indicated constructs, and cell lysates were co-immunoprecipitated (IP) with anti-HA monoclonal antibody; the anti-Myc monoclonal Ab was used for detection. The immunoblot was probed using the indicated antibodies. (C) At 24 hpf, zccndx mRNA was observed to be expressed in the motor neurons of hindbrain and spinal cord (a and a′). At 48 hpf, zccndx mRNA was also expressed in the hindbrain, but not in the spinal cord (b and b′). At 72 hpf, zccndx mRNA was weakly expressed in the hindbrain, with no signal in the spinal cord (c and c′). Dorsal views of zccndx mRNA signal in the hindbrain are shown (a′–c′). Scale bar, 100 mm. (D) Sections were made in the hindbrain at 22 hpf. The zccndx signal is shown in green and the isl1 (panel a), olig2 (panel b), and vsx1 (panel c), and nkx6.1 (panel d) mRNA signals are shown in red. The cartoon (panel e) shows the location of zccndx (green), isl1 (purple), olig2 (orange), vsx1 (blue), and nkx6.1 (red) signals. The Shh (blue) signaling gradient has been created from the floor plate to diffuse along the ventral tube while BMP/Wnt (purple) gradient shows opposite direction. The interaction of the dorsalizing and ventralizing signals defines the proper position of motoneurons. Scale bar, 100 mm.|
|Figure 3. The role of zccndx in cell proliferation and regulation of the numbers of motor neuron progenitors (pMNs).(A) Confocal images showing lateral views of the heads of control-MO-injected (panel a) or ccndx-MO-injected (panel b) embryos stained with anti-pH3 (red in panels a and b) or anti-BrdU (green in d and e). Panels c and f: graphs showing the number of proliferating cells (pH3 or BrdU positive) at 24 hpf. The asterisks indicate significant differences between the control and experimental morphants. The n value is indicated. (B) Whole–mount in situ hybridization of motor neurons and interneuron markers in control-MO and zccndx ATG-MO-injected embryos. Probes against olig2 (a and b), nkx6.1(c and d), isl1 (e and f), and vsx1 (g and h) were used for detection. control MO-injected embryos are shown in panels a, c, e and g while ccndx ATG-MO-injected embryos are shown in panels b, d, f and h. Motor neurons (MN) were lost (panels b, f, lost MNs indicated by arrowheads), while interneurons (IN) (panels d and h) were unaffected. Scale bar, 100 mm.|
|Figure 4. Expression of the ccndx gene is regulated by the HIF2α transcription factor.(A) The promoter region of the ccndx gene contains several HRE sites, as indicated in the schematic diagram (top left). The 2.5-kb, 1-kb, and 0.7-kb promoters are all activated by HIF2α and arnt1a transcription factors. (B) The HIF2α and ARNT1α proteins co-activate ccndx gene expression through two HRE sites in the 700bp promoter region. Data represent mean ± s.d. (n = 3). (C) The DNA binding ability of HIF2α and the ARNT1α complex were confirmed by EMSA. Nuclear extract (NE) from pcDNA3-HA (Con) or pcDNA3-Hif2α-HA and pcDNA3-Arnt1α-HA (HIF2α + ARNT1α) transfection were indicated. Two HRE oligonucleotides (as indicated) were used as probes and normal (Cold) or mutant (Mut) oligonucleotides were used for competition. The asterisks in lanes 2, 4, 6, and 8 indicate the specific binding complex.|