Journal: PLoS Biology

Article Title: PLZF Regulates Fibroblast Growth Factor Responsiveness and Maintenance of Neural Progenitors

doi: 10.1371/journal.pbio.1001676

Figure Lengend Snippet: (A, B, F, G) Stable transfection of NPCs with PLZF expression plasmids at e3 (HH 17) directs most cells to form SOX9 + glial progenitors when analyzed at e15 (HH 41) instead of NEUN + neurons. The frequency of cells expressing the undifferentiated NPC marker NESTIN is unchanged. Yellow lines indicate the midline of the spinal cord. (L) Chart displays the mean fraction of control and PLZF-transfected cells expressing these markers ± SEM. (C–E, H–J) Sustained PLZF expression enhances the formation of OLIG2 + oligodendrocyte progenitors, FGFR3 + astrocyte progenitors, and GFAP + astrocytes. (M) Chart displays the mean number of PLZF-transfected cells expressing the indicated markers ± SEM relative to cells electroporated with the empty control vector. Transfected cell counts were based on at least 10 images taken from ≥8 electroporated embryos. In all panels, ** p <0.01 and **** p <0.0001. (K) Location of panels (A–J) within the e15 (HH 41) spinal cord. WM, white matter; GM, grey matter; VZ ventricular zone. (N) Schematic model depicting the suppressive effects of PLZF on neurogenesis and enhancement of gliogenesis. ASTs, astrocytes; OLs, oligodendrocytes.

Article Snippet: Plasmid expression vectors were generated by cloning cDNAs of interest into a Gateway cloning-compatible variant of the vector pCIG , as follows: PLZF, full-length chick clone isolated by PCR from e4 chick cDNA; EnR-PLZF and VP16-PLZF were created by respectively fusing either the Drosophila Engrailed repressor domain or the herpes simplex VP16 transactivation domain to aa 300–665 of chick PLZF; FGFR3, WT form of the human FGFR3 ; caFGFR3, myristoylated and constitutively activated (K650E) form of the human FGFR3 cytoplasmic domain (aa 399–806) ; STAT3-C, mouse STAT3 containing two activating mutations (A662C, N664C) obtained from Addgene; and dnSTAT3 was created by incorporating into the mouse STAT3 nonphosphorylatable Y705F mutant , obtained from Addgene, an additional H332Y mutation that disrupts DNA binding .

Techniques: Stable Transfection, Expressing, Marker, Control, Transfection, Plasmid Preparation

Journal: PLoS Biology

Article Title: PLZF Regulates Fibroblast Growth Factor Responsiveness and Maintenance of Neural Progenitors

doi: 10.1371/journal.pbio.1001676

Figure Lengend Snippet: (A–F) The pattern of PLZF expression closely matches that of FGFR3 in the developing spinal cord. (G–J) PLZF misexpression is sufficient to induce the ectopic expression of FGFR3 in the dorsal spinal cord, while PLZF knockdown reduces FGFR3 expression in the intermediate spinal cord. (K) Chart displays the mean level of FGFR3 mRNA ± SEM in spinal cords electroporated with the indicated constructs relative to the contralateral control sides. (L–M, O–Q, S–T) NPCs transfected with either FGFR3 or STAT3-C expression plasmids display an increased propensity for SOX2 expression and reduced expression of NEUN. (N, R) Disruption of endogenous FGFR3 function through the expression of a dominant negative FGFR promotes the formation of NEUN + neurons. (T) Chart displays the mean number of cells expressing SOX2 ± SEM among the indicated experimental conditions, relative to empty vector controls. All electroporations except those shown in (I–K) were performed at e3 (HH 17) and collected at e5 (HH 25). Embryos in (I–K) were electroporated at e2 (HH 10) and collected at e4 (HH 21). Counts were based on at least 12 images taken from ≥8 electroporated embryos. **** p <0.0001. (U) Summary of results highlighting the similarities of PLZF, FGFR3, and STAT3-C misexpression on neural progenitor maintenance, and their presumed hierarchical relationship. PLZF repressor function (solid line) indirectly elevates FGFR3 expression levels, resulting in increased activation of STAT3 (STAT3*) and enhanced progenitor maintenance.

Article Snippet: Plasmid expression vectors were generated by cloning cDNAs of interest into a Gateway cloning-compatible variant of the vector pCIG , as follows: PLZF, full-length chick clone isolated by PCR from e4 chick cDNA; EnR-PLZF and VP16-PLZF were created by respectively fusing either the Drosophila Engrailed repressor domain or the herpes simplex VP16 transactivation domain to aa 300–665 of chick PLZF; FGFR3, WT form of the human FGFR3 ; caFGFR3, myristoylated and constitutively activated (K650E) form of the human FGFR3 cytoplasmic domain (aa 399–806) ; STAT3-C, mouse STAT3 containing two activating mutations (A662C, N664C) obtained from Addgene; and dnSTAT3 was created by incorporating into the mouse STAT3 nonphosphorylatable Y705F mutant , obtained from Addgene, an additional H332Y mutation that disrupts DNA binding .

Techniques: Expressing, Knockdown, Construct, Control, Transfection, Disruption, Dominant Negative Mutation, Plasmid Preparation, Activation Assay

Journal: PLoS Biology

Article Title: PLZF Regulates Fibroblast Growth Factor Responsiveness and Maintenance of Neural Progenitors

doi: 10.1371/journal.pbio.1001676

Figure Lengend Snippet: (A–L) The ability of ectopic PLZF to hold cells in a SOX2 + progenitor state and suppress neurogenesis is blocked by the coexpression of either dnFGFR or dnSTAT3. These electroporations were performed at e3 (HH 17) and analyzed at e5 (HH 25). (M–X) The reduced intensity of SOX2 expression and increased numbers of cells expressing NEUROG2 following PLZF knockdown are restored by coexpression with either FGFR3 or STAT3-C. These electroporations were performed at e2 (HH 10) and analyzed at e4 (HH 21). (Y, AA) Charts display the mean number of cells expressing SOX2 or NEUROG2 ± SEM between the indicated experimental conditions relative to empty vector controls. (Z) Chart displays the mean pixel intensity of SOX2 staining ± SEM relative to empty vector controls. Counts were based on at least 12 images taken from ≥8 electroporated embryos. * p <0.05, *** p <0.001, and **** p <0.0001. (AB) Summary of the epistasis tests used to show that FGFR3 acts downstream of PLZF.

Article Snippet: Plasmid expression vectors were generated by cloning cDNAs of interest into a Gateway cloning-compatible variant of the vector pCIG , as follows: PLZF, full-length chick clone isolated by PCR from e4 chick cDNA; EnR-PLZF and VP16-PLZF were created by respectively fusing either the Drosophila Engrailed repressor domain or the herpes simplex VP16 transactivation domain to aa 300–665 of chick PLZF; FGFR3, WT form of the human FGFR3 ; caFGFR3, myristoylated and constitutively activated (K650E) form of the human FGFR3 cytoplasmic domain (aa 399–806) ; STAT3-C, mouse STAT3 containing two activating mutations (A662C, N664C) obtained from Addgene; and dnSTAT3 was created by incorporating into the mouse STAT3 nonphosphorylatable Y705F mutant , obtained from Addgene, an additional H332Y mutation that disrupts DNA binding .

Techniques: Expressing, Knockdown, Plasmid Preparation, Staining

Journal: PLoS Biology

Article Title: PLZF Regulates Fibroblast Growth Factor Responsiveness and Maintenance of Neural Progenitors

doi: 10.1371/journal.pbio.1001676

Figure Lengend Snippet: (A–N) Electroporation of FGF8 expression plasmids elicits a heightened progenitor proliferation response and reduced neurogenesis in the PLZF + FGFR3 + PAX6 high central region of the spinal cord (yellow brackets) relative to the PLZF − FGFR3 − PAX6 low dorsal spinal cord (blue brackets). (O–U) Coexpression of FGF8 with PLZF further increases progenitor proliferation and decreases neuronal differentiation. (V–X) Charts indicate the change in the number of cells ± SEM expressing the indicated markers following transfection with FGF8 plasmids alone or in combination with PLZF, relative to the contralateral control sides of the spinal cord. All electroporations were performed at e3 (HH 17) and collected at e5 (HH 25). Counts were based on at least 10 images taken from ≥8 electroporated embryos. * p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001. (Y) Summary model depicting the regional differences between PLZF + FGFR3 high neural progenitors in the central spinal cord, which exhibit a heightened responsiveness to FGF stimulation, compared to PLZF − FGFR3 low progenitors in the ventral and dorsal spinal cord.

Article Snippet: Plasmid expression vectors were generated by cloning cDNAs of interest into a Gateway cloning-compatible variant of the vector pCIG , as follows: PLZF, full-length chick clone isolated by PCR from e4 chick cDNA; EnR-PLZF and VP16-PLZF were created by respectively fusing either the Drosophila Engrailed repressor domain or the herpes simplex VP16 transactivation domain to aa 300–665 of chick PLZF; FGFR3, WT form of the human FGFR3 ; caFGFR3, myristoylated and constitutively activated (K650E) form of the human FGFR3 cytoplasmic domain (aa 399–806) ; STAT3-C, mouse STAT3 containing two activating mutations (A662C, N664C) obtained from Addgene; and dnSTAT3 was created by incorporating into the mouse STAT3 nonphosphorylatable Y705F mutant , obtained from Addgene, an additional H332Y mutation that disrupts DNA binding .

Techniques: Electroporation, Expressing, Transfection, Control