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Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord.

Ramos C, Rocha S, Gaspar C, Henrique D - PLoS ONE (2010)

Bottom Line: In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains.However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Lisboa, Portugal.

ABSTRACT

Background: Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.

Methodology/principal findings: In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.

Conclusions/significance: Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.

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Related in: MedlinePlus

Jag1 deletion can be compensated by Dll1-signalling from adjacent domains.In double mutant Dll1f/f;Jag1f/f;NesCre embryos (A), both Foxd3+ V1 INs and Chx10+ V2a INs are strongly increased, while one functional copy of Dll1 (B) rescues the V2a phenotype completely and the V1 phenotype partially. On the contrary, one functional copy of Jag1, in the absence of Dll1 (C), rescues the V1 phenotype but fails to revert the excess of V2a INs. (D–I) A neurogenic phenotype in the dI6 domain can only be observed in the absence of both ligands, using either bHLHb5 (D–F) or Pax2 (G–I) to identify dI6 neurons, located dorsally to the Evx1+ V0V INs (indicated with asterisk). The presence of one functional copy of Dll1 (E,H) or of Jag1 (F,I) is enough to prevent excessive dI6 neurogenesis. The number of Evx1+ V0V INs is only increased in the complete absence of Dll1 (D,F,G,I) as one functional copy of Dll1 is enough to revert the V0V neurogenic phenotype, even in the absence of Jag1 (E,H). Although one functional copy of Jag1 (F) is enough to revert the excess of Bhlhb5+ dI6 INs detected in Dll1f/f;Jag1f/f;NesCre embryos (D), an excess of Pax2+ INs located dorsally to Evx1+ V0V INs (indicated with asterisk) can still be detected in Dll1f/f;Jag1f/+;NesCre embryos (I), when compared to Dll1f/f;Jag1f/f;NesCre (G). The excess of Pax2+ INs arises from the Dll1-dependent V0D domain (Pax2+/Evx1−) and not from the Jag1-expressing dI6 domain (Pax2+/Evx1−/Bhlhb5+). On the contrary, one functional copy of Dll1 is enough to rescue both the V0D and dI6 neurogenic phenotypes (E,H). Scale bar 50 µm. (J–N) Graphics depicting the quantification of various types of INs in different allelic combinations of Dll1 and Jag1. The percentage of positive cells for each marker is relative to the total number of cells, detected by DAPI staining of the entire spinal cord sections where the counts were done. Error bars represent s.d. for at least three biological replicates. Student's t-test * p<0.05; ** p<0.05; *** p<0.001.
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pone-0015515-g006: Jag1 deletion can be compensated by Dll1-signalling from adjacent domains.In double mutant Dll1f/f;Jag1f/f;NesCre embryos (A), both Foxd3+ V1 INs and Chx10+ V2a INs are strongly increased, while one functional copy of Dll1 (B) rescues the V2a phenotype completely and the V1 phenotype partially. On the contrary, one functional copy of Jag1, in the absence of Dll1 (C), rescues the V1 phenotype but fails to revert the excess of V2a INs. (D–I) A neurogenic phenotype in the dI6 domain can only be observed in the absence of both ligands, using either bHLHb5 (D–F) or Pax2 (G–I) to identify dI6 neurons, located dorsally to the Evx1+ V0V INs (indicated with asterisk). The presence of one functional copy of Dll1 (E,H) or of Jag1 (F,I) is enough to prevent excessive dI6 neurogenesis. The number of Evx1+ V0V INs is only increased in the complete absence of Dll1 (D,F,G,I) as one functional copy of Dll1 is enough to revert the V0V neurogenic phenotype, even in the absence of Jag1 (E,H). Although one functional copy of Jag1 (F) is enough to revert the excess of Bhlhb5+ dI6 INs detected in Dll1f/f;Jag1f/f;NesCre embryos (D), an excess of Pax2+ INs located dorsally to Evx1+ V0V INs (indicated with asterisk) can still be detected in Dll1f/f;Jag1f/+;NesCre embryos (I), when compared to Dll1f/f;Jag1f/f;NesCre (G). The excess of Pax2+ INs arises from the Dll1-dependent V0D domain (Pax2+/Evx1−) and not from the Jag1-expressing dI6 domain (Pax2+/Evx1−/Bhlhb5+). On the contrary, one functional copy of Dll1 is enough to rescue both the V0D and dI6 neurogenic phenotypes (E,H). Scale bar 50 µm. (J–N) Graphics depicting the quantification of various types of INs in different allelic combinations of Dll1 and Jag1. The percentage of positive cells for each marker is relative to the total number of cells, detected by DAPI staining of the entire spinal cord sections where the counts were done. Error bars represent s.d. for at least three biological replicates. Student's t-test * p<0.05; ** p<0.05; *** p<0.001.

Mentions: If Dll1 signalling from adjacent domains is able to control neurogenesis in the V1 domain of Jag1 mutants, the prediction is that the mild V1 phenotype detected in Jag1f/f;NesCre embryos would become more pronounced in the absence of the two ligands. Indeed, quantification of Foxd3+ V1 INs in E11.5 full conditional Dll1;Jag1 double mutants (Dll1f/f;Jag1f/f;NesCre) revealed the highest increase, when compared to all other genotypes. For instance, single Jag1 mutants displayed a 23% increase in Foxd3+ V1 INs (p<0.05), while Dll1f/f;Jag1f/f;NesCre mutants exhibited a 49% increase (p<0.005) (Fig. 6 A,J). This excess in V1 neurogenesis was further confirmed by the analysis of En1+ V1 INs in Dll1f/f;Jag1f/f;NesCre mutants (Fig. S3). In addition, Dll1f/f;Jag1f/f;NesCre mutants showed a marked increase in V1-derived Calbindin+ Renshaw cells and FoxP2+ non-Renshaw cells (Fig. S4), in contrast to single Jag1f/f;NesCre mutant embryos (Fig. S2).


Two Notch ligands, Dll1 and Jag1, are differently restricted in their range of action to control neurogenesis in the mammalian spinal cord.

Ramos C, Rocha S, Gaspar C, Henrique D - PLoS ONE (2010)

Jag1 deletion can be compensated by Dll1-signalling from adjacent domains.In double mutant Dll1f/f;Jag1f/f;NesCre embryos (A), both Foxd3+ V1 INs and Chx10+ V2a INs are strongly increased, while one functional copy of Dll1 (B) rescues the V2a phenotype completely and the V1 phenotype partially. On the contrary, one functional copy of Jag1, in the absence of Dll1 (C), rescues the V1 phenotype but fails to revert the excess of V2a INs. (D–I) A neurogenic phenotype in the dI6 domain can only be observed in the absence of both ligands, using either bHLHb5 (D–F) or Pax2 (G–I) to identify dI6 neurons, located dorsally to the Evx1+ V0V INs (indicated with asterisk). The presence of one functional copy of Dll1 (E,H) or of Jag1 (F,I) is enough to prevent excessive dI6 neurogenesis. The number of Evx1+ V0V INs is only increased in the complete absence of Dll1 (D,F,G,I) as one functional copy of Dll1 is enough to revert the V0V neurogenic phenotype, even in the absence of Jag1 (E,H). Although one functional copy of Jag1 (F) is enough to revert the excess of Bhlhb5+ dI6 INs detected in Dll1f/f;Jag1f/f;NesCre embryos (D), an excess of Pax2+ INs located dorsally to Evx1+ V0V INs (indicated with asterisk) can still be detected in Dll1f/f;Jag1f/+;NesCre embryos (I), when compared to Dll1f/f;Jag1f/f;NesCre (G). The excess of Pax2+ INs arises from the Dll1-dependent V0D domain (Pax2+/Evx1−) and not from the Jag1-expressing dI6 domain (Pax2+/Evx1−/Bhlhb5+). On the contrary, one functional copy of Dll1 is enough to rescue both the V0D and dI6 neurogenic phenotypes (E,H). Scale bar 50 µm. (J–N) Graphics depicting the quantification of various types of INs in different allelic combinations of Dll1 and Jag1. The percentage of positive cells for each marker is relative to the total number of cells, detected by DAPI staining of the entire spinal cord sections where the counts were done. Error bars represent s.d. for at least three biological replicates. Student's t-test * p<0.05; ** p<0.05; *** p<0.001.
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Related In: Results  -  Collection

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pone-0015515-g006: Jag1 deletion can be compensated by Dll1-signalling from adjacent domains.In double mutant Dll1f/f;Jag1f/f;NesCre embryos (A), both Foxd3+ V1 INs and Chx10+ V2a INs are strongly increased, while one functional copy of Dll1 (B) rescues the V2a phenotype completely and the V1 phenotype partially. On the contrary, one functional copy of Jag1, in the absence of Dll1 (C), rescues the V1 phenotype but fails to revert the excess of V2a INs. (D–I) A neurogenic phenotype in the dI6 domain can only be observed in the absence of both ligands, using either bHLHb5 (D–F) or Pax2 (G–I) to identify dI6 neurons, located dorsally to the Evx1+ V0V INs (indicated with asterisk). The presence of one functional copy of Dll1 (E,H) or of Jag1 (F,I) is enough to prevent excessive dI6 neurogenesis. The number of Evx1+ V0V INs is only increased in the complete absence of Dll1 (D,F,G,I) as one functional copy of Dll1 is enough to revert the V0V neurogenic phenotype, even in the absence of Jag1 (E,H). Although one functional copy of Jag1 (F) is enough to revert the excess of Bhlhb5+ dI6 INs detected in Dll1f/f;Jag1f/f;NesCre embryos (D), an excess of Pax2+ INs located dorsally to Evx1+ V0V INs (indicated with asterisk) can still be detected in Dll1f/f;Jag1f/+;NesCre embryos (I), when compared to Dll1f/f;Jag1f/f;NesCre (G). The excess of Pax2+ INs arises from the Dll1-dependent V0D domain (Pax2+/Evx1−) and not from the Jag1-expressing dI6 domain (Pax2+/Evx1−/Bhlhb5+). On the contrary, one functional copy of Dll1 is enough to rescue both the V0D and dI6 neurogenic phenotypes (E,H). Scale bar 50 µm. (J–N) Graphics depicting the quantification of various types of INs in different allelic combinations of Dll1 and Jag1. The percentage of positive cells for each marker is relative to the total number of cells, detected by DAPI staining of the entire spinal cord sections where the counts were done. Error bars represent s.d. for at least three biological replicates. Student's t-test * p<0.05; ** p<0.05; *** p<0.001.
Mentions: If Dll1 signalling from adjacent domains is able to control neurogenesis in the V1 domain of Jag1 mutants, the prediction is that the mild V1 phenotype detected in Jag1f/f;NesCre embryos would become more pronounced in the absence of the two ligands. Indeed, quantification of Foxd3+ V1 INs in E11.5 full conditional Dll1;Jag1 double mutants (Dll1f/f;Jag1f/f;NesCre) revealed the highest increase, when compared to all other genotypes. For instance, single Jag1 mutants displayed a 23% increase in Foxd3+ V1 INs (p<0.05), while Dll1f/f;Jag1f/f;NesCre mutants exhibited a 49% increase (p<0.005) (Fig. 6 A,J). This excess in V1 neurogenesis was further confirmed by the analysis of En1+ V1 INs in Dll1f/f;Jag1f/f;NesCre mutants (Fig. S3). In addition, Dll1f/f;Jag1f/f;NesCre mutants showed a marked increase in V1-derived Calbindin+ Renshaw cells and FoxP2+ non-Renshaw cells (Fig. S4), in contrast to single Jag1f/f;NesCre mutant embryos (Fig. S2).

Bottom Line: In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains.However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Lisboa, Portugal.

ABSTRACT

Background: Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.

Methodology/principal findings: In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.

Conclusions/significance: Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression.

Show MeSH
Related in: MedlinePlus