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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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Marker analysis of the LFP domain.(A) Schematic drawings of the zebrafish ventral spinal cord at 24 hpf in a dorsal view (a), lateral view (b), and cross-section (c). The lateral floor plate domain, consisting of LFP cells and KA″ interneurons, flanks the medial floor plate (MFP). The motor neuron domain locates dorsal to the LFP domain, which contains motor neuron progenitors (pMN), motor neurons (MN), and Kolmer-Agduhr′ interneurons (KA′). KA interneurons, including KA′ and KA″ cells, represent a unique class of GABAergic interneurons that are in contact with the cerebrospinal fluid and are implicated in regulating the swimming behavior in zebrafish [35], [47], [48]. Marker expression in MFP, LFP and KA″ cells are indicated on the right. N: notochord. (B) KA″ cells, labeled by tal2 expression (arrows, red), are discontinuously distributed flanking the medial floor plate (arrowhead) along the anterior-posterior axis. Cell nuclei were labeled by Draq5 staining (green). (C) All tal2-positive cells (arrows, red) in the LFP domain also express pan-neuronal marker HuC (green). (D–E) All GFP-expressing cells in the LFP region (arrows, green) in Gata2-GFP fish also co-express tal2 (D, red) and GABA (E, red). (F–G) Wild type embryos were co-labeled with nkx2.9 (F, green) or nkx2.2a (G, green) together with tal2 (red). tal2-expressing KA″ cells (arrows) show lower expression of nkx2.9 and nkx2.2a compared to their neighboring cells. Embryos shown are at 21 hpf (B), 30 hpf (C), 25 hpf (D), 27 hpf (E), and 18 hpf (F, G). B, D, F, and G show dorsal views, and C and E represent lateral views. Scale bars: 20 µm.
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pgen-1002762-g001: Marker analysis of the LFP domain.(A) Schematic drawings of the zebrafish ventral spinal cord at 24 hpf in a dorsal view (a), lateral view (b), and cross-section (c). The lateral floor plate domain, consisting of LFP cells and KA″ interneurons, flanks the medial floor plate (MFP). The motor neuron domain locates dorsal to the LFP domain, which contains motor neuron progenitors (pMN), motor neurons (MN), and Kolmer-Agduhr′ interneurons (KA′). KA interneurons, including KA′ and KA″ cells, represent a unique class of GABAergic interneurons that are in contact with the cerebrospinal fluid and are implicated in regulating the swimming behavior in zebrafish [35], [47], [48]. Marker expression in MFP, LFP and KA″ cells are indicated on the right. N: notochord. (B) KA″ cells, labeled by tal2 expression (arrows, red), are discontinuously distributed flanking the medial floor plate (arrowhead) along the anterior-posterior axis. Cell nuclei were labeled by Draq5 staining (green). (C) All tal2-positive cells (arrows, red) in the LFP domain also express pan-neuronal marker HuC (green). (D–E) All GFP-expressing cells in the LFP region (arrows, green) in Gata2-GFP fish also co-express tal2 (D, red) and GABA (E, red). (F–G) Wild type embryos were co-labeled with nkx2.9 (F, green) or nkx2.2a (G, green) together with tal2 (red). tal2-expressing KA″ cells (arrows) show lower expression of nkx2.9 and nkx2.2a compared to their neighboring cells. Embryos shown are at 21 hpf (B), 30 hpf (C), 25 hpf (D), 27 hpf (E), and 18 hpf (F, G). B, D, F, and G show dorsal views, and C and E represent lateral views. Scale bars: 20 µm.

Mentions: We used the lateral floor plate as a model system to study how differentiated cells are generated from progenitor cells. First, we confirmed the heterogeneous nature of the LFP domain using in situ hybridization and immunohistochemistry (Figure 1). By 1 day post fertilization (dpf), two distinct cell types can be distinguished by specific marker expression (Figure 1A). KA″ cells are marked by the expression of gata2, tal2, the neurotransmitter GABA, and HuC, a marker of differentiated neurons (Figure 1B–1E). They are discontinuously distributed along the anterior-posterior axis. By contrast, nkx2.9 and nkx2.2a are expressed in a pattern complementary to tal2 expression, with little expression in tal2-positive KA″ cells (Figure 1F and 1G). Since both nkx2.9 and nkx2.2a are required for the induction of KA″ cells [5], these results suggest that nkx2.9 and nkx2.2a label undifferentiated LFP progenitor cells and are downregulated in differentiated KA″ cells marked by HuC and GABA.


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)

Marker analysis of the LFP domain.(A) Schematic drawings of the zebrafish ventral spinal cord at 24 hpf in a dorsal view (a), lateral view (b), and cross-section (c). The lateral floor plate domain, consisting of LFP cells and KA″ interneurons, flanks the medial floor plate (MFP). The motor neuron domain locates dorsal to the LFP domain, which contains motor neuron progenitors (pMN), motor neurons (MN), and Kolmer-Agduhr′ interneurons (KA′). KA interneurons, including KA′ and KA″ cells, represent a unique class of GABAergic interneurons that are in contact with the cerebrospinal fluid and are implicated in regulating the swimming behavior in zebrafish [35], [47], [48]. Marker expression in MFP, LFP and KA″ cells are indicated on the right. N: notochord. (B) KA″ cells, labeled by tal2 expression (arrows, red), are discontinuously distributed flanking the medial floor plate (arrowhead) along the anterior-posterior axis. Cell nuclei were labeled by Draq5 staining (green). (C) All tal2-positive cells (arrows, red) in the LFP domain also express pan-neuronal marker HuC (green). (D–E) All GFP-expressing cells in the LFP region (arrows, green) in Gata2-GFP fish also co-express tal2 (D, red) and GABA (E, red). (F–G) Wild type embryos were co-labeled with nkx2.9 (F, green) or nkx2.2a (G, green) together with tal2 (red). tal2-expressing KA″ cells (arrows) show lower expression of nkx2.9 and nkx2.2a compared to their neighboring cells. Embryos shown are at 21 hpf (B), 30 hpf (C), 25 hpf (D), 27 hpf (E), and 18 hpf (F, G). B, D, F, and G show dorsal views, and C and E represent lateral views. Scale bars: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002762-g001: Marker analysis of the LFP domain.(A) Schematic drawings of the zebrafish ventral spinal cord at 24 hpf in a dorsal view (a), lateral view (b), and cross-section (c). The lateral floor plate domain, consisting of LFP cells and KA″ interneurons, flanks the medial floor plate (MFP). The motor neuron domain locates dorsal to the LFP domain, which contains motor neuron progenitors (pMN), motor neurons (MN), and Kolmer-Agduhr′ interneurons (KA′). KA interneurons, including KA′ and KA″ cells, represent a unique class of GABAergic interneurons that are in contact with the cerebrospinal fluid and are implicated in regulating the swimming behavior in zebrafish [35], [47], [48]. Marker expression in MFP, LFP and KA″ cells are indicated on the right. N: notochord. (B) KA″ cells, labeled by tal2 expression (arrows, red), are discontinuously distributed flanking the medial floor plate (arrowhead) along the anterior-posterior axis. Cell nuclei were labeled by Draq5 staining (green). (C) All tal2-positive cells (arrows, red) in the LFP domain also express pan-neuronal marker HuC (green). (D–E) All GFP-expressing cells in the LFP region (arrows, green) in Gata2-GFP fish also co-express tal2 (D, red) and GABA (E, red). (F–G) Wild type embryos were co-labeled with nkx2.9 (F, green) or nkx2.2a (G, green) together with tal2 (red). tal2-expressing KA″ cells (arrows) show lower expression of nkx2.9 and nkx2.2a compared to their neighboring cells. Embryos shown are at 21 hpf (B), 30 hpf (C), 25 hpf (D), 27 hpf (E), and 18 hpf (F, G). B, D, F, and G show dorsal views, and C and E represent lateral views. Scale bars: 20 µm.
Mentions: We used the lateral floor plate as a model system to study how differentiated cells are generated from progenitor cells. First, we confirmed the heterogeneous nature of the LFP domain using in situ hybridization and immunohistochemistry (Figure 1). By 1 day post fertilization (dpf), two distinct cell types can be distinguished by specific marker expression (Figure 1A). KA″ cells are marked by the expression of gata2, tal2, the neurotransmitter GABA, and HuC, a marker of differentiated neurons (Figure 1B–1E). They are discontinuously distributed along the anterior-posterior axis. By contrast, nkx2.9 and nkx2.2a are expressed in a pattern complementary to tal2 expression, with little expression in tal2-positive KA″ cells (Figure 1F and 1G). Since both nkx2.9 and nkx2.2a are required for the induction of KA″ cells [5], these results suggest that nkx2.9 and nkx2.2a label undifferentiated LFP progenitor cells and are downregulated in differentiated KA″ cells marked by HuC and GABA.

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