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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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Prolonged Hh response interferes with KA″ specification.(A) Quantification of KA″ specification in embryos overexpressing GFP-Gli1. hsp-GFP-Gli1 embryos and their non-transgenic sibling controls were heat-shocked at indicated stages, and stained at 25 hpf for the expression of tal2. (B) hsp-GFP-Gli1 and control embryos were heat-shocked at 14 hpf, and stained at 24 hpf for the expression of tal2 and nkx2.9. Induction of GFP-Gli1 results in a reduction of tal2-positive KA″ cells (arrows) and expansion of nkx2.9-expressing LFPs. (C) Ptc1-Kaede control embryos, and Ptc1-Kaede; hsp-GFP-Gli1 embryos were heat-shocked at 14 hpf, photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Induced expression of GFP-Gli1 results in a reduction of KA″ cells (arrows). Note that at 36 hpf, GFP-Gli1 expression has minimal contribution to the green fluorescence. (D) Ptc1-Kaede control embryos, and Ptc1-Kaede embryos injected with Shh mRNA, Ptc1 and Ptc2 morpholinos, or dnPKA mRNA were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Arrows indicated GABA-positive cells in the LFP domain. Note that Shh and dnPKA overexpression induced many ectopic GABAergic neurons (arrowheads) throughout the dorsal-ventral axis of the spinal cord, and most of them appeared to lose Hh response by 24 hpf as indicated by the expression of Ptc1-Kaedered. Scale bars: 20 µm.
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pgen-1002762-g005: Prolonged Hh response interferes with KA″ specification.(A) Quantification of KA″ specification in embryos overexpressing GFP-Gli1. hsp-GFP-Gli1 embryos and their non-transgenic sibling controls were heat-shocked at indicated stages, and stained at 25 hpf for the expression of tal2. (B) hsp-GFP-Gli1 and control embryos were heat-shocked at 14 hpf, and stained at 24 hpf for the expression of tal2 and nkx2.9. Induction of GFP-Gli1 results in a reduction of tal2-positive KA″ cells (arrows) and expansion of nkx2.9-expressing LFPs. (C) Ptc1-Kaede control embryos, and Ptc1-Kaede; hsp-GFP-Gli1 embryos were heat-shocked at 14 hpf, photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Induced expression of GFP-Gli1 results in a reduction of KA″ cells (arrows). Note that at 36 hpf, GFP-Gli1 expression has minimal contribution to the green fluorescence. (D) Ptc1-Kaede control embryos, and Ptc1-Kaede embryos injected with Shh mRNA, Ptc1 and Ptc2 morpholinos, or dnPKA mRNA were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Arrows indicated GABA-positive cells in the LFP domain. Note that Shh and dnPKA overexpression induced many ectopic GABAergic neurons (arrowheads) throughout the dorsal-ventral axis of the spinal cord, and most of them appeared to lose Hh response by 24 hpf as indicated by the expression of Ptc1-Kaedered. Scale bars: 20 µm.

Mentions: The loss of Hh response in KA″ cells raised the question whether termination of Hh signaling is important for cell fate specification. To test this idea, we prolonged Hh response in KA″ cells by expressing a GFP-Gli1 transgene under the control of a heat-shock inducible promoter (hsp-GFP-Gli1) [31]. Strikingly, forced expression of GFP-Gli1 reduced the number of KA″ cells with an accompanying increase in nkx2.9-expressing LFPs (Figure 5A and 5B). The strongest phenotype was observed when GFP-Gli1 was induced around 16.5 hpf, a stage when most KA″ cells are born but before terminal differentiation markers such as GABA and HuC are expressed (Figure 5A). Since most KA″ cells no longer require active Hh signaling by 16 hpf (Figure 4A), this result suggests that sustained Hh response in differentiating KA″ cells converts them to an LFP-like identity. To test this idea, we induced GFP-Gli1 expression in Ptc1-Kaede fish at 14 hpf, and performed photoconversion at 24 hpf. At 36 hpf (12 hours-post-conversion), there was a marked reduction in the number of Ptc1-Kaedered KA″ cells in the LFP domain (Figure 5C). By contrast, the remainder of the LFP domain expressed Ptc1-Kaedered+green, indicating continuously active Hh response (Figure 5C). These results suggest that termination of Hh response is essential for KA″ differentiation.


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)

Prolonged Hh response interferes with KA″ specification.(A) Quantification of KA″ specification in embryos overexpressing GFP-Gli1. hsp-GFP-Gli1 embryos and their non-transgenic sibling controls were heat-shocked at indicated stages, and stained at 25 hpf for the expression of tal2. (B) hsp-GFP-Gli1 and control embryos were heat-shocked at 14 hpf, and stained at 24 hpf for the expression of tal2 and nkx2.9. Induction of GFP-Gli1 results in a reduction of tal2-positive KA″ cells (arrows) and expansion of nkx2.9-expressing LFPs. (C) Ptc1-Kaede control embryos, and Ptc1-Kaede; hsp-GFP-Gli1 embryos were heat-shocked at 14 hpf, photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Induced expression of GFP-Gli1 results in a reduction of KA″ cells (arrows). Note that at 36 hpf, GFP-Gli1 expression has minimal contribution to the green fluorescence. (D) Ptc1-Kaede control embryos, and Ptc1-Kaede embryos injected with Shh mRNA, Ptc1 and Ptc2 morpholinos, or dnPKA mRNA were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Arrows indicated GABA-positive cells in the LFP domain. Note that Shh and dnPKA overexpression induced many ectopic GABAergic neurons (arrowheads) throughout the dorsal-ventral axis of the spinal cord, and most of them appeared to lose Hh response by 24 hpf as indicated by the expression of Ptc1-Kaedered. Scale bars: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002762-g005: Prolonged Hh response interferes with KA″ specification.(A) Quantification of KA″ specification in embryos overexpressing GFP-Gli1. hsp-GFP-Gli1 embryos and their non-transgenic sibling controls were heat-shocked at indicated stages, and stained at 25 hpf for the expression of tal2. (B) hsp-GFP-Gli1 and control embryos were heat-shocked at 14 hpf, and stained at 24 hpf for the expression of tal2 and nkx2.9. Induction of GFP-Gli1 results in a reduction of tal2-positive KA″ cells (arrows) and expansion of nkx2.9-expressing LFPs. (C) Ptc1-Kaede control embryos, and Ptc1-Kaede; hsp-GFP-Gli1 embryos were heat-shocked at 14 hpf, photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Induced expression of GFP-Gli1 results in a reduction of KA″ cells (arrows). Note that at 36 hpf, GFP-Gli1 expression has minimal contribution to the green fluorescence. (D) Ptc1-Kaede control embryos, and Ptc1-Kaede embryos injected with Shh mRNA, Ptc1 and Ptc2 morpholinos, or dnPKA mRNA were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 36 hpf. Arrows indicated GABA-positive cells in the LFP domain. Note that Shh and dnPKA overexpression induced many ectopic GABAergic neurons (arrowheads) throughout the dorsal-ventral axis of the spinal cord, and most of them appeared to lose Hh response by 24 hpf as indicated by the expression of Ptc1-Kaedered. Scale bars: 20 µm.
Mentions: The loss of Hh response in KA″ cells raised the question whether termination of Hh signaling is important for cell fate specification. To test this idea, we prolonged Hh response in KA″ cells by expressing a GFP-Gli1 transgene under the control of a heat-shock inducible promoter (hsp-GFP-Gli1) [31]. Strikingly, forced expression of GFP-Gli1 reduced the number of KA″ cells with an accompanying increase in nkx2.9-expressing LFPs (Figure 5A and 5B). The strongest phenotype was observed when GFP-Gli1 was induced around 16.5 hpf, a stage when most KA″ cells are born but before terminal differentiation markers such as GABA and HuC are expressed (Figure 5A). Since most KA″ cells no longer require active Hh signaling by 16 hpf (Figure 4A), this result suggests that sustained Hh response in differentiating KA″ cells converts them to an LFP-like identity. To test this idea, we induced GFP-Gli1 expression in Ptc1-Kaede fish at 14 hpf, and performed photoconversion at 24 hpf. At 36 hpf (12 hours-post-conversion), there was a marked reduction in the number of Ptc1-Kaedered KA″ cells in the LFP domain (Figure 5C). By contrast, the remainder of the LFP domain expressed Ptc1-Kaedered+green, indicating continuously active Hh response (Figure 5C). These results suggest that termination of Hh response is essential for KA″ differentiation.

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