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Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord.

Huang P, Xiong F, Megason SG, Schier AF - PLoS Genet. (2012)

Bottom Line: Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation.Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells.These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Center for Brain Science, Harvard Stem Cell Institute, Broad Institute, Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA. huang@mcb.harvard.edu

ABSTRACT
During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.

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Interaction between Notch and Hh signaling in KA″ specification.(A) Embryos treated with DMSO alone, compound E, cyclopamine, or both compound E and cyclopamine from 14 hpf to 25 hpf, were stained for the expression of tal2. (B) Quantification of total number of KA″ cells in experiments shown in A. Note that the fewer data points in compound E treatment is due to the fact that most compound E-treated embryos had many tal2-positive cells in close clusters which prevents reliable scoring. The two data points thus underestimate the total number of KA″ cells in E-treated embryos. Cyc: cyclopamine. (C) hsp-Gal4; UAS-NICD embryos and non-transgenic sibling controls were left uninjected or injected with Shh mRNA, heat-shocked at 11.5 hpf, and stained at 24 hpf for the expression of tal2. Brackets in A and C indicate the dorsal-ventral extent of the LFP domain. Scale bars: 50 µm. (D) hsp-Gli1 embryos and non-transgenic controls were injected with compound E at 15 hpf, and heat-shocked at 16 hpf, and stained at 22 hpf for the expression of nkx2.9. Arrows denote the two rows of LFP domains in dorsal views. (E) Model of KA″ specification. At the early stage (t0), LFP progenitors have high level of Notch signaling activity and thereby maintain the progenitor state and Hh responsiveness (NotchON HhON). Active Hh signaling in progenitor cells is required for specifying the KA″ identity in subsequent cell divisions. LFP progenitors can undergo three different types of divisions: symmetric LFP/LFP divisions, asymmetric KA″/LFP divisions (shown here), and symmetric KA″/KA″ divisions. Divisions of LFP progenitors at t1 generate daughter cells with similar competence to either acquire the KA″ fate or maintain the LFP progenitor fate. Cell-cell interactions or stochastic fluctuations in Notch signaling result in cells with different levels of Notch signaling (t2). Cells that maintain high levels of Notch signaling will remain as LFP progenitors and continue to respond Shh (NotchON HhON). In contrast, cells that have attenuated Notch signaling will lose Hh response and differentiate into KA″ interneurons (NotchOFF HhOFF). Since sustained Notch or Hh signaling disrupt the differentiation of KA″ cells, formation of KA″ cells initially depends on the activation and then the attenuation of Notch and Hh signaling.
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pgen-1002762-g007: Interaction between Notch and Hh signaling in KA″ specification.(A) Embryos treated with DMSO alone, compound E, cyclopamine, or both compound E and cyclopamine from 14 hpf to 25 hpf, were stained for the expression of tal2. (B) Quantification of total number of KA″ cells in experiments shown in A. Note that the fewer data points in compound E treatment is due to the fact that most compound E-treated embryos had many tal2-positive cells in close clusters which prevents reliable scoring. The two data points thus underestimate the total number of KA″ cells in E-treated embryos. Cyc: cyclopamine. (C) hsp-Gal4; UAS-NICD embryos and non-transgenic sibling controls were left uninjected or injected with Shh mRNA, heat-shocked at 11.5 hpf, and stained at 24 hpf for the expression of tal2. Brackets in A and C indicate the dorsal-ventral extent of the LFP domain. Scale bars: 50 µm. (D) hsp-Gli1 embryos and non-transgenic controls were injected with compound E at 15 hpf, and heat-shocked at 16 hpf, and stained at 22 hpf for the expression of nkx2.9. Arrows denote the two rows of LFP domains in dorsal views. (E) Model of KA″ specification. At the early stage (t0), LFP progenitors have high level of Notch signaling activity and thereby maintain the progenitor state and Hh responsiveness (NotchON HhON). Active Hh signaling in progenitor cells is required for specifying the KA″ identity in subsequent cell divisions. LFP progenitors can undergo three different types of divisions: symmetric LFP/LFP divisions, asymmetric KA″/LFP divisions (shown here), and symmetric KA″/KA″ divisions. Divisions of LFP progenitors at t1 generate daughter cells with similar competence to either acquire the KA″ fate or maintain the LFP progenitor fate. Cell-cell interactions or stochastic fluctuations in Notch signaling result in cells with different levels of Notch signaling (t2). Cells that maintain high levels of Notch signaling will remain as LFP progenitors and continue to respond Shh (NotchON HhON). In contrast, cells that have attenuated Notch signaling will lose Hh response and differentiate into KA″ interneurons (NotchOFF HhOFF). Since sustained Notch or Hh signaling disrupt the differentiation of KA″ cells, formation of KA″ cells initially depends on the activation and then the attenuation of Notch and Hh signaling.

Mentions: Our results indicate that attenuation of both Notch and Hh signaling is essential for KA″ differentiation. To further clarify the interactions between Notch and Hh signaling in KA″ specification, we first tested whether the induction of ectopic KA″ cells by Notch inhibition depends on active Hh signaling. Inhibition of Notch signaling by compound E from 14 hpf to 25 hpf lead to a more than 50% increase in the number of KA″ cells (Figure 7A and 7B). Concurrent treatment with compound E and cyclopamine at 14 hpf resulted in ∼50% reduction in KA″ cells at 25 hpf similar to cyclopamine treatment alone (Figure 7A and 7B). In contrast, blocking late Hh signaling at 18 hpf with cyclopamine, following compound E treatment at 14 hpf, had no effects on the induction of ectopic KA″ cells (data not shown). These results are consistent with the observation that KA″ cells no longer require Hh signaling after 18 hpf and indicate that the generation of KA″ cells by Notch inhibition depends on early Hh signaling.


Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord.

Huang P, Xiong F, Megason SG, Schier AF - PLoS Genet. (2012)

Interaction between Notch and Hh signaling in KA″ specification.(A) Embryos treated with DMSO alone, compound E, cyclopamine, or both compound E and cyclopamine from 14 hpf to 25 hpf, were stained for the expression of tal2. (B) Quantification of total number of KA″ cells in experiments shown in A. Note that the fewer data points in compound E treatment is due to the fact that most compound E-treated embryos had many tal2-positive cells in close clusters which prevents reliable scoring. The two data points thus underestimate the total number of KA″ cells in E-treated embryos. Cyc: cyclopamine. (C) hsp-Gal4; UAS-NICD embryos and non-transgenic sibling controls were left uninjected or injected with Shh mRNA, heat-shocked at 11.5 hpf, and stained at 24 hpf for the expression of tal2. Brackets in A and C indicate the dorsal-ventral extent of the LFP domain. Scale bars: 50 µm. (D) hsp-Gli1 embryos and non-transgenic controls were injected with compound E at 15 hpf, and heat-shocked at 16 hpf, and stained at 22 hpf for the expression of nkx2.9. Arrows denote the two rows of LFP domains in dorsal views. (E) Model of KA″ specification. At the early stage (t0), LFP progenitors have high level of Notch signaling activity and thereby maintain the progenitor state and Hh responsiveness (NotchON HhON). Active Hh signaling in progenitor cells is required for specifying the KA″ identity in subsequent cell divisions. LFP progenitors can undergo three different types of divisions: symmetric LFP/LFP divisions, asymmetric KA″/LFP divisions (shown here), and symmetric KA″/KA″ divisions. Divisions of LFP progenitors at t1 generate daughter cells with similar competence to either acquire the KA″ fate or maintain the LFP progenitor fate. Cell-cell interactions or stochastic fluctuations in Notch signaling result in cells with different levels of Notch signaling (t2). Cells that maintain high levels of Notch signaling will remain as LFP progenitors and continue to respond Shh (NotchON HhON). In contrast, cells that have attenuated Notch signaling will lose Hh response and differentiate into KA″ interneurons (NotchOFF HhOFF). Since sustained Notch or Hh signaling disrupt the differentiation of KA″ cells, formation of KA″ cells initially depends on the activation and then the attenuation of Notch and Hh signaling.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369957&req=5

pgen-1002762-g007: Interaction between Notch and Hh signaling in KA″ specification.(A) Embryos treated with DMSO alone, compound E, cyclopamine, or both compound E and cyclopamine from 14 hpf to 25 hpf, were stained for the expression of tal2. (B) Quantification of total number of KA″ cells in experiments shown in A. Note that the fewer data points in compound E treatment is due to the fact that most compound E-treated embryos had many tal2-positive cells in close clusters which prevents reliable scoring. The two data points thus underestimate the total number of KA″ cells in E-treated embryos. Cyc: cyclopamine. (C) hsp-Gal4; UAS-NICD embryos and non-transgenic sibling controls were left uninjected or injected with Shh mRNA, heat-shocked at 11.5 hpf, and stained at 24 hpf for the expression of tal2. Brackets in A and C indicate the dorsal-ventral extent of the LFP domain. Scale bars: 50 µm. (D) hsp-Gli1 embryos and non-transgenic controls were injected with compound E at 15 hpf, and heat-shocked at 16 hpf, and stained at 22 hpf for the expression of nkx2.9. Arrows denote the two rows of LFP domains in dorsal views. (E) Model of KA″ specification. At the early stage (t0), LFP progenitors have high level of Notch signaling activity and thereby maintain the progenitor state and Hh responsiveness (NotchON HhON). Active Hh signaling in progenitor cells is required for specifying the KA″ identity in subsequent cell divisions. LFP progenitors can undergo three different types of divisions: symmetric LFP/LFP divisions, asymmetric KA″/LFP divisions (shown here), and symmetric KA″/KA″ divisions. Divisions of LFP progenitors at t1 generate daughter cells with similar competence to either acquire the KA″ fate or maintain the LFP progenitor fate. Cell-cell interactions or stochastic fluctuations in Notch signaling result in cells with different levels of Notch signaling (t2). Cells that maintain high levels of Notch signaling will remain as LFP progenitors and continue to respond Shh (NotchON HhON). In contrast, cells that have attenuated Notch signaling will lose Hh response and differentiate into KA″ interneurons (NotchOFF HhOFF). Since sustained Notch or Hh signaling disrupt the differentiation of KA″ cells, formation of KA″ cells initially depends on the activation and then the attenuation of Notch and Hh signaling.
Mentions: Our results indicate that attenuation of both Notch and Hh signaling is essential for KA″ differentiation. To further clarify the interactions between Notch and Hh signaling in KA″ specification, we first tested whether the induction of ectopic KA″ cells by Notch inhibition depends on active Hh signaling. Inhibition of Notch signaling by compound E from 14 hpf to 25 hpf lead to a more than 50% increase in the number of KA″ cells (Figure 7A and 7B). Concurrent treatment with compound E and cyclopamine at 14 hpf resulted in ∼50% reduction in KA″ cells at 25 hpf similar to cyclopamine treatment alone (Figure 7A and 7B). In contrast, blocking late Hh signaling at 18 hpf with cyclopamine, following compound E treatment at 14 hpf, had no effects on the induction of ectopic KA″ cells (data not shown). These results are consistent with the observation that KA″ cells no longer require Hh signaling after 18 hpf and indicate that the generation of KA″ cells by Notch inhibition depends on early Hh signaling.

Bottom Line: Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation.Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells.These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Center for Brain Science, Harvard Stem Cell Institute, Broad Institute, Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA. huang@mcb.harvard.edu

ABSTRACT
During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.

Show MeSH
Related in: MedlinePlus