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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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KA″ specification depends on Hh signaling.(A) Quantification of the total number of KA″ cells at 24 hpf under different conditions. Cyclopamine (Cyc) was treated from indicated stage until 24 hpf. Embryos injected with dnPKA or Shh mRNA showed increase in total number of KA″ cells compared to uninjected control embryos. (B) Cyclopamine-treated embryos (from 8 hpf to 24 hpf) abolished tal2 expression in the LFP domain, while overexpression of dnPKA and Shh induced ectopic tal2+ cells. Note that the few tal2+ cells in cyclopamine-treated embryos are located dorsal to the LFP domain. Images shown are lateral views of embryos at 24 hpf. The dorsal-ventral extent of the LFP domain is indicated by brackets. Scale bar: 50 µm.
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pgen-1002762-g004: KA″ specification depends on Hh signaling.(A) Quantification of the total number of KA″ cells at 24 hpf under different conditions. Cyclopamine (Cyc) was treated from indicated stage until 24 hpf. Embryos injected with dnPKA or Shh mRNA showed increase in total number of KA″ cells compared to uninjected control embryos. (B) Cyclopamine-treated embryos (from 8 hpf to 24 hpf) abolished tal2 expression in the LFP domain, while overexpression of dnPKA and Shh induced ectopic tal2+ cells. Note that the few tal2+ cells in cyclopamine-treated embryos are located dorsal to the LFP domain. Images shown are lateral views of embryos at 24 hpf. The dorsal-ventral extent of the LFP domain is indicated by brackets. Scale bar: 50 µm.

Mentions: PHRESH analysis suggested that Hh signaling is active in progenitors of KA″ cells but absent in differentiated KA″ interneurons. To determine when Hh signaling is required for the generation of KA″ cells, we inhibited Hh signaling using cyclopamine at different stages of development. Inhibition of Hh signaling from 8 hpf to 25 hpf completely abolished tal2 expression within the LFP domain at 25 hpf (Figure 4A and 4B), suggesting that Hh signaling is required for KA″ induction. By contrast, more than half of KA″ cells were generated when Hh signaling was blocked from mid-somitogenesis (15 hpf) until 25 hpf, while a normal number of KA″ cells formed upon cyclopamine treatment after 18 hpf (Figure 4A). These results suggest that Hh signaling is required in the LFP progenitors before 18 hpf to specify the KA″ fate and that differentiated KA″ cells no longer depend on active Hh signaling. Notably, we did not observe an increase in the number of KA″ cells upon cyclopamine treatment (Figure 4A), indicating that early termination of Hh signaling in LFP cells is not sufficient to transform them into KA″ cells.


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)

KA″ specification depends on Hh signaling.(A) Quantification of the total number of KA″ cells at 24 hpf under different conditions. Cyclopamine (Cyc) was treated from indicated stage until 24 hpf. Embryos injected with dnPKA or Shh mRNA showed increase in total number of KA″ cells compared to uninjected control embryos. (B) Cyclopamine-treated embryos (from 8 hpf to 24 hpf) abolished tal2 expression in the LFP domain, while overexpression of dnPKA and Shh induced ectopic tal2+ cells. Note that the few tal2+ cells in cyclopamine-treated embryos are located dorsal to the LFP domain. Images shown are lateral views of embryos at 24 hpf. The dorsal-ventral extent of the LFP domain is indicated by brackets. Scale bar: 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002762-g004: KA″ specification depends on Hh signaling.(A) Quantification of the total number of KA″ cells at 24 hpf under different conditions. Cyclopamine (Cyc) was treated from indicated stage until 24 hpf. Embryos injected with dnPKA or Shh mRNA showed increase in total number of KA″ cells compared to uninjected control embryos. (B) Cyclopamine-treated embryos (from 8 hpf to 24 hpf) abolished tal2 expression in the LFP domain, while overexpression of dnPKA and Shh induced ectopic tal2+ cells. Note that the few tal2+ cells in cyclopamine-treated embryos are located dorsal to the LFP domain. Images shown are lateral views of embryos at 24 hpf. The dorsal-ventral extent of the LFP domain is indicated by brackets. Scale bar: 50 µm.
Mentions: PHRESH analysis suggested that Hh signaling is active in progenitors of KA″ cells but absent in differentiated KA″ interneurons. To determine when Hh signaling is required for the generation of KA″ cells, we inhibited Hh signaling using cyclopamine at different stages of development. Inhibition of Hh signaling from 8 hpf to 25 hpf completely abolished tal2 expression within the LFP domain at 25 hpf (Figure 4A and 4B), suggesting that Hh signaling is required for KA″ induction. By contrast, more than half of KA″ cells were generated when Hh signaling was blocked from mid-somitogenesis (15 hpf) until 25 hpf, while a normal number of KA″ cells formed upon cyclopamine treatment after 18 hpf (Figure 4A). These results suggest that Hh signaling is required in the LFP progenitors before 18 hpf to specify the KA″ fate and that differentiated KA″ cells no longer depend on active Hh signaling. Notably, we did not observe an increase in the number of KA″ cells upon cyclopamine treatment (Figure 4A), indicating that early termination of Hh signaling in LFP cells is not sufficient to transform them into KA″ cells.

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