January 1, 2019;
Cdc42 Effector Protein 3 Interacts With Cdc42 in Regulating Xenopus Somite Segmentation.
Somitogenesis is a critical process during vertebrate development that establishes the segmented body plan and gives rise to the vertebra
, skeletal muscles, and dermis
. While segmentation clock
and wave front mechanisms have been elucidated to control the size and time of somite
formation, regulation of the segmentation process that physically separates somites
is not understood in detail. Here, we identified a cytoskeletal player, Cdc42
effector protein 3 (Cdc42ep3, CEP3) that is required for somite
segmentation in Xenopus embryos. CEP3 is specifically expressed in somite tissue
segmentation. Loss-of-function experiments showed that CEP3 is not required for the specification of paraxial mesoderm
, nor the differentiation of muscle
cells, but is required for the segmentation process. Live imaging analysis further revealed that CEP3 is required for cell shape changes and alignment during somitogenesis. When CEP3 was knocked down, somitic cells did not elongate efficiently along the mediolateral axis and failed to undertake the 90° rotation. As a result, cells remained in a continuous sheet without an apparent segmentation cleft. CEP3 likely interacts with Cdc42
during this process, and both increased and decreased Cdc42
activity led to defective somite
segmentation. Segmentation defects caused by Cdc42
knockdown can be partially rescued by the overexpression of CEP3. Conversely, loss of CEP3 resulted in the maintenance of high levels of Cdc42
activity at the cell membrane, which is normally reduced during and after somite
segmentation. These results suggest that there is a feedback regulation between Cdc42
and CEP3 during somite
segmentation and the activity of Cdc42
needs to be fine-tuned to control the coordinated cell shape changes and movement required for somite
[+] show captions
Figure 1. Expression of cdc42ep3 during Xenopus embryogenesis. In situ hybridization analysis shows that cdc42ep3 (CEP3) is specifically expressed in paraxial mesoderm and the developing somite. At stage 17, CEP3 is broadly expressed in the paraxial mesoderm before somite segmentation takes place. During somite segmentation, CEP3 is specifically expressed in segmented somites.
Figure 2. Cdc42ep3-MO affected the expression of somitic genes. (A–C) Embyros were injected with 10 ng of CEP3-MO on one side, and the expression of somitic genes were compared between injected and uninjected sides. At the late gastrula stage (stage 15), CEP3-MO did not affect the expression of mesodermal genes myf5 or myod1 or pan-mesodermal gene tbxt (Xbra). At the early neurula stage (stage 22), the expression of mef2d and myod1 shows a segmented pattern in the uninjected side, but not in the CEP3-MO-injected side (arrows). At late tailbud stages, the segmented expression pattern, but not the overall expression level of myogenic genes myog and myf6 was affected by CEP3-MO. Similar segmentation defect was observed in embryos stained with 12/101 antibody. (D) While 0.3ng CEP3 alone did not induce obvious segmentation defect, coinjection of CEP3 with CEP3-MO partially rescued the segmentation defects. Dorsal view (A, top panels in B, and top panels in D) or lateral view (bottom panels in B,C, and bottom panels in D, showing both sides of the same embryo) of the embryos was shown, with anterior to the left. (E) The percentage of defective embryos were summarized in the bar graph. χ2 test was performed and both CEP3-MO mediated knockdown and CEP3 rescue led to significant difference in the phenotype. *P < 0.01.
Figure 3. Loss of Cdc42ep3 disrupted somite segmentation and cell shape changes. Wilson explants were dissected from embryos receiving membrane-tethered EGFP (EGFP-CAAX) on one side, and CEP3-MO plus EGFP-CAAX on the other side, and the segmentation process was followed by time-lapse microscopy. (A–F) Time frames at 2-h intervals. The dashed lines in (A) marks the boundaries between pre-somitic mesoderm and the notochord. Arrowheads marked segmentation furrows. Two cells on each side of the explant were followed by magenta shade. CEP3-MO not only impaired somite segmentation, but also affected the mediolateral elongation of cells. Scale bar = 100 μm.
Figure 4. Cdc42 needs to be tightly controlled during somite segmentation. (A) 0.05 ng of constitutively active (CA) or dominant negative (DN) Cdc42 was injected into one side of the embryo, and their effect on somite segmentation was examined by the expression of myod1. Comparing to the uninjected control side, the segmented expression pattern of myod1 was lost on the injected side (arrows), suggesting that both increased and decreased activity of Cdc42 will affect somite segmentation. (B) CA-Cdc42 was co-injected with CEP3-MO, and CEP3 or CEP3-MO was co-injected with DN-Cdc42 to determine whether they can rescue segmentation defects caused by CEP3 or Cdc42 knockdown. The percentage of embryos with segmentation defect was summarized in the bar graph. χ2 test indicates that CA-Cdc42 failed to rescue segmentation defects caused by CEP3-MO (p = 0.52), but CEP3 significantly rescued segmentation defects caused by DN-Cdc42 (*p < 0.01). Coinjection of CEP3-MO with DN-Cdc42, on the other hand, further inhibited somite segmentation (p = 0.02 when comparing with DN-Cdc42 alone).
Figure 5. CEP3-MO regulates Cdc42 activity in the developing somite. GFP-wGBD that binds to active Cdc42 was expressed in the paraxial mesoderm alone or together with CEP3-MO. (A) While GFP-wGBD was largely internalized and distributed in a diffused manner in control somitic cells, it was localized to the cell membrane in CEP3-MO-injected cells, reflecting a higher level of Cdc42 activity. (B) An intensity plot of GFP-wGBD across the mediolateral axis of cells was generated. There was a significantly higher level of GFP-wGBD at the cell periphery in CEP3-MO-injected cells and a significantly lower level of GFP-wGBD in the center of CEP3-MO-injected cells. Error bars: standard error of the mean. Student t-test was performed, and the gray area marks places where p > 0.01.