Nie S and Chang C (2007), Regulation of Xenopus gastrulation by ErbB sign...

XB-ART-34976

Dev Biol 2007 Mar 01;3031:93-107. doi: 10.1016/j.ydbio.2006.10.039.

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Regulation of Xenopus gastrulation by ErbB signaling.

Nie S , Chang C .

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Species referenced: Xenopus
Genes referenced: chrd egfr erbb2 erbb3 erbb4 fn1 gsc sox3 tbxt
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	Fig. 2. Inhibition of ErbB signaling did not interfere with mesodermal induction in early frog embryos. (A) Expression of the mesodermal marker Brachyury (Xbra), the dorsal mesodermal marker Chordin and the head mesodermal marker Goosecoid was not blocked by loss of ErbB signaling in frog gastrulae. (B) At neurula stages, transcription of the notochord markers Xbra and chordin as well as the neural marker Sox3 was not impaired. The pattern of dorsal gene expression was wider and shorter in ErbB-inhibited embryos compared with that in control embryos.
	Fig. 1. ErbB signaling regulates Xenopus gastrulation. (A) ErbB4-specific MO blocks induction of ectopic structures by Xenopus, but not by human, ErbB4. RNAs encoding ErbB4 (1–2 ng), with or without 20 ng ErbB4-MO, were injected into the animal region of two-cell stage embryos. (B) Injection of ErbB4-MO (20 ng) into dorsal marginal zone (DMZ) of four-cell stage embryos induced gastrulation defects. Morphants showed delayed blastopore closure, open back, and axial and head defects. (C) Human ErbB4 (5–8 pg) partially rescued gastrulation defects induced by ErbB4-MO. (D) Rescue of gastrulation defects induced by DN-ErbBs (2–4 ng) with wild-type ErbBs (0.2 ng). In panels C and D, embryos with different phenotypes were counted and summarized in the bar graph.


	Fig. 5. Blocking ErbB signaling interfered with bipolar cell morphology and inhibited mediolateral cell intercalation. The membrane-tethered fluorescent proteins EGFP and tdTomato were injected separately into different dorsal blastomeres of two- to four-cell stage embryos, alone or with RNAs encoding DN-ErbBs (2 ng). Dorsal explants were dissected at late gastrula stages (∼ stage 12) and plated on fibronectin-coated coverslip. The explants were then examined at neurula stages. DN-ErbBs blocked bipolar spindle cell shape and cell mixing in the dorsal region.
	Fig. 6. Inhibition of ErbB signaling impaired head mesoderm migration on fibronectin (FN) substratum. (A) Anterior DMZ explants were dissected from stage 10.5–11 embryos and plated on FN-coated dish. Migration of head mesoderm was recorded at 1-h intervals for 6 h. A continuous sheet of cells was seen to migrate away from the control explants, but cells from DN-ErbB-expressing explants did not migrate efficiently and often moved as unconnected individual cells. (B) Quantification of anterior mesendoderm migration. The number and percentage of explants with migratory cells were listed under each sample, and the distances of furthest cell migration in explants containing migratory cells were shown in the bar graph. Student t-test suggested that the differences between the control and the injected samples were significant, with the p-values less than 0.0001. (C) Migration of head mesoderm from ErbB4 morphant embryos was less affected than those from DN-ErbB-expressing explants, but cells dissociated during the course of migration. The cell adhesion defects were rescued by human ErbB4. (D) Quantification of head mesoderm migration from ErbB4 morphant embryos. The percentage of explants maintaining cell adhesion was calculated among the migrated explants. Student t-test indicated that the difference in average migration distances between the control and the ErbB4-MO explants was significant, with the p-value of 1.3E-6.
	Fig. 7. Blocking ErbB signaling affected cell–cell adhesion. DMZ explants were dissected at early gastrula stages and dissociated in CMFB for an hour before reaggregation in MBSH with horizontal shaking. Cells from control explants reaggregated to form large clusters. Cells expressing DN-ErbBs (A) or ErbB4-MO (B) still reaggregated, but they formed smaller and looser clusters.
	Fig. 8. ErbB signaling modulated adhesion of mesodermal cells to fibronectin. Dissociated DMZ cells were plated on FN-coated coverslip for an hour before loose, non-adherent cells were removed with gentle wash. Pictures were taken before and after the wash and the number of the cells on the coverslip was counted. While about 92% of cells from control explants remained attached to the coverslip, 64–82% of cells from explants injected with DN-ErbBs or ErbB4-MO adhered to the glass after the wash. The effect of ErbB4-MO on cell adhesion was rescued by coexpression of human ErbB4 (89% of adherent cells).
	Fig. 9. ErbB signaling controlled cell spreading on fibronectin. (A) Cells from control DMZ explants spread upon binding to FN, but cells from explants injected with DN-ErbBs or ErbB4-MO showed reduced ability to spread. (B) Quantification of cell spreading on FN-coated coverslip. Control samples showed 58% of cells spreading on FN, but 35%, 34%, 27% and 33% of cells expressing DN-EGFR, DN-ErbB2, DN-ErbB3 and DN-ErbB4 spread on FN respectively. 30% of cells expressing ErbB4-MO spread, and the number increased to 52% when human ErbB4 was coexpressed with the MO.
	Fig. 10. ErbB signaling influenced formation of membrane protrusions. Embryos were injected with membrane-tethered EGFP with or without ErbB4-MO and human ErbB4. DMZ explants were dissected and dissociated in CMFB for an hour, and dissociated cells were plated on FN-coated coverslip in MBSH for an hour to allow cell adhesion. Time-lapse movies were made to record the membrane protrusive activities. (A) Control cells showed dynamic lamellipodia and filopodia formation, whereas cells expressing ErbB4-MO did not form these protrusions efficiently. Instead, they had dynamic membrane blebs. The defects in protrusive activities were rescued when ErbB4-MO was coexpressed with human ErbB4 RNA. (B) Quantification of membrane protrusions in different samples. Lamellipodia, filopodia and membrane blebs were counted separately and summarized in the bar graph. An average of 1.3 lamellipodia, 1.7 filopodia and 0.3 membrane blebs per minute were observed in control cells. Expression of ErbB4-MO decreased both lamellipodia and filopodia to 0.3 times per minute, but increased membrane blebs to 0.7 times per minute. Coinjection of human ErbB4 shifted the frequencies of these membrane protrusions back to 0.9, 1.3 and 0.3 times per minute respectively. Student t-test indicated that the differences in protrusive activities between ErbB-MO and control cells were significant (p-values of 2.4E-24, 1.3E-9 and 1.7E-2 for the three different protrusions), while the differences between control and the rescued samples were not significant (p-values of 0.08, 0.19 and 0.35 for the three protrusions). The total numbers of cells counted were given under the bar graph.

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