Faulkner RL , Wishard TJ , Thompson CK , Liu HH , Cline HT .

F32 NS071807 NINDS NIH HHS , K99 ES022992 NIEHS NIH HHS , R01 EY011261 NEI NIH HHS , T34 GM087193 NIGMS NIH HHS , R00 ES022992 NIEHS NIH HHS

	Figure 1. FMRP is expressed in Xenopus optic tectal progenitors and neurons. A, Schematic of the Xenopus tadpole optic tectum showing the location of neural progenitor cells (purple) and neurons (green) extending processes into the neuropil. B, A single optical confocal section of stage 47 Xenopus optic tectum shows widespread FMRP immunoreactivity. Scale bar, 100 μm. C, A higher magnification view from a single optical section in a different animal shows FMRP immunoreactivity across all cell layers and throughout the neuropil. Scale bar, 50 μm.
	Figure 2. Validation of fmr1a morpholino-mediated knockdown. Antibody-dependent and -independent strategies to validate knockdown of FMRP by translation-blocking antisense morpholinos. A, Confocal Z-projections of FMRP immunoreactivity in 40 μm sections through optic tectum. B, HIGH (0.1 mM) fmr1a MO results in a 60% decrease in FMRP immunoreactivity (***p < 0.001). C, Antibody-independent strategy to validate in vivo knockdown by morpholinos. Animals are co-electroporated with Sox2bd::gal4-UAS::fmr1-t2A-eGFP and UAS::tRFPnls plasmids and either control morpholino (CMO) or fmr1a MO. In the presence of CMO, the electroporated plasmids will all be translated resulting in expression of FMRP, eGFP, and tRFPnls. In the presence of fmr1a MO, translation is inhibited resulting in a lack of FMRP and eGFP, while tRFPnls is expressed. The fluorescence intensity of eGFP is correlated with the expression of FMRP. D, Confocal Z-projections of optic tecta electroporated with the expression constructs and morpholinos in C and imaged in vivo show that LOW (0.05 mM) fmr1a MO and HIGH (0.1 mM) fmr1a MO decrease the expression of eGFP. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. E, fmr1a MO significantly increases the percentage of cells in which only tRFPnls is detected (**p < 0.01, ***p < 0.001). F, eGFP/tRFP ratios in cells that had detectable eGFP. fmr1a MO significantly reduced the eGFP/tRFP ratio compared to CMO and the decrease with HIGH fmr1a MO is larger than that of LOW fmr1a MO (*p < 0.05, ***p < 0.001). Scale bars, 50 μm.
	Figure 3. Validation of FMRP rescue and overexpression. A, Strategy for validating that the Δfmr1 Rescue construct is morpholino-insensitive. Point mutations in the fmr1 expression construct prevent translational inhibition by fmr1a MO resulting in control levels of FMRP, eGFP, and tRFPnls. B, Confocal Z-projections of optic tecta electroporated with the 2 μg/μl Δfmr1-t2A-eGFP, 1 μg/μl UAS::tRFPnls and LOW (0.05 mM) fmr1a MO as depicted in A and imaged in vivo. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. C, D, Quantification of the percentage of cells expressing tRFP-only (C) and the eGFP/tRFP ratio (D) were no different between CMO and LOW fmr1a MO. E, Western blots of Xenopus tadpole midbrain lysate labeled with anti-FMRP yields a band of approximately 72 kD, which is higher in intensity when FMRP is overexpressed with 1 μg/μl Δfmr1-t2A-eGFP (HIGH FMRP OE) compared to 1 μg/μl Sox2bd::eGFP (Ctrl) in two independent experiments. β-tubulin was used as a loading control. F, HIGH FMRP OE increases the intensity of the FMRP band by 1.6-fold compared to control. G, Z-projections from in vivo two-photon imaging of cells expressing 0.5 μg/μl Δfmr1-t2A-eGFP (LOW FMRP OE) or HIGH FMRP OE. H, eGFP fluorescence intensity is more than three times greater for HIGH FMRP OE compared to LOW FMRP OE (*p < 0.05). This reflects the difference in FMRP expression from these two construct concentrations since FMRP and eGFP are made from a single transcript. Scale bars, 50 μm.
	Figure 4. Knockdown and overexpression of FMRP decrease proliferation. A, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.05 mM fmr1a MO (LOW fmr1a MO), and 1 μg/μl Δfmr1-t2A-eGFP + 0.05 mM fmr1a MO (LOW MO HIGH Δfmr1 Rescue) taken at 1 and 3 dfe. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, The percent change in the number of eGFP+ cells increases over 3 d in CMO animals. FMRP knockdown with LOW fmr1a MO blocks the increase in cell number between 1 − 3 dfe. Coexpression of LOW fmr1a MO and HIGH Δfmr1 (LOW MO HIGH Δfmr1 Rescue) rescues the normal increase in cell number from 1 − 3 dfe (*p < 0.05, **p < 0.01). C, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.1 mM fmr1a MO (HIGH fmr1a MO), and 1 μg/μl Δfmr1-t2A-eGFP + 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue). Dashed lines outline the optic tectum. D, FMRP knockdown with HIGH fmr1a MO results in a negative percent change in cell number between 1 − 3 dfe, suggesting that proliferation and cell survival are affected with a higher concentration of morpholino. This decrease was rescued by co-electroporation of HIGH Δfmr1 (**p < 0.01, ***p < 0.001). E, A 2 h pulse of the thymidine analog CldU delivered at 3 dfe confirms that cell proliferation is decreased by HIGH fmr1a MO (*p < 0.05). F, Z-projections from in vivo confocal time-lapse images of Sox2bd::eGFP+ (Control) and 1 μg/μl Δfmr1-t2A-eGFP+ (HIGH FMRP OE) cells collected at 1 and 3 dfe. Dashed lines outline the optic tectum. G, H, The percent change in the number of eGFP+ cells increases over 3 d in control animals. HIGH FMRP OE significantly reduced the percent change in cell number between 1 − 3 dfe. Data from individual animals (G) and the mean ± SEM (H; ***p < 0.001). Scale bar, 50 μm.
	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).
	Figure 6. FMRP regulates differentiation. In vivo confocal time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 3 dfe and quantification of the changes in neural progenitor cells (NPCs) and neurons over time. A, Confocal Z-projections show the numbers of NPCs (purple arrows), neurons (green arrows), and unidentifiable cells (yellow arrows) in optic tecta expressing CMO, LOW (0.05 mM) fmr1a MO, and HIGH (0.1 mM) fmr1a MO. Dashed lines outline the optic tectum and inset shows a schematic of the optic tectum. B, C, Over 3 d of imaging, there is a decrease in the number of NPCs (B) and an increase in the number of neurons (C) in control animals. LOW and HIGH fmr1a MO decrease the number of NPCs, and HIGH fmr1a MO also decreases the number of neurons (*p < 0.05, **p < 0.01, ***p < 0.001). D, Knockdown of FMRP with HIGH fmr1a MO decreases the proportion of NPCs and increases the proportion of neurons (*p < 0.05). E, Z-projections from in vivo confocal time-lapse images of cells expressing Sox2bd::eGFP + CMO (CMO) or 0.1 mM fmr1a MO (HIGH fmr1a MO), or 1 μg/μl Δfmr1-t2A-eGFP alone (HIGH FMRP OE) or with 0.1 mM fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) at 3 dfe. Dashed lines outline the optic tectum. F, HIGH fmr1a MO and HIGH FMRP OE decrease the number of NPCs and co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP and HIGH fmr1a MO (HIGH MO HIGH Δfmr1 Rescue) partially rescues the defect at 3 dfe with HIGH FMRP OE alone, but does not rescue to control levels (*p < 0.05, **p < 0.01, ***p < 0.001). G, Neuron numbers decrease with HIGH fmr1a MO and this decrease is rescued by co-electroporation of 1 μg/μl Δfmr1-t2A-eGFP (HIGH MO HIGH Δfmr1 Rescue; *p < 0.05). H, HIGH fmr1a MO and HIGH FMRP OE both decrease the proportion of NPCs, and HIGH FMRP OE also increases the proportion of neurons and unidentifiable cells. At 3 dfe, coexpression of HIGH fmr1a MO and 1 μg/μl Δfmr1 partially rescues the HIGH FMRP OE-mediated decrease in NPC proportion, but other defects are not rescued (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 50 μm.
	Figure 7. FMRP regulates dendritic development. In vivo two-photon time-lapse images of cells expressing Sox2bd::eGFP and CMO or fmr1a MO collected at 2 and 3 dfe. A, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 2 and 3 dfe for cells with FMRP knockdown compared to control. B, HIGH (0.1 mM) fmr1a MO decreased total dendritic length at 3 dfe (**p < 0.01). C, HIGH fmr1a MO decreased total dendritic branch tip number at 2 and 3 dfe (*p < 0.05, **p < 0.01). D, Branch density was unchanged between the groups. E, Two-photon Z-projection and reconstructed dendritic arbor of a cell expressing 1 μg/μl Δfmr1-t2A-eGFP (HIGH FMRP OE) at 3 dfe. F, G, HIGH FMRP OE decreased total dendritic length (F) and total dendritic branch tip number (G) compared to control (Sox2bd::eGFP; ***p < 0.001). H, Two-photon Z-projections of imaged cells and their reconstructed dendritic arbors at 3 dfe for cells when FMRP is knocked down (HIGH fmr1a MO), overexpressed with 0.5 μg/μl Δfmr1-t2A-eGFP (LOW FMRP OE), and rescued (HIGH MO LOW Δfmr1 Rescue) compared to control (CMO). I, J, Co-electroporation of LOW Δfmr1-t2A-eGFP rescued HIGH fmr1a MO-mediated decreases in total dendritic length (I) and dendritic branch tip number (J) (*p < 0.05, **p <0.01). Scale bars, 20 μm.
	Figure 8. Neurogenesis is sensitive to FMRP levels. Summary diagram showing the consequences of perturbing FMRP levels on the labeled cell population. The numbers and proportions of neural progenitor cells (purple) and neurons (green), as well as the dendritic arbor morphology of neurons are altered in the presence of fmr1a MO or overexpression of FMRP. LOW fmr1a MO increases NPC apoptosis, leading to a reduction in the progenitor pool and a lower total number of cells present at 3 dfe compared to control. The neurons generated with LOW fmr1a MO have a trend toward deficient dendritic arbor development. HIGH fmr1a MO increases apoptosis compared to LOW fmr1a MO. In addition, HIGH fmr1a MO decreases proliferation and increases NPC differentiation into neurons. This leads to a greater reduction of the progenitor pool, a lower total number of cells present at 3 dfe, and a larger proportion of neurons among the cell population at 3 dfe. In addition, those neurons have a persistent defect in dendrite arbor elaboration. LOW FMRP overexpression does not result in defects in cell proliferation, cell death, differentiation, or dendritic morphology. HIGH FMRP overexpression increases cell death, decreases proliferation, and increases differentiation leading to a complete loss of the progenitor pool at 3 dfe. Neuron numbers are at control levels at 3 dfe because of the dramatic increase in differentiation, but those neurons have a defect in dendritic arbor development.

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