Velloso I et al. (2026), Cell division during Xenopus gastrulation influ...

XB-ART-61841

Front Cell Dev Biol 2026 Apr 22;14:1798565. doi: 10.3389/fcell.2026.1798565.

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Cell division during Xenopus gastrulation influences neuroectoderm patterning.

Velloso I, Araujo R, Horb M, Abreu JG.

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Oriented cell division is emerging as a fundamental morphogenetic mechanism driving tissue elongation and axis formation. Although Xenopus laevis embryos can undergo gastrulation and neurulation in the absence of cell division, the specific contribution of mitosis during gastrulation remains poorly defined. Here, we systematically examine the role of cell division during gastrulation in neural plate formation and anterior-posterior (A-P) patterning. Using Hydroxyurea and Aphidicolin (HUA) to block cell division, combined with in situ hybridization and time-lapse imaging, we analyzed division dynamics and developmental outcomes in the dorsal mesoderm and neuroectoderm. Consistent with previous work, we find that cell division is dispensable for dorsal mesoderm patterning and neural tube closure. Strikingly, however, inhibition of cell division during gastrulation profoundly disrupts neural plate patterning, leading to severe head and trunk defects that manifest at tailbud stages. This phenotype is accompanied by markedly reduced expression of key anterior neural markers (bf1 and krox20), indicating a failure to properly specify the forebrain, midbrain, and hindbrain domains. These defects reveal an essential role for cell division in early neural regionalization rather than in tissue formation per se. Moreover, quantitative analysis shows that cell divisions are abundant throughout the gastrulating ectoderm and exhibit strong A-P orientation, particularly in the dorsal posterior region. Together, our results identify A-P oriented cell division as a conserved and previously underappreciated mechanism required for neural plate elongation and anterior neural patterning during vertebrate development.

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Species referenced: Xenopus laevis
Genes referenced: egr2 foxg1 myod1 not sox2
GO keywords: regulation of gastrulation [+]
???displayArticle.antibodies??? H3f3a Ab45
Lines/Strains:

Xla.Tg(CMV:hist2h2be-GFP;CMV:mRFPFlc

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	Graphical Abstract
	FIGURE 1. Blocking cell division during gastrulation. (A) Embryos were treated with a combination of Hydroxyurea and Aphidicolin (HUA) or DMSO from stage 9.5 to stage 12.5 and then transferred to Barth’s 0.1X solution starting at stage 12.5. Wash off moment is indicated in red (sages illustrated according to Nieuwkoop and Faber, 1994). (B) Dorsal view of a representative embryo treated with DMSO, showing the neural tube closure steps from stage 14 to stage 20. (C) Dorsal view of a representative embryo treated with HUA, showing the neural tube closure steps from stage 14 to stage 20. 14 out of 17 HUA-treated embryos showed the phenotype presented in C (embryos in B and C placed anterior right and posterior left). Three embryos showed a normal phenotype, similar to all DMSO-treated embryos (D) Dorsal view of an embryo treated with DMSO at stage 13 and immunostained for Histone H2B, showing cells in division. (E) Dorsal view of an embryo treated with HUA at stage 13 and immunostained for Histone H2B. (F) Transversal section of a DMSO-treated embryo at stage 20, showing notochord, paraxial mesoderm, and neural tube. (G) Transversal section of an HUA-treated embryo at stage 20, showing notochord, paraxial mesoderm, and neural tube. The vertical black line in A and B indicates the site where the transversal section was made.
	FIGURE 2. Blocking cell division during gastrulation leads to head and trunk defects. (A) Embryos were treated with a combination of Hydroxyurea and Aphidicolin (HUA) or DMSO from stage 9.5 to stage 12.5 and then transferred to Barth’s 0.1X solution starting at stage 12.5 (stages illustrated according to Nieuwkoop and Faber, 1994) (B) Lateral view of a representative DMSO-treated embryo at stage 25. Wash off moment is indicated in red. (B′) Ventral-anterior view of a representative DMSO-treated embryo at stage 25. (C) Lateral view of a representative DMSO-treated embryo at stage 25. (C′) Ventral-anterior view of a representative HUA-treated embryo at stage 25. (D) Phenotype quantification at stage 25. (E) Lateral view of a representative DMSO-treated embryo at stage 43. (E′) Dorsal-anterior view of a representative DMSO-treated embryo at stage 43. (E′′) Ventral-anterior view of a representative DMSO-treated embryo at stage 43. (F) Lateral view of a representative HUA-treated embryo at stage 43. (F′) Dorsal-anterior view of a representative HUA-treated embryo at stage 43. (F′′) Ventral-anterior view of a representative HUA-treated embryo at stage 43. (G) Phenotype quantification at stage 43.
	FIGURE 3. Blocking cell division during neurulation and tailbud stages does not cause head or trunk defects. (A) Embryos were kept in Barth’s 0.1X solution until stage 12.5, then transferred to HUA or DMSO solution, where they remained from stage 12.5 to stage 25, before being returned to Barth’s 0.1X solution. Wash off moment is indicated in red (sages illustrated according to Nieuwkoop and Faber, 1994). (B) Lateral view of a representative DMSO-treated embryo at stage 25. (B′) Ventral-anterior view of a representative DMSO-treated embryo at stage 25. (C) Lateral view of a representative HUA-treated embryo at stage 25. (C′) Ventral-anterior view of a representative HUA-treated embryo at stage 25. (D) Phenotype quantification at stage 25. (E) Lateral view of a representative DMSO-treated embryo at stage 46. (E′) Dorsal-anterior view of a representative DMSO-treated embryo at stage 46. (E′′) Ventral-anterior view of a representative DMSO-treated embryo at stage 46. (F) Lateral view of a representative HUA-treated embryo at stage 46. (F′) Dorsal-anterior view of a representative HUA-treated embryo at stage 46. (F′′) Ventral-anterior view of a representative HUA-treated embryo at stage 46. (G) Phenotype quantification at stage 46. (stages illustrated according to Nieuwkoop and Faber, 1994).
	FIGURE 4. Blocking cell division during gastrulation affects neural plate shaping and patterning. (A) Dorsal view of five embryos at stage 13 treated with DMSO during gastrulation. (B) Dorsal view of five embryos at stage 13 treated with HUA during gastrulation. Arrowheads indicate the lateral hinges of the neural plate. Anterior is always at the top and posterior at the bottom. In situ hybridizations were conducted with embryos at stage 13 treated with DMSO (C,E,G,G′,I) or HUA (D,F,H,H′,J). The embryos were marked for xnot (C,D), myod (E,F), Sox2 (G,G′,H,H′), and krox20/bf1 (I,J). Two head arrows in (G) and (H) indicate the Sox2 expression domain width at the most dorsal portion of the embryo and are the same size. Dashed lines in (G′) and (H′) indicate the width of the most anterior region of the sox2 domain in each condition. Purple arrows in (I) indicate Krox20 expression domains, while black arrows in (I) and (J) indicate the Bf1 domain. Quantification of each expression pattern is shown in (K) for xnot, in (L) for myoD, in (M) for Sox2 and in (N) for krox20/bf1. All embryos (with the exceptions of (G′,H′,I,J)) are placed anterior up and posterior down.
	FIGURE 5. Mitotic cell divisions show strong anterior-posterior (A–P) orientation across the embryo’s ectoderm with higher strength at dorsal region. (A) The three distinct areas that were imaged are indicated in panel as the embryo goes throughout gastrulation and neurulation. Pink indicates the dorsal region; green denotes the left-ventral region; and blue represents the right-ventral region (sages illustrated according to Nieuwkoop and Faber, 1994). (B) The rate of cell divisions during the stages of gastrulation and neurulation, categorized by areas. (C) Rose plot showing the percentage (radial axis) of cell divisions in the left-ventral area that fall into each angular orientation. 90°–270° corresponds to the Anterior-Posterior (A–P) axis. (D) Rose plot showing the percentage (radial axis) of cell divisions in the dorsal area that fall into each angular orientation. (E) Rose plot showing the percentage (radial axis) of cell divisions in the right-ventral area that fall into each angular orientation. n: number of cell divisions; M.A.: Mean angle; v: Signed polarity index; (C). SD: Circular standard deviation. (F) Comparison of the signed polarity index calculated for each area were made using two-tailed Welch’s t-test and Bonferroni correction. (p < 0.01); (p < 0.001). The graphics from (B–E) reflect the three time-lapses performed from different angles of the same embryo exhibiting fluorescent nucleus-NXR_0139 Xla.Tg (CMV:hist2h2be-GFP; CMV:mRFP. (G) Keller explant performed from embryos with fluorescent nucleus-NXR_0139 Xla.Tg (CMV:hist2h2be-GFP; CMV:mRFP. Dorsal involuting marginal zone (DIMZ) and Dorsal non-involuting marginal zone (DNIMZ) are indicated. (H) Comparison of the signed polarity index calculated for each area. **(p < 0.001). Other parameters: DIMZ (number of cell divisions = 54; mean angle = 56.2°; Circular standard deviation = 36.9°) and DNIMZ (number of cell divisions = 141; mean angle = 84.9°; Circular standard deviation = 19.5°).
	SUPPLEMENTARY FIGURE 1. Blocking cell division during gastrulation leads to head and trunk defects. (A) Embryos were treated with a combination of Hydroxyurea and Aphidicolin (HUA) or DMSO from stage 9.5 to stage 11.5 and then transferred to Barth’s 0.1X solution starting at stage 11.5. Wash off moment is indicated in red. (B) Lateral view of a representative DMSO-treated embryo at stage 22. (B′) Dorsal view of a representative DMSO-treated embryo at stage 22. (C) Lateral view of a representative HUA-treated embryo at stage 22. (C′) Dorsal view of a representative HUA-treated embryo at stage 22. (D) Lateral view of a representative DMSO-treated embryo at stage 28. (D′) Ventral-anterior view of a representative DMSO-treated embryo at stage 28. (E) Lateral view of a representative HUA-treated embryo at stage 28. (E′) Ventral-anterior view of a representative HUA-treated embryo at stage 28. (F) Phenotype quantification at stage 28.
	SUPPLEMENTARY FIGURE 2. Methodology used to count cell divisions (A). The three recording angles (green, pink and blue) were each divided into four quadrants. These quadrants were used to count the number of cell divisions and the orientation of these cell divisions during the gastrulation and neurulation process. (B) The three recording angles illustrated in the embryo at stage 11.5. (C) A 21 min period of the time lapse (8 frames out of 200) within the quadrant 7 showing 4 cell divisions taking place. The cell that is about to divide (single arrowheads) shows chromosome alignment before separation (coupled arrowheads). Metaphase is always observable before separation of the chromosomes (note nucleus morphology pointed by green arrowhead at 0 min; Orange arrowhead at 6 and 9 min; Pink arrowhead at 9 and 12 min; and Red arrowhead at 21 min). Cell division cannot be mistaken by radial intercalation, which is pointed by the blue arrow. In this case a cell from a deeper layer emerges to the more superficial layer without showing any sign of cell division (note how its signal is almost imperceptible at 15 min, than it becomes stronger at 18 min and even more at 21 min). The case of cell intercalation taking place close to a cell division was specifically chosen to show how those two kinds of events are easy to be distinguished. The images were made in a 20X increase.
	SUPPLEMENTARY FIGURE 5. Anterior and posterior halves of each area recorded. (A) anterior left-ventral. (B) anterior dorsal. (C) anterior right-ventral. (D) posterior left-ventral. (E) posterior dorsal. (F) posterior right-ventral. In green, dorsal in pink and right ventral in blue). In each rose plot, radial axis indicates absolute number of cell divisions.90°–270° corresponds to anterior-posterior (A–P) body axis. n: number of cell divisions; M.A.: Mean angle; V: Signed polarity index; C. SD: Circular standard deviation.