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FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles.

Faulkner RL, Wishard TJ, Thompson CK, Liu HH, Cline HT - eNeuro (2015 Jan-Feb)

Bottom Line: Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable.Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival.These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California 92037.

ABSTRACT

Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction, however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles which express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or over-expression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

No MeSH data available.


Related in: MedlinePlus

Knockdown of FMRP increases cell death. A, Confocal Z-projections through five optical sections of tectum with caspase-3 (Casp3) immunoreactivity and SYTOX Orange staining. Twenty-four hour incubation in staurosporine (STS) increases the number of apoptotic cells that are immunoreactive for Casp3 and brightly stained for SYTOX Orange. Scale bar, 100 μm. B, High-magnification single-optical sections from different animals demonstrate the staining variations of apoptotic cells. The majority of positively labeled cells are stained for both Casp3 and SYTOX Orange (white arrows). The remaining cells are positive for only SYTOX (yellow arrow) or only Casp3 (blue arrow). Scale bar, 20 μm. C, Quantification of total apoptotic cells in the presence or absence of STS demonstrates that SYTOX Orange and Casp3 detect the STS-induced increase in cell death. SYTOX stains a larger dying cell population than Casp3. D, SYTOX Green staining in whole-mount optic tecta was used to identify cells undergoing apoptosis in the presence of fmr1a MO. Bright, apoptotic SYTOX Green+ cells are marked by blue and yellow arrows in confocal Z-projections through the dorsal 30 optical sections of tectum. Cells marked by blue arrows are shown at higher magnification (right) in single-optical sections of the areas highlighted to the left (yellow arrows in the Z-projection to the left are out of the plane of focus in the single-optical section to the right). Scale bars, 50 μm. E, Quantification of the total number of apoptotic SYTOX Green+ cells at 1 dfe shows that both concentrations of fmr1a MO increase cell death compared to CMO (**p < 0.01, ***p < 0.001).
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Figure 5: Knockdown of FMRP increases cell death. A, Confocal Z-projections through five optical sections of tectum with caspase-3 (Casp3) immunoreactivity and SYTOX Orange staining. Twenty-four hour incubation in staurosporine (STS) increases the number of apoptotic cells that are immunoreactive for Casp3 and brightly stained for SYTOX Orange. Scale bar, 100 μm. B, High-magnification single-optical sections from different animals demonstrate the staining variations of apoptotic cells. The majority of positively labeled cells are stained for both Casp3 and SYTOX Orange (white arrows). The remaining cells are positive for only SYTOX (yellow arrow) or only Casp3 (blue arrow). Scale bar, 20 μm. C, Quantification of total apoptotic cells in the presence or absence of STS demonstrates that SYTOX Orange and Casp3 detect the STS-induced increase in cell death. SYTOX stains a larger dying cell population than Casp3. D, SYTOX Green staining in whole-mount optic tecta was used to identify cells undergoing apoptosis in the presence of fmr1a MO. Bright, apoptotic SYTOX Green+ cells are marked by blue and yellow arrows in confocal Z-projections through the dorsal 30 optical sections of tectum. Cells marked by blue arrows are shown at higher magnification (right) in single-optical sections of the areas highlighted to the left (yellow arrows in the Z-projection to the left are out of the plane of focus in the single-optical section to the right). Scale bars, 50 μm. E, Quantification of the total number of apoptotic SYTOX Green+ cells at 1 dfe shows that both concentrations of fmr1a MO increase cell death compared to CMO (**p < 0.01, ***p < 0.001).

Mentions: Next, we developed a sensitive in vivo assay to assess the ability of MOs to block translation in Xenopus that does not require antibody detection. For this assay, we electroporated a reporter construct into the Xenopus optic tectum, which generates two discrete proteins from a single transcript: the protein of interest and a fluorescent protein reporter (FP) linked by a t2A sequence. When MO and the reporter construct are co-electroporated, the MO prevents translation of the transcript, decreasing expression of both the protein of interest and the FP. Measurements of FP intensity can be used as a proxy for knockdown of the protein of interest, in this case, FMRP. Here, we used a plasmid that contains a promoter with the Sox2 and Oct3/4-binding domain of the FGF minipromoter that requires binding of endogenous Sox2 to express eGFP and FMRP in Sox2-expressing NPCs and their neuronal progeny (Bestman et al., 2012). FMRP and eGFP are separated by a t2A sequence, producing two discrete proteins from a single transcript. Expression from this plasmid is amplified using the gal4/UAS system. This plasmid is called Sox2bd::gal4-UAS::fmr1-t2A-eGFP and will be referred to as fmr1-t2A-eGFP (Fig. 2C). In addition, we co-expressed a UAS-driven turboRFP tagged with a nuclear localization sequence (UAS::tRFPnls) to visualize labeled cells. We anticipated that when CMO is co-electroporated with fmr1-t2A-eGFP and UAS::tRFPnls, CMO would not affect translation and FMRP, eGFP, and tRFPnls would all be expressed. In contrast, when fmr1a MO is co-electroporated, translation of FMRP and eGFP would be inhibited, but expression of tRFPnls would be unaffected. We electroporated stage 46 − 47 animals with fmr1-t2A-eGFP, UAS::tRFPnls, and either CMO, LOW fmr1a MO, or HIGH fmr1a MO and then imaged labeled cells in vivo using a spinning-disk confocal microscope (Fig. 2D). When we imaged control cells 1 dfe, we found that cells expressed tRFPnls but very little eGFP (data not shown). This is most likely explained by differences in the timing of expression of tRFP and eGFP, because tRFP matures more rapidly than eGFP. When we imaged control cells at 2 dfe, we found that eGFP and tRFPnls were both highly expressed in electroporated cells (Fig. 2D). Therefore, we imaged animals at 2 dfe to test the effectiveness of the two concentrations of fmr1a MO (Fig. 2D−F). We quantified the percentage of cells that lacked detectable eGFP expression, an indicator of strong knockdown. Both concentrations of fmr1a MO yielded a higher percentage of cells that lacked detectable eGFP expression compared to CMO (Fig. 2E; CMO: N = 27 animals; LOW fmr1a MO: N = 30 animals, p < 0.0001b compared to CMO; HIGH fmr1a MO: N = 20 animals, p = 0.0016b compared to CMO). We did not detect any significant differences between LOW and HIGH fmr1a MO on the percentage of tRFP-only cells (p = 0.31b). However, animals electroporated with HIGH fmr1a MO tended to have fewer labeled cells and more debris from what we suspect are dying cells. Therefore, it is likely that cells with the most severe knockdown in the presence of HIGH fmr1a MO did not survive. We address the potential effect of FMRP knockdown on cell survival in Fig. 5. In cells where eGFP was visible, the ratio of eGFP/tRFP was significantly reduced with fmr1a MO compared to CMO, and HIGH fmr1a MO had a greater reduction than LOW fmr1a MO (Fig. 2F; CMO: N = 253 cells; LOW fmr1a MO: N = 275 cells, p < 0.0001c compared to CMO; HIGH fmr1a MO: N = 172 cells, p < 0.0001c compared to CMO, p = 0.020c compared to LOW fmr1a MO). Electroporation of a lower concentration of fmr1a MO (0.01 mM) resulted in no significant knockdown (data not shown). Together, these two assays demonstrate that fmr1a MO is effective at knocking down FMRP expression and that LOW and HIGH fmr1a MO reflect different levels of knockdown. We used both concentrations of fmr1a MO in our experiments to test how sensitive tectal cells are to the reduction in FMRP.


FMRP regulates neurogenesis in vivo in Xenopus laevis tadpoles.

Faulkner RL, Wishard TJ, Thompson CK, Liu HH, Cline HT - eNeuro (2015 Jan-Feb)

Knockdown of FMRP increases cell death. A, Confocal Z-projections through five optical sections of tectum with caspase-3 (Casp3) immunoreactivity and SYTOX Orange staining. Twenty-four hour incubation in staurosporine (STS) increases the number of apoptotic cells that are immunoreactive for Casp3 and brightly stained for SYTOX Orange. Scale bar, 100 μm. B, High-magnification single-optical sections from different animals demonstrate the staining variations of apoptotic cells. The majority of positively labeled cells are stained for both Casp3 and SYTOX Orange (white arrows). The remaining cells are positive for only SYTOX (yellow arrow) or only Casp3 (blue arrow). Scale bar, 20 μm. C, Quantification of total apoptotic cells in the presence or absence of STS demonstrates that SYTOX Orange and Casp3 detect the STS-induced increase in cell death. SYTOX stains a larger dying cell population than Casp3. D, SYTOX Green staining in whole-mount optic tecta was used to identify cells undergoing apoptosis in the presence of fmr1a MO. Bright, apoptotic SYTOX Green+ cells are marked by blue and yellow arrows in confocal Z-projections through the dorsal 30 optical sections of tectum. Cells marked by blue arrows are shown at higher magnification (right) in single-optical sections of the areas highlighted to the left (yellow arrows in the Z-projection to the left are out of the plane of focus in the single-optical section to the right). Scale bars, 50 μm. E, Quantification of the total number of apoptotic SYTOX Green+ cells at 1 dfe shows that both concentrations of fmr1a MO increase cell death compared to CMO (**p < 0.01, ***p < 0.001).
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Figure 5: Knockdown of FMRP increases cell death. A, Confocal Z-projections through five optical sections of tectum with caspase-3 (Casp3) immunoreactivity and SYTOX Orange staining. Twenty-four hour incubation in staurosporine (STS) increases the number of apoptotic cells that are immunoreactive for Casp3 and brightly stained for SYTOX Orange. Scale bar, 100 μm. B, High-magnification single-optical sections from different animals demonstrate the staining variations of apoptotic cells. The majority of positively labeled cells are stained for both Casp3 and SYTOX Orange (white arrows). The remaining cells are positive for only SYTOX (yellow arrow) or only Casp3 (blue arrow). Scale bar, 20 μm. C, Quantification of total apoptotic cells in the presence or absence of STS demonstrates that SYTOX Orange and Casp3 detect the STS-induced increase in cell death. SYTOX stains a larger dying cell population than Casp3. D, SYTOX Green staining in whole-mount optic tecta was used to identify cells undergoing apoptosis in the presence of fmr1a MO. Bright, apoptotic SYTOX Green+ cells are marked by blue and yellow arrows in confocal Z-projections through the dorsal 30 optical sections of tectum. Cells marked by blue arrows are shown at higher magnification (right) in single-optical sections of the areas highlighted to the left (yellow arrows in the Z-projection to the left are out of the plane of focus in the single-optical section to the right). Scale bars, 50 μm. E, Quantification of the total number of apoptotic SYTOX Green+ cells at 1 dfe shows that both concentrations of fmr1a MO increase cell death compared to CMO (**p < 0.01, ***p < 0.001).
Mentions: Next, we developed a sensitive in vivo assay to assess the ability of MOs to block translation in Xenopus that does not require antibody detection. For this assay, we electroporated a reporter construct into the Xenopus optic tectum, which generates two discrete proteins from a single transcript: the protein of interest and a fluorescent protein reporter (FP) linked by a t2A sequence. When MO and the reporter construct are co-electroporated, the MO prevents translation of the transcript, decreasing expression of both the protein of interest and the FP. Measurements of FP intensity can be used as a proxy for knockdown of the protein of interest, in this case, FMRP. Here, we used a plasmid that contains a promoter with the Sox2 and Oct3/4-binding domain of the FGF minipromoter that requires binding of endogenous Sox2 to express eGFP and FMRP in Sox2-expressing NPCs and their neuronal progeny (Bestman et al., 2012). FMRP and eGFP are separated by a t2A sequence, producing two discrete proteins from a single transcript. Expression from this plasmid is amplified using the gal4/UAS system. This plasmid is called Sox2bd::gal4-UAS::fmr1-t2A-eGFP and will be referred to as fmr1-t2A-eGFP (Fig. 2C). In addition, we co-expressed a UAS-driven turboRFP tagged with a nuclear localization sequence (UAS::tRFPnls) to visualize labeled cells. We anticipated that when CMO is co-electroporated with fmr1-t2A-eGFP and UAS::tRFPnls, CMO would not affect translation and FMRP, eGFP, and tRFPnls would all be expressed. In contrast, when fmr1a MO is co-electroporated, translation of FMRP and eGFP would be inhibited, but expression of tRFPnls would be unaffected. We electroporated stage 46 − 47 animals with fmr1-t2A-eGFP, UAS::tRFPnls, and either CMO, LOW fmr1a MO, or HIGH fmr1a MO and then imaged labeled cells in vivo using a spinning-disk confocal microscope (Fig. 2D). When we imaged control cells 1 dfe, we found that cells expressed tRFPnls but very little eGFP (data not shown). This is most likely explained by differences in the timing of expression of tRFP and eGFP, because tRFP matures more rapidly than eGFP. When we imaged control cells at 2 dfe, we found that eGFP and tRFPnls were both highly expressed in electroporated cells (Fig. 2D). Therefore, we imaged animals at 2 dfe to test the effectiveness of the two concentrations of fmr1a MO (Fig. 2D−F). We quantified the percentage of cells that lacked detectable eGFP expression, an indicator of strong knockdown. Both concentrations of fmr1a MO yielded a higher percentage of cells that lacked detectable eGFP expression compared to CMO (Fig. 2E; CMO: N = 27 animals; LOW fmr1a MO: N = 30 animals, p < 0.0001b compared to CMO; HIGH fmr1a MO: N = 20 animals, p = 0.0016b compared to CMO). We did not detect any significant differences between LOW and HIGH fmr1a MO on the percentage of tRFP-only cells (p = 0.31b). However, animals electroporated with HIGH fmr1a MO tended to have fewer labeled cells and more debris from what we suspect are dying cells. Therefore, it is likely that cells with the most severe knockdown in the presence of HIGH fmr1a MO did not survive. We address the potential effect of FMRP knockdown on cell survival in Fig. 5. In cells where eGFP was visible, the ratio of eGFP/tRFP was significantly reduced with fmr1a MO compared to CMO, and HIGH fmr1a MO had a greater reduction than LOW fmr1a MO (Fig. 2F; CMO: N = 253 cells; LOW fmr1a MO: N = 275 cells, p < 0.0001c compared to CMO; HIGH fmr1a MO: N = 172 cells, p < 0.0001c compared to CMO, p = 0.020c compared to LOW fmr1a MO). Electroporation of a lower concentration of fmr1a MO (0.01 mM) resulted in no significant knockdown (data not shown). Together, these two assays demonstrate that fmr1a MO is effective at knocking down FMRP expression and that LOW and HIGH fmr1a MO reflect different levels of knockdown. We used both concentrations of fmr1a MO in our experiments to test how sensitive tectal cells are to the reduction in FMRP.

Bottom Line: Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable.Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival.These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California 92037.

ABSTRACT

Fragile X Syndrome (FXS) is the leading known monogenic form of autism and the most common form of inherited intellectual disability. FXS results from silencing the FMR1 gene during embryonic development, leading to loss of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates mRNA transport, stability, and translation. FXS is commonly thought of as a disease of synaptic dysfunction, however, FMRP expression is lost early in embryonic development, well before most synaptogenesis occurs. Recent studies suggest that loss of FMRP results in aberrant neurogenesis, but neurogenic defects have been variable. We investigated whether FMRP affects neurogenesis in Xenopus laevis tadpoles which express a homolog of FMR1. We used in vivo time-lapse imaging of neural progenitor cells and their neuronal progeny to evaluate the effect of acute loss or over-expression of FMRP on neurogenesis in the developing optic tectum. We complimented the time-lapse studies with SYTOX labeling to quantify apoptosis and CldU labeling to measure cell proliferation. Animals with increased or decreased levels of FMRP have significantly decreased neuronal proliferation and survival. They also have increased neuronal differentiation, but deficient dendritic arbor elaboration. The presence and severity of these defects was highly sensitive to FMRP levels. These data demonstrate that FMRP plays an important role in neurogenesis and suggest that endogenous FMRP levels are carefully regulated. These studies show promise in using Xenopus as an experimental system to study fundamental deficits in brain development with loss of FMRP and give new insight into the pathophysiology of FXS.

No MeSH data available.


Related in: MedlinePlus