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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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Related in: MedlinePlus

Lineage analysis of the LFP domain.(A) Schematic drawings of scatter labeling and time-lapse imaging. Gata2-GFP fish were injected with H2B-mCherry mRNA (red) into a single blastomere at 16- to 32-cell stage. Scatter labeled embryos with nuclear mCherry expression (red) were imaged in the dorsal view starting at 3-somite stage (11 hpf) for about 11 hours. At the end of the time-lapse, an image with both the green and red channels was acquired to identify Gata2-GFP-expressing KA″ cells (green). (B) Observed division patterns in the LFP domain. Of a total of 25 cell divisions tracked, 16 were LFP/LFP divisions, 6 were KA″/LFP divisions, and 3 were KA″/KA″ divisions. KA″ cells (green) were identified base on the expression of Gata2-GFP reporter. (C) Gata2-GFP fish (green) was scatter labeled by H2B-mCherry (red) and imaged from 11 hpf for about 11 hours. Two examples are shown. Top panel: merged images with both the red channel and the bright field of a single optical slice at the start of the movie at 11 hpf. The underlying notochord (brackets) is visible but out of focus. Middle and bottom panels: the merged image with both green and red channels and the green channel alone of a confocal projection at the end of the movie around 22 hpf. KA″ cells (arrows) can be distinguished from LFP cells (arrowheads) based on the expression of Gata2-GFP (green) at 22 hpf. Medial floor plate cells are indicated by white dots. Lineage related cells confirmed by cell tracking are indicated (also see Figure S1 and Video S1). For example, cell 1 generates a KA″ cell (cell 1a) and an LFP cell (cell 1b). Of the 7 examples shown here, cells 1 and 6 undergo KA″/LFP divisions, cell 7 undergoes KA″/KA″ division, and cells 2–5 divide symmetrically giving rise to two LFP cells (LFP/LFP divisions). Note that cells 4 and 6 are more dorsally located and therefore not in focus in images at 11 hpf (top panels). Scale bars: 20 µm.
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pgen-1002762-g002: Lineage analysis of the LFP domain.(A) Schematic drawings of scatter labeling and time-lapse imaging. Gata2-GFP fish were injected with H2B-mCherry mRNA (red) into a single blastomere at 16- to 32-cell stage. Scatter labeled embryos with nuclear mCherry expression (red) were imaged in the dorsal view starting at 3-somite stage (11 hpf) for about 11 hours. At the end of the time-lapse, an image with both the green and red channels was acquired to identify Gata2-GFP-expressing KA″ cells (green). (B) Observed division patterns in the LFP domain. Of a total of 25 cell divisions tracked, 16 were LFP/LFP divisions, 6 were KA″/LFP divisions, and 3 were KA″/KA″ divisions. KA″ cells (green) were identified base on the expression of Gata2-GFP reporter. (C) Gata2-GFP fish (green) was scatter labeled by H2B-mCherry (red) and imaged from 11 hpf for about 11 hours. Two examples are shown. Top panel: merged images with both the red channel and the bright field of a single optical slice at the start of the movie at 11 hpf. The underlying notochord (brackets) is visible but out of focus. Middle and bottom panels: the merged image with both green and red channels and the green channel alone of a confocal projection at the end of the movie around 22 hpf. KA″ cells (arrows) can be distinguished from LFP cells (arrowheads) based on the expression of Gata2-GFP (green) at 22 hpf. Medial floor plate cells are indicated by white dots. Lineage related cells confirmed by cell tracking are indicated (also see Figure S1 and Video S1). For example, cell 1 generates a KA″ cell (cell 1a) and an LFP cell (cell 1b). Of the 7 examples shown here, cells 1 and 6 undergo KA″/LFP divisions, cell 7 undergoes KA″/KA″ division, and cells 2–5 divide symmetrically giving rise to two LFP cells (LFP/LFP divisions). Note that cells 4 and 6 are more dorsally located and therefore not in focus in images at 11 hpf (top panels). Scale bars: 20 µm.

Mentions: The discontinuous distribution of KA″ cells in the LFP domain suggests two models for KA″ formation. First, KA″ cells and LFP cells might be generated separately from distinct pools of progenitor cells, and subsequent cell intercalation results in the characteristic “salt-and-pepper” pattern. Alternatively, a LFP progenitor might give rise to both KA″ cells and LFP cells. To test these models, we carried out confocal time-lapse microscopy to assess cell lineages within the LFP domain (Figure 2, Figure S1, and Video S1). Cells were tracked at high temporal resolution using the nuclear marker, H2B-mCherry. A Gata2-GFP transgenic reporter [27] was used to identify KA″ cells at the end of the time-lapse movie (Figure 1 and Figure S2). LFP cells were identified based on their lateral juxtaposition to the medial floor plate and their anterior or posterior juxtaposition to KA″ cells (Figure 1 and Figure S2). To achieve reliable cell tracing, we generated scatter labeled embryos by injecting H2B-mCherry RNA into a single blastomere at the 16-cell to 32-cell stage (Figure 2A). This method allowed us to unambiguously identify cell divisions, trace cell movements and determine the fate of daughter cells (Video S1). By retrospective cell tracing, we identified the distribution of LFP progenitor cells at the early somite stage (11 hpf). At this stage LFP progenitors were positioned both medial-laterally and dorsal-ventrally (Figure 2C, top panels). Progeny of LFP progenitor cells eventually converged to align as two rows of one-cell-wide domains flanking medial floor plate cells (Figure 2C, middle and bottom panels). At 22 hpf, the positions of the two daughter cells generated by a LFP progenitor cell division were quite variable. Some siblings remained immediate neighbors, whereas others were separated by a few cells or even by the midline (Figure 2C, middle and bottom panels). Of 25 divisions imaged within the LFP domain, 64% generated two Gata2-GFP− LFP cells (LFP/LFP), 24% gave rise to one KA″ cell and one LFP cell (KA″/LFP), and 12% generated two Gata2-GFP+ KA″ cells (KA″/KA″) (Figure 2B). These results reveal that KA″ cells can be generated by both symmetric and asymmetric cell divisions from LFP progenitors.


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)

Lineage analysis of the LFP domain.(A) Schematic drawings of scatter labeling and time-lapse imaging. Gata2-GFP fish were injected with H2B-mCherry mRNA (red) into a single blastomere at 16- to 32-cell stage. Scatter labeled embryos with nuclear mCherry expression (red) were imaged in the dorsal view starting at 3-somite stage (11 hpf) for about 11 hours. At the end of the time-lapse, an image with both the green and red channels was acquired to identify Gata2-GFP-expressing KA″ cells (green). (B) Observed division patterns in the LFP domain. Of a total of 25 cell divisions tracked, 16 were LFP/LFP divisions, 6 were KA″/LFP divisions, and 3 were KA″/KA″ divisions. KA″ cells (green) were identified base on the expression of Gata2-GFP reporter. (C) Gata2-GFP fish (green) was scatter labeled by H2B-mCherry (red) and imaged from 11 hpf for about 11 hours. Two examples are shown. Top panel: merged images with both the red channel and the bright field of a single optical slice at the start of the movie at 11 hpf. The underlying notochord (brackets) is visible but out of focus. Middle and bottom panels: the merged image with both green and red channels and the green channel alone of a confocal projection at the end of the movie around 22 hpf. KA″ cells (arrows) can be distinguished from LFP cells (arrowheads) based on the expression of Gata2-GFP (green) at 22 hpf. Medial floor plate cells are indicated by white dots. Lineage related cells confirmed by cell tracking are indicated (also see Figure S1 and Video S1). For example, cell 1 generates a KA″ cell (cell 1a) and an LFP cell (cell 1b). Of the 7 examples shown here, cells 1 and 6 undergo KA″/LFP divisions, cell 7 undergoes KA″/KA″ division, and cells 2–5 divide symmetrically giving rise to two LFP cells (LFP/LFP divisions). Note that cells 4 and 6 are more dorsally located and therefore not in focus in images at 11 hpf (top panels). 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-g002: Lineage analysis of the LFP domain.(A) Schematic drawings of scatter labeling and time-lapse imaging. Gata2-GFP fish were injected with H2B-mCherry mRNA (red) into a single blastomere at 16- to 32-cell stage. Scatter labeled embryos with nuclear mCherry expression (red) were imaged in the dorsal view starting at 3-somite stage (11 hpf) for about 11 hours. At the end of the time-lapse, an image with both the green and red channels was acquired to identify Gata2-GFP-expressing KA″ cells (green). (B) Observed division patterns in the LFP domain. Of a total of 25 cell divisions tracked, 16 were LFP/LFP divisions, 6 were KA″/LFP divisions, and 3 were KA″/KA″ divisions. KA″ cells (green) were identified base on the expression of Gata2-GFP reporter. (C) Gata2-GFP fish (green) was scatter labeled by H2B-mCherry (red) and imaged from 11 hpf for about 11 hours. Two examples are shown. Top panel: merged images with both the red channel and the bright field of a single optical slice at the start of the movie at 11 hpf. The underlying notochord (brackets) is visible but out of focus. Middle and bottom panels: the merged image with both green and red channels and the green channel alone of a confocal projection at the end of the movie around 22 hpf. KA″ cells (arrows) can be distinguished from LFP cells (arrowheads) based on the expression of Gata2-GFP (green) at 22 hpf. Medial floor plate cells are indicated by white dots. Lineage related cells confirmed by cell tracking are indicated (also see Figure S1 and Video S1). For example, cell 1 generates a KA″ cell (cell 1a) and an LFP cell (cell 1b). Of the 7 examples shown here, cells 1 and 6 undergo KA″/LFP divisions, cell 7 undergoes KA″/KA″ division, and cells 2–5 divide symmetrically giving rise to two LFP cells (LFP/LFP divisions). Note that cells 4 and 6 are more dorsally located and therefore not in focus in images at 11 hpf (top panels). Scale bars: 20 µm.
Mentions: The discontinuous distribution of KA″ cells in the LFP domain suggests two models for KA″ formation. First, KA″ cells and LFP cells might be generated separately from distinct pools of progenitor cells, and subsequent cell intercalation results in the characteristic “salt-and-pepper” pattern. Alternatively, a LFP progenitor might give rise to both KA″ cells and LFP cells. To test these models, we carried out confocal time-lapse microscopy to assess cell lineages within the LFP domain (Figure 2, Figure S1, and Video S1). Cells were tracked at high temporal resolution using the nuclear marker, H2B-mCherry. A Gata2-GFP transgenic reporter [27] was used to identify KA″ cells at the end of the time-lapse movie (Figure 1 and Figure S2). LFP cells were identified based on their lateral juxtaposition to the medial floor plate and their anterior or posterior juxtaposition to KA″ cells (Figure 1 and Figure S2). To achieve reliable cell tracing, we generated scatter labeled embryos by injecting H2B-mCherry RNA into a single blastomere at the 16-cell to 32-cell stage (Figure 2A). This method allowed us to unambiguously identify cell divisions, trace cell movements and determine the fate of daughter cells (Video S1). By retrospective cell tracing, we identified the distribution of LFP progenitor cells at the early somite stage (11 hpf). At this stage LFP progenitors were positioned both medial-laterally and dorsal-ventrally (Figure 2C, top panels). Progeny of LFP progenitor cells eventually converged to align as two rows of one-cell-wide domains flanking medial floor plate cells (Figure 2C, middle and bottom panels). At 22 hpf, the positions of the two daughter cells generated by a LFP progenitor cell division were quite variable. Some siblings remained immediate neighbors, whereas others were separated by a few cells or even by the midline (Figure 2C, middle and bottom panels). Of 25 divisions imaged within the LFP domain, 64% generated two Gata2-GFP− LFP cells (LFP/LFP), 24% gave rise to one KA″ cell and one LFP cell (KA″/LFP), and 12% generated two Gata2-GFP+ KA″ cells (KA″/KA″) (Figure 2B). These results reveal that KA″ cells can be generated by both symmetric and asymmetric cell divisions from LFP progenitors.

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