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Regulation of Hoxb4 induction after neurulation by somite signal and neural competence.

Amirthalingam GS, Howard S, Alvarez S, de Lera AR, Itasaki N - BMC Dev. Biol. (2009)

Bottom Line: We also show that the dorsal side of the neural tube has a greater susceptibility to expressing Hoxb4 than the ventral region, a feature associated with dorsalization of the neural tube by BMP signals.BMP4 is additionally able to up-regulate Hoxb4 ventrally, but the effect is restricted to the axial levels at which Hoxb4 is normally expressed, and only in the presence of retinoic acid (RA) or somites, suggesting a role for BMP in rendering the neural tube competent to express Hoxb4 in response to RA or somite signals.In identifying the collaboration between somites and neural tube competence in the induction of Hoxb4, this study demonstrates interplay between A-P and dorsal-ventral (D-V) patterning systems, whereby a specific feature of D-V polarity may be a prerequisite for proper A-P patterning by Hox genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, London, NW7 1AA, UK. saroshi7@yahoo.co.uk

ABSTRACT

Background: While the body axis is largely patterned along the anterior-posterior (A-P) axis during gastrulation, the central nervous system (CNS) shows dynamic changes in the expression pattern of Hox genes during neurulation, suggesting that the CNS refines the A-P pattern continuously after neural tube formation. This study aims at clarifying the role of somites in up-regulating Hoxb4 expression to eventually establish its final pattern and how the neural tube develops a competence to respond to extrinsic signals.

Results: We show that somites are required for the up-regulation of Hoxb4 in the neural tube at the level of somites 1 to 5, the anterior-most domain of expression. However, each somite immediately adjacent to the neural tube is not sufficient at each level; planar signaling is additionally required particularly at the anterior-most segments of the expression domain. We also show that the dorsal side of the neural tube has a greater susceptibility to expressing Hoxb4 than the ventral region, a feature associated with dorsalization of the neural tube by BMP signals. BMP4 is additionally able to up-regulate Hoxb4 ventrally, but the effect is restricted to the axial levels at which Hoxb4 is normally expressed, and only in the presence of retinoic acid (RA) or somites, suggesting a role for BMP in rendering the neural tube competent to express Hoxb4 in response to RA or somite signals.

Conclusion: In identifying the collaboration between somites and neural tube competence in the induction of Hoxb4, this study demonstrates interplay between A-P and dorsal-ventral (D-V) patterning systems, whereby a specific feature of D-V polarity may be a prerequisite for proper A-P patterning by Hox genes.

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Dorsal expression of Hoxb4 is preceded by dorsalization of the neural tube by BMP signals. (A-D) Flat-mounted neural tubes from embryos electroporated with BMP4, incubated for 6–8 hours and assayed for Hoxb4 (A) and Msx1 (C). Electroporation of BMP4 results in a ventral expansion of Hoxb4 (A, n = 25/30) and Msx1 (C, n = 14/15) within 6–8 hours. Scale bar; 100 μm. (E-H) Electroporation of Smad6 followed by 24 hours of incubation does not cause any obvious changes in Hoxb4 expression pattern (E, n = 23/25. 2/25 showed down-regulation). Msx1 expression is down-regulated at the electroporated side (G, n = 19/20). Scale bar; 200 μm. (I-L) Electroporation of Smad6 followed by 48 hours of incubation causes down-regulation of both Hoxb4 (I, n = 19/21) and Msx1 (K, n = 18/20). Arrows indicate changes in the expression compared to the contralateral side. (B, D, F, H, J, L) shows electroporated, GFP-positive cells in (A, C, E, G, I, K), respectively. Arrowheads in (A, E, I) show the rhombomere 6/7 boundary (r6/7). Scale bar; 200 μm.
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Figure 8: Dorsal expression of Hoxb4 is preceded by dorsalization of the neural tube by BMP signals. (A-D) Flat-mounted neural tubes from embryos electroporated with BMP4, incubated for 6–8 hours and assayed for Hoxb4 (A) and Msx1 (C). Electroporation of BMP4 results in a ventral expansion of Hoxb4 (A, n = 25/30) and Msx1 (C, n = 14/15) within 6–8 hours. Scale bar; 100 μm. (E-H) Electroporation of Smad6 followed by 24 hours of incubation does not cause any obvious changes in Hoxb4 expression pattern (E, n = 23/25. 2/25 showed down-regulation). Msx1 expression is down-regulated at the electroporated side (G, n = 19/20). Scale bar; 200 μm. (I-L) Electroporation of Smad6 followed by 48 hours of incubation causes down-regulation of both Hoxb4 (I, n = 19/21) and Msx1 (K, n = 18/20). Arrows indicate changes in the expression compared to the contralateral side. (B, D, F, H, J, L) shows electroporated, GFP-positive cells in (A, C, E, G, I, K), respectively. Arrowheads in (A, E, I) show the rhombomere 6/7 boundary (r6/7). Scale bar; 200 μm.

Mentions: We further examined whether the up-regulation of Hoxb4 by BMP4 is a direct effect of activation of BMP pathway, or as a consequence of the dorsalized feature of the neural tube. Consistent with the result of embryo cultures with exogenous BMP4, electroporation of BMP4 in the neural tube at the 5 somite stage followed by 6–8 hours of incubation caused a noticeable up-regulation of Hoxb4 expanded toward the ventral side (Fig. 8A, B). This was accompanied by up-regulation of other dorsal neural markers such as Msx1 (Fig. 8C, D) and Pax7 (data not shown). Electroporation of a GFP construct did not show any changes (data not shown). Hence, the D-V pattern of the neural tube has already been altered by the time we observe the changes in the Hoxb4 expression. Next, the same stage of neural tube was electroporated with Smad6 to test the requirement of BMP signals for Hoxb4 expression. Smad6 blocks transduction of BMP and TGFβ signals at the intracellular level [43]. Smad6 successfully inhibited Hoxb4 expression at the dorsal side of the neural tube (Fig. 8I, J). However, the down-regulation was not seen at 6 (data not shown) or 24 hours (Fig. 8E, F) but at 48 hours of incubation (Fig. 8I, J), which was presumably due to the late onset of exogenous Smad6 expression, which might not be prompt enough to override the endogenous programme. In fact, Smad6 electroporation caused down-regulation of Msx1/2 not at 6 hours, but after 24 (Fig. 8G, H and data not shown) and 48 hours (Fig. 8K, L and data not shown). These data suggest that Hoxb4 expression is preceded by the BMP signal-dependent dorsalization of the neural tube. It should be noted that the dorsalizing activity of exogenous BMP4 could be mediated by other members of the TGFβ super family whose function BMP4 can mimic. Because of the delay in the change in Hoxb4 following Smad6 electroporation, and the fact that no known Smad binding sites have been identified in Hox gene enhancer elements, it is likely that up-regulation of Hoxb4 is due to the dorsal feature of the neural tube induced by BMP signals, rather than the direct effect of activation of the BMP pathway.


Regulation of Hoxb4 induction after neurulation by somite signal and neural competence.

Amirthalingam GS, Howard S, Alvarez S, de Lera AR, Itasaki N - BMC Dev. Biol. (2009)

Dorsal expression of Hoxb4 is preceded by dorsalization of the neural tube by BMP signals. (A-D) Flat-mounted neural tubes from embryos electroporated with BMP4, incubated for 6–8 hours and assayed for Hoxb4 (A) and Msx1 (C). Electroporation of BMP4 results in a ventral expansion of Hoxb4 (A, n = 25/30) and Msx1 (C, n = 14/15) within 6–8 hours. Scale bar; 100 μm. (E-H) Electroporation of Smad6 followed by 24 hours of incubation does not cause any obvious changes in Hoxb4 expression pattern (E, n = 23/25. 2/25 showed down-regulation). Msx1 expression is down-regulated at the electroporated side (G, n = 19/20). Scale bar; 200 μm. (I-L) Electroporation of Smad6 followed by 48 hours of incubation causes down-regulation of both Hoxb4 (I, n = 19/21) and Msx1 (K, n = 18/20). Arrows indicate changes in the expression compared to the contralateral side. (B, D, F, H, J, L) shows electroporated, GFP-positive cells in (A, C, E, G, I, K), respectively. Arrowheads in (A, E, I) show the rhombomere 6/7 boundary (r6/7). Scale bar; 200 μm.
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Figure 8: Dorsal expression of Hoxb4 is preceded by dorsalization of the neural tube by BMP signals. (A-D) Flat-mounted neural tubes from embryos electroporated with BMP4, incubated for 6–8 hours and assayed for Hoxb4 (A) and Msx1 (C). Electroporation of BMP4 results in a ventral expansion of Hoxb4 (A, n = 25/30) and Msx1 (C, n = 14/15) within 6–8 hours. Scale bar; 100 μm. (E-H) Electroporation of Smad6 followed by 24 hours of incubation does not cause any obvious changes in Hoxb4 expression pattern (E, n = 23/25. 2/25 showed down-regulation). Msx1 expression is down-regulated at the electroporated side (G, n = 19/20). Scale bar; 200 μm. (I-L) Electroporation of Smad6 followed by 48 hours of incubation causes down-regulation of both Hoxb4 (I, n = 19/21) and Msx1 (K, n = 18/20). Arrows indicate changes in the expression compared to the contralateral side. (B, D, F, H, J, L) shows electroporated, GFP-positive cells in (A, C, E, G, I, K), respectively. Arrowheads in (A, E, I) show the rhombomere 6/7 boundary (r6/7). Scale bar; 200 μm.
Mentions: We further examined whether the up-regulation of Hoxb4 by BMP4 is a direct effect of activation of BMP pathway, or as a consequence of the dorsalized feature of the neural tube. Consistent with the result of embryo cultures with exogenous BMP4, electroporation of BMP4 in the neural tube at the 5 somite stage followed by 6–8 hours of incubation caused a noticeable up-regulation of Hoxb4 expanded toward the ventral side (Fig. 8A, B). This was accompanied by up-regulation of other dorsal neural markers such as Msx1 (Fig. 8C, D) and Pax7 (data not shown). Electroporation of a GFP construct did not show any changes (data not shown). Hence, the D-V pattern of the neural tube has already been altered by the time we observe the changes in the Hoxb4 expression. Next, the same stage of neural tube was electroporated with Smad6 to test the requirement of BMP signals for Hoxb4 expression. Smad6 blocks transduction of BMP and TGFβ signals at the intracellular level [43]. Smad6 successfully inhibited Hoxb4 expression at the dorsal side of the neural tube (Fig. 8I, J). However, the down-regulation was not seen at 6 (data not shown) or 24 hours (Fig. 8E, F) but at 48 hours of incubation (Fig. 8I, J), which was presumably due to the late onset of exogenous Smad6 expression, which might not be prompt enough to override the endogenous programme. In fact, Smad6 electroporation caused down-regulation of Msx1/2 not at 6 hours, but after 24 (Fig. 8G, H and data not shown) and 48 hours (Fig. 8K, L and data not shown). These data suggest that Hoxb4 expression is preceded by the BMP signal-dependent dorsalization of the neural tube. It should be noted that the dorsalizing activity of exogenous BMP4 could be mediated by other members of the TGFβ super family whose function BMP4 can mimic. Because of the delay in the change in Hoxb4 following Smad6 electroporation, and the fact that no known Smad binding sites have been identified in Hox gene enhancer elements, it is likely that up-regulation of Hoxb4 is due to the dorsal feature of the neural tube induced by BMP signals, rather than the direct effect of activation of the BMP pathway.

Bottom Line: We also show that the dorsal side of the neural tube has a greater susceptibility to expressing Hoxb4 than the ventral region, a feature associated with dorsalization of the neural tube by BMP signals.BMP4 is additionally able to up-regulate Hoxb4 ventrally, but the effect is restricted to the axial levels at which Hoxb4 is normally expressed, and only in the presence of retinoic acid (RA) or somites, suggesting a role for BMP in rendering the neural tube competent to express Hoxb4 in response to RA or somite signals.In identifying the collaboration between somites and neural tube competence in the induction of Hoxb4, this study demonstrates interplay between A-P and dorsal-ventral (D-V) patterning systems, whereby a specific feature of D-V polarity may be a prerequisite for proper A-P patterning by Hox genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, London, NW7 1AA, UK. saroshi7@yahoo.co.uk

ABSTRACT

Background: While the body axis is largely patterned along the anterior-posterior (A-P) axis during gastrulation, the central nervous system (CNS) shows dynamic changes in the expression pattern of Hox genes during neurulation, suggesting that the CNS refines the A-P pattern continuously after neural tube formation. This study aims at clarifying the role of somites in up-regulating Hoxb4 expression to eventually establish its final pattern and how the neural tube develops a competence to respond to extrinsic signals.

Results: We show that somites are required for the up-regulation of Hoxb4 in the neural tube at the level of somites 1 to 5, the anterior-most domain of expression. However, each somite immediately adjacent to the neural tube is not sufficient at each level; planar signaling is additionally required particularly at the anterior-most segments of the expression domain. We also show that the dorsal side of the neural tube has a greater susceptibility to expressing Hoxb4 than the ventral region, a feature associated with dorsalization of the neural tube by BMP signals. BMP4 is additionally able to up-regulate Hoxb4 ventrally, but the effect is restricted to the axial levels at which Hoxb4 is normally expressed, and only in the presence of retinoic acid (RA) or somites, suggesting a role for BMP in rendering the neural tube competent to express Hoxb4 in response to RA or somite signals.

Conclusion: In identifying the collaboration between somites and neural tube competence in the induction of Hoxb4, this study demonstrates interplay between A-P and dorsal-ventral (D-V) patterning systems, whereby a specific feature of D-V polarity may be a prerequisite for proper A-P patterning by Hox genes.

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