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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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Temporal profiles of Hh response visualized by a Ptc1-Kaede reporter.(A) Schematic drawing of the Ptc1-Kaede BAC reporter. A cassette containing Kaede and Kanamycin resistant gene was recombined to replace the first exon of ptc1. (B) Ptc1-Kaede fish showed kaede expression in a pattern similar to the expression of ptc1 in wild type embryos at 19-som stage. (C) Inhibition of Hh signaling using cyclopamine blocked Ptc1-Kaede expression, while overexpression of dnPKA mRNA induced ectopic expression of the transgene. Fluorescent signal in cyclopamine-treated fish is due to auto-fluorescence of the yolk (asterisk). (D) Schematic drawings of PHRESH analysis. Photoconversion of the Ptc1-Kaede reporter can be used to determine the timing of Hh response (see text for details). (E) Ptc1-Kaede fish were photoconverted at 25 hpf, and imaged at 36 hpf. Single optical sections of a lateral view, dorsal view, and cross-section are shown. Arrows indicate Ptc1-Kaedered cells in the lateral floor domain. Note that dorsally located KA′ cells also only express Ptc1-Kaedered (arrowheads). (F) Ptc1-Kaede fish were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 35 hpf. Arrows indicate GABA-positive KA″ cells. (G) Gata2-GFP fish were co-labeled with ptc1 (green), and the GFP antibody (red). Images shown are the dorsal view of an 18 hpf embryo (top) and the lateral view of a 24 hpf embryo (bottom). KA″ cells are indicated by arrows. Scale bars: 200 µm in B–C and 20 µm in E–G.
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pgen-1002762-g003: Temporal profiles of Hh response visualized by a Ptc1-Kaede reporter.(A) Schematic drawing of the Ptc1-Kaede BAC reporter. A cassette containing Kaede and Kanamycin resistant gene was recombined to replace the first exon of ptc1. (B) Ptc1-Kaede fish showed kaede expression in a pattern similar to the expression of ptc1 in wild type embryos at 19-som stage. (C) Inhibition of Hh signaling using cyclopamine blocked Ptc1-Kaede expression, while overexpression of dnPKA mRNA induced ectopic expression of the transgene. Fluorescent signal in cyclopamine-treated fish is due to auto-fluorescence of the yolk (asterisk). (D) Schematic drawings of PHRESH analysis. Photoconversion of the Ptc1-Kaede reporter can be used to determine the timing of Hh response (see text for details). (E) Ptc1-Kaede fish were photoconverted at 25 hpf, and imaged at 36 hpf. Single optical sections of a lateral view, dorsal view, and cross-section are shown. Arrows indicate Ptc1-Kaedered cells in the lateral floor domain. Note that dorsally located KA′ cells also only express Ptc1-Kaedered (arrowheads). (F) Ptc1-Kaede fish were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 35 hpf. Arrows indicate GABA-positive KA″ cells. (G) Gata2-GFP fish were co-labeled with ptc1 (green), and the GFP antibody (red). Images shown are the dorsal view of an 18 hpf embryo (top) and the lateral view of a 24 hpf embryo (bottom). KA″ cells are indicated by arrows. Scale bars: 200 µm in B–C and 20 µm in E–G.

Mentions: The discontinuous organization of KA″ cells along the LFP domain raises the question how cells with common progenitors acquire different fates. Hh signaling is important in patterning the ventral spinal cord in zebrafish [26], [28]. Since cells in the LFP domain appear to be exposed to similar levels of Shh, it is possible that different duration of Hh signaling in sibling cells results in different fates. To visualize Hh signaling dynamics in vivo, we generated a reporter line for ptc1, a direct target of Hh signaling [29]. The cDNA encoding the photoconvertible fluorescent protein Kaede was engineered into a BAC (bacteria artificial chromosome) containing the ptc1 genomic region, including 150 kb upstream and 20 kb downstream regulatory sequences (Figure 3A). The Ptc1-Kaede reporter faithfully recapitulated endogenous ptc1 expression and responded to Hh signaling (Figure 3B and 3C). Inhibition of Hh signaling using cyclopamine, a potent antagonist of Smo [30], inhibited Ptc1-Kaede expression, while overexpression of dnPKA mRNA induced substantial expansion of the expression domain (Figure 3C). These results indicate that the Ptc1-Kaede reporter is a sensitive readout for Hh response in vivo.


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)

Temporal profiles of Hh response visualized by a Ptc1-Kaede reporter.(A) Schematic drawing of the Ptc1-Kaede BAC reporter. A cassette containing Kaede and Kanamycin resistant gene was recombined to replace the first exon of ptc1. (B) Ptc1-Kaede fish showed kaede expression in a pattern similar to the expression of ptc1 in wild type embryos at 19-som stage. (C) Inhibition of Hh signaling using cyclopamine blocked Ptc1-Kaede expression, while overexpression of dnPKA mRNA induced ectopic expression of the transgene. Fluorescent signal in cyclopamine-treated fish is due to auto-fluorescence of the yolk (asterisk). (D) Schematic drawings of PHRESH analysis. Photoconversion of the Ptc1-Kaede reporter can be used to determine the timing of Hh response (see text for details). (E) Ptc1-Kaede fish were photoconverted at 25 hpf, and imaged at 36 hpf. Single optical sections of a lateral view, dorsal view, and cross-section are shown. Arrows indicate Ptc1-Kaedered cells in the lateral floor domain. Note that dorsally located KA′ cells also only express Ptc1-Kaedered (arrowheads). (F) Ptc1-Kaede fish were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 35 hpf. Arrows indicate GABA-positive KA″ cells. (G) Gata2-GFP fish were co-labeled with ptc1 (green), and the GFP antibody (red). Images shown are the dorsal view of an 18 hpf embryo (top) and the lateral view of a 24 hpf embryo (bottom). KA″ cells are indicated by arrows. Scale bars: 200 µm in B–C and 20 µm in E–G.
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Related In: Results  -  Collection

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pgen-1002762-g003: Temporal profiles of Hh response visualized by a Ptc1-Kaede reporter.(A) Schematic drawing of the Ptc1-Kaede BAC reporter. A cassette containing Kaede and Kanamycin resistant gene was recombined to replace the first exon of ptc1. (B) Ptc1-Kaede fish showed kaede expression in a pattern similar to the expression of ptc1 in wild type embryos at 19-som stage. (C) Inhibition of Hh signaling using cyclopamine blocked Ptc1-Kaede expression, while overexpression of dnPKA mRNA induced ectopic expression of the transgene. Fluorescent signal in cyclopamine-treated fish is due to auto-fluorescence of the yolk (asterisk). (D) Schematic drawings of PHRESH analysis. Photoconversion of the Ptc1-Kaede reporter can be used to determine the timing of Hh response (see text for details). (E) Ptc1-Kaede fish were photoconverted at 25 hpf, and imaged at 36 hpf. Single optical sections of a lateral view, dorsal view, and cross-section are shown. Arrows indicate Ptc1-Kaedered cells in the lateral floor domain. Note that dorsally located KA′ cells also only express Ptc1-Kaedered (arrowheads). (F) Ptc1-Kaede fish were photoconverted at 24 hpf, and stained with the GABA antibody (blue) at 35 hpf. Arrows indicate GABA-positive KA″ cells. (G) Gata2-GFP fish were co-labeled with ptc1 (green), and the GFP antibody (red). Images shown are the dorsal view of an 18 hpf embryo (top) and the lateral view of a 24 hpf embryo (bottom). KA″ cells are indicated by arrows. Scale bars: 200 µm in B–C and 20 µm in E–G.
Mentions: The discontinuous organization of KA″ cells along the LFP domain raises the question how cells with common progenitors acquire different fates. Hh signaling is important in patterning the ventral spinal cord in zebrafish [26], [28]. Since cells in the LFP domain appear to be exposed to similar levels of Shh, it is possible that different duration of Hh signaling in sibling cells results in different fates. To visualize Hh signaling dynamics in vivo, we generated a reporter line for ptc1, a direct target of Hh signaling [29]. The cDNA encoding the photoconvertible fluorescent protein Kaede was engineered into a BAC (bacteria artificial chromosome) containing the ptc1 genomic region, including 150 kb upstream and 20 kb downstream regulatory sequences (Figure 3A). The Ptc1-Kaede reporter faithfully recapitulated endogenous ptc1 expression and responded to Hh signaling (Figure 3B and 3C). Inhibition of Hh signaling using cyclopamine, a potent antagonist of Smo [30], inhibited Ptc1-Kaede expression, while overexpression of dnPKA mRNA induced substantial expansion of the expression domain (Figure 3C). These results indicate that the Ptc1-Kaede reporter is a sensitive readout for Hh response in vivo.

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