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Hox genes control vertebrate body elongation by collinear Wnt repression.

Denans N, Iimura T, Pourquié O - Elife (2015)

Bottom Line: Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength.This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process.Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, University of Strasbourg, Illkirch, France.

ABSTRACT
In vertebrates, the total number of vertebrae is precisely defined. Vertebrae derive from embryonic somites that are continuously produced posteriorly from the presomitic mesoderm (PSM) during body formation. We show that in the chicken embryo, activation of posterior Hox genes (paralogs 9-13) in the tail-bud correlates with the slowing down of axis elongation. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength. This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process. Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size. This ultimately brings the retinoic acid (RA)-producing segmented region in close vicinity to the tail bud, potentially accounting for the termination of segmentation and axis elongation.

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Epiblast cells overexpressing Hox genes do not convertto a neural fate.(A) Transverse section of a stage 7 HH chicken embryo labeledwith phalloidin (white) to highlight the actin network and with laminin(red) to identify the epiblast basal membrane. Colored boxes indicate thedifferent phases of differentiation of the mesoderm: epiblast (purple),ingressing cells (yellow), and mesoderm (blue).(B–E″) Transverse sections at thePSM progenitors level 5 hr after electroporation of a control Venus or ofHoxa13. (B-C”) Lamininimmunolabeling (red) after Venus(B–B″) or Hoxa13over-expression (C–C″).(D–E″) Acetylatedα-tubulin immunolabeling (red) after Venus(D–D″) or Hoxa13(E–E″) over-expression.(F–G) Transverse cryosections of theanterior primitive streak of an embryo electroporated with Venus(F) or with Venus and Hoxa13(G). White arrow: cells ingressed in the primitive streak(F) and non-ingressed epiblast cells (G).Green: Venus; red: laminin; blue: nuclei. (H) Quantification ofingression in embryos electroporated with control orHoxa13-expressing constructs.(I–J) In situ hybridization of 2-day oldchicken embryos electroporated with Venus (I) orHoxa13-Venus (J) expressing vectors. Leftpanel shows Sox2 expression in the neural tube andtail-bud, and right panels show GFP immunohistochemistry. (K)Chicken embryo consecutively electroporated with a control (Cherry, red) anda mix of Hoxa13+DN-RhoA (Venus, green). Arrowheads:anterior boundary of Cherry (red) and Venus (green) domains. Stars: p-valueof two-tailed Student's t-test applied between thedifferent conditions. ***p < 0.005. Error bars:standard error to the mean (SEM).DOI:http://dx.doi.org/10.7554/eLife.04379.010
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fig4: Epiblast cells overexpressing Hox genes do not convertto a neural fate.(A) Transverse section of a stage 7 HH chicken embryo labeledwith phalloidin (white) to highlight the actin network and with laminin(red) to identify the epiblast basal membrane. Colored boxes indicate thedifferent phases of differentiation of the mesoderm: epiblast (purple),ingressing cells (yellow), and mesoderm (blue).(B–E″) Transverse sections at thePSM progenitors level 5 hr after electroporation of a control Venus or ofHoxa13. (B-C”) Lamininimmunolabeling (red) after Venus(B–B″) or Hoxa13over-expression (C–C″).(D–E″) Acetylatedα-tubulin immunolabeling (red) after Venus(D–D″) or Hoxa13(E–E″) over-expression.(F–G) Transverse cryosections of theanterior primitive streak of an embryo electroporated with Venus(F) or with Venus and Hoxa13(G). White arrow: cells ingressed in the primitive streak(F) and non-ingressed epiblast cells (G).Green: Venus; red: laminin; blue: nuclei. (H) Quantification ofingression in embryos electroporated with control orHoxa13-expressing constructs.(I–J) In situ hybridization of 2-day oldchicken embryos electroporated with Venus (I) orHoxa13-Venus (J) expressing vectors. Leftpanel shows Sox2 expression in the neural tube andtail-bud, and right panels show GFP immunohistochemistry. (K)Chicken embryo consecutively electroporated with a control (Cherry, red) anda mix of Hoxa13+DN-RhoA (Venus, green). Arrowheads:anterior boundary of Cherry (red) and Venus (green) domains. Stars: p-valueof two-tailed Student's t-test applied between thedifferent conditions. ***p < 0.005. Error bars:standard error to the mean (SEM).DOI:http://dx.doi.org/10.7554/eLife.04379.010

Mentions: To analyze the effect of posterior Hox genes on ingression, PMprecursors were electroporated with Venus and a Hoxa13 or a controlconstruct and harvested after 5 hr when the electroporated cells start to ingress. Noectopic expression of laminin (Figure4A,B–C″), acetylated tubulin (Figure 4A,D–E″), or E-cadherin (data not shown) was observedafter Hoxa13 over-expression. We compared the number ofVenus-positive cells in epiblast vs primitive streak and mesoderm in embryo sections.The majority of Hoxa13-expressing cells were still found in theepiblast while control cells have ingressed into the primitive streak and mesodermindicating that Hoxa13 delays ingression by retaining cells in theepiblast (Figure 4F–H, n = 4embryos for each condition). No up-regulation of the neural markerSox2 was observed in the tail-bud of embryos electroporated withHoxa13 (Figure4I–J, n = 8 embryos for each condition) and very few cells wereobserved in the neural tube of embryos electroporated with Hox constructs (see Figure 3B, Figure4I–J, Figure 6A–B, Figure 7D–J and Figure 9A andVideos 4–8). This indicates that the effect on ingression is not caused by therecruitment of PM precursor cells to a neural fate. Ingression of cells from theepiblast to the primitive streak occurs via an epithelium to mesenchyme transitionwhich involves first destabilization and then complete loss of basal microtubules inthese cells. This process has been shown to be regulated by a basally localizedactivity of the small GTPase RhoA (Nakaya et al.,2008). In order to test if the effect of the posterior Hoxgenes on delaying PSM progenitors ingression could involve regulation of microtubulestability, we used a dominant negative form of RhoA (DN-RhoA) as atool to destabilize basal microtubules in the epiblast (Nakaya et al., 2008). We performed consecutive electroporationsat stage 5 HH to overexpress a control Cherry vector in one population of PSMprogenitors and Hoxa13 with DN-RhoA vectors inanother population and allowed the embryos to develop for 20 hr. We observed that thetwo populations of cells reach the same anterior level (Figure 4K, n = 5/5 embryos) indicating that these cellsingressed at the same time. Altogether, theseresults suggest that Hox genes control PSM progenitors ingressionthrough the regulation of basal microtubule stability in the epiblast.10.7554/eLife.04379.010Figure 4.Epiblast cells overexpressing Hox genes do not convertto a neural fate.


Hox genes control vertebrate body elongation by collinear Wnt repression.

Denans N, Iimura T, Pourquié O - Elife (2015)

Epiblast cells overexpressing Hox genes do not convertto a neural fate.(A) Transverse section of a stage 7 HH chicken embryo labeledwith phalloidin (white) to highlight the actin network and with laminin(red) to identify the epiblast basal membrane. Colored boxes indicate thedifferent phases of differentiation of the mesoderm: epiblast (purple),ingressing cells (yellow), and mesoderm (blue).(B–E″) Transverse sections at thePSM progenitors level 5 hr after electroporation of a control Venus or ofHoxa13. (B-C”) Lamininimmunolabeling (red) after Venus(B–B″) or Hoxa13over-expression (C–C″).(D–E″) Acetylatedα-tubulin immunolabeling (red) after Venus(D–D″) or Hoxa13(E–E″) over-expression.(F–G) Transverse cryosections of theanterior primitive streak of an embryo electroporated with Venus(F) or with Venus and Hoxa13(G). White arrow: cells ingressed in the primitive streak(F) and non-ingressed epiblast cells (G).Green: Venus; red: laminin; blue: nuclei. (H) Quantification ofingression in embryos electroporated with control orHoxa13-expressing constructs.(I–J) In situ hybridization of 2-day oldchicken embryos electroporated with Venus (I) orHoxa13-Venus (J) expressing vectors. Leftpanel shows Sox2 expression in the neural tube andtail-bud, and right panels show GFP immunohistochemistry. (K)Chicken embryo consecutively electroporated with a control (Cherry, red) anda mix of Hoxa13+DN-RhoA (Venus, green). Arrowheads:anterior boundary of Cherry (red) and Venus (green) domains. Stars: p-valueof two-tailed Student's t-test applied between thedifferent conditions. ***p < 0.005. Error bars:standard error to the mean (SEM).DOI:http://dx.doi.org/10.7554/eLife.04379.010
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Related In: Results  -  Collection

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fig4: Epiblast cells overexpressing Hox genes do not convertto a neural fate.(A) Transverse section of a stage 7 HH chicken embryo labeledwith phalloidin (white) to highlight the actin network and with laminin(red) to identify the epiblast basal membrane. Colored boxes indicate thedifferent phases of differentiation of the mesoderm: epiblast (purple),ingressing cells (yellow), and mesoderm (blue).(B–E″) Transverse sections at thePSM progenitors level 5 hr after electroporation of a control Venus or ofHoxa13. (B-C”) Lamininimmunolabeling (red) after Venus(B–B″) or Hoxa13over-expression (C–C″).(D–E″) Acetylatedα-tubulin immunolabeling (red) after Venus(D–D″) or Hoxa13(E–E″) over-expression.(F–G) Transverse cryosections of theanterior primitive streak of an embryo electroporated with Venus(F) or with Venus and Hoxa13(G). White arrow: cells ingressed in the primitive streak(F) and non-ingressed epiblast cells (G).Green: Venus; red: laminin; blue: nuclei. (H) Quantification ofingression in embryos electroporated with control orHoxa13-expressing constructs.(I–J) In situ hybridization of 2-day oldchicken embryos electroporated with Venus (I) orHoxa13-Venus (J) expressing vectors. Leftpanel shows Sox2 expression in the neural tube andtail-bud, and right panels show GFP immunohistochemistry. (K)Chicken embryo consecutively electroporated with a control (Cherry, red) anda mix of Hoxa13+DN-RhoA (Venus, green). Arrowheads:anterior boundary of Cherry (red) and Venus (green) domains. Stars: p-valueof two-tailed Student's t-test applied between thedifferent conditions. ***p < 0.005. Error bars:standard error to the mean (SEM).DOI:http://dx.doi.org/10.7554/eLife.04379.010
Mentions: To analyze the effect of posterior Hox genes on ingression, PMprecursors were electroporated with Venus and a Hoxa13 or a controlconstruct and harvested after 5 hr when the electroporated cells start to ingress. Noectopic expression of laminin (Figure4A,B–C″), acetylated tubulin (Figure 4A,D–E″), or E-cadherin (data not shown) was observedafter Hoxa13 over-expression. We compared the number ofVenus-positive cells in epiblast vs primitive streak and mesoderm in embryo sections.The majority of Hoxa13-expressing cells were still found in theepiblast while control cells have ingressed into the primitive streak and mesodermindicating that Hoxa13 delays ingression by retaining cells in theepiblast (Figure 4F–H, n = 4embryos for each condition). No up-regulation of the neural markerSox2 was observed in the tail-bud of embryos electroporated withHoxa13 (Figure4I–J, n = 8 embryos for each condition) and very few cells wereobserved in the neural tube of embryos electroporated with Hox constructs (see Figure 3B, Figure4I–J, Figure 6A–B, Figure 7D–J and Figure 9A andVideos 4–8). This indicates that the effect on ingression is not caused by therecruitment of PM precursor cells to a neural fate. Ingression of cells from theepiblast to the primitive streak occurs via an epithelium to mesenchyme transitionwhich involves first destabilization and then complete loss of basal microtubules inthese cells. This process has been shown to be regulated by a basally localizedactivity of the small GTPase RhoA (Nakaya et al.,2008). In order to test if the effect of the posterior Hoxgenes on delaying PSM progenitors ingression could involve regulation of microtubulestability, we used a dominant negative form of RhoA (DN-RhoA) as atool to destabilize basal microtubules in the epiblast (Nakaya et al., 2008). We performed consecutive electroporationsat stage 5 HH to overexpress a control Cherry vector in one population of PSMprogenitors and Hoxa13 with DN-RhoA vectors inanother population and allowed the embryos to develop for 20 hr. We observed that thetwo populations of cells reach the same anterior level (Figure 4K, n = 5/5 embryos) indicating that these cellsingressed at the same time. Altogether, theseresults suggest that Hox genes control PSM progenitors ingressionthrough the regulation of basal microtubule stability in the epiblast.10.7554/eLife.04379.010Figure 4.Epiblast cells overexpressing Hox genes do not convertto a neural fate.

Bottom Line: Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength.This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process.Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, University of Strasbourg, Illkirch, France.

ABSTRACT
In vertebrates, the total number of vertebrae is precisely defined. Vertebrae derive from embryonic somites that are continuously produced posteriorly from the presomitic mesoderm (PSM) during body formation. We show that in the chicken embryo, activation of posterior Hox genes (paralogs 9-13) in the tail-bud correlates with the slowing down of axis elongation. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength. This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process. Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size. This ultimately brings the retinoic acid (RA)-producing segmented region in close vicinity to the tail bud, potentially accounting for the termination of segmentation and axis elongation.

Show MeSH
Related in: MedlinePlus