Intracellular FGF14 (iFGF14) is required for spontaneous and evoked firing in cerebellar Purkinje neurons and for motor coordination and balance

Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia (SCA27). In addition, mice lacking Fgf14 (Fgf14(−/−)) exhibit an ataxia phenotype resembling SCA27, accompanied by marked changes in the excitability of cerebellar granule and Purkinje neurons. It is not known, however, whether these phenotypes result from defects in neuronal development or if they reflect a physiological requirement for iFGF14 in the adult cerebellum. Here, we demonstrate that the acute and selective Fgf14-targeted short hairpin RNA (shRNA)-mediated in vivo “knock-down” of iFGF14 in adult Purkinje neurons attenuates spontaneous and evoked action potential firing without measurably affecting the expression or localization of voltage-gated Na(+) (Nav) channels at Purkinje neuron axon initial segments. The selective shRNA-mediated in vivo “knock-down” of iFGF14 in adult Purkinje neurons also impairs motor coordination and balance. Repetitive firing can be restored in Fgf14-targeted shRNA-expressing Purkinje neurons, as well as in Fgf14(−/−) Purkinje neurons, by prior membrane hyperpolarization, suggesting that the iFGF14-mediated regulation of the excitability of mature Purkinje neurons depends on membrane potential. Further experiments revealed that the loss of iFGF14 results in a marked hyperpolarizing shift in the voltage dependence of steady-state inactivation of the Nav currents in adult Purkinje neurons. We also show here that expressing iFGF14 selectively in adult Fgf14(−/−) Purkinje neurons rescues spontaneous firing and improves motor performance. Together, these results demonstrate that iFGF14 is required for spontaneous and evoked action potential firing in adult Purkinje neurons, thereby controlling the output of these cells and the regulation of motor coordination and balance

FGF14 Is Functionally Significant in Adult Mice

Mentor: David Ornitz From the Washington University Undergraduate Research Digest: WUURD, Volume 9, Issue 1, Fall 2013. Published by the Office of Undergraduate Research. Joy Zalis Kiefer Director of Undergraduate Research and Assistant Dean in the College of Arts & Sciences

Fibroblast Growth Factor 9 Regulation by MicroRNAs Controls Lung Development and Links DICER1 Loss to the Pathogenesis of Pleuropulmonary Blastoma

Pleuropulmonary Blastoma (PPB) is the primary neoplastic manifestation of a pediatric cancer predisposition syndrome that is associated with several diseases including cystic nephroma, Wilms tumor, neuroblastoma, rhabdomyosarcoma, medulloblastoma, and ovarian Sertoli-Leydig cell tumor. The primary pathology of PPB, epithelial cysts with stromal hyperplasia and risk for progression to a complex primitive sarcoma, is associated with familial heterozygosity and lesion-associated epithelial loss-of-heterozygosity of DICER1. It has been hypothesized that loss of heterozygosity of DICER1 in lung epithelium is a non-cell autonomous etiology of PPB and a critical pathway that regulates lung development; however, there are no known direct targets of epithelial microRNAs (miRNAs) in the lung. Fibroblast Growth Factor 9 (FGF9) is expressed in the mesothelium and epithelium during lung development and primarily functions to regulate lung mesenchyme; however, there are no known mechanisms that regulate FGF9 expression during lung development. Using mouse genetics and molecular phenotyping of human PPB tissue, we show that FGF9 is overexpressed in lung epithelium in the initial multicystic stage of Type I PPB and that in mice lacking epithelial Dicer1, or induced to overexpress epithelial Fgf9, increased Fgf9 expression results in pulmonary mesenchymal hyperplasia and a multicystic architecture that is histologically and molecularly indistinguishable from Type I PPB. We further show that miR-140 is expressed in lung epithelium, regulates epithelial Fgf9 expression, and regulates pseudoglandular stages of lung development. These studies identify an essential miRNA-FGF9 pathway for lung development and a non-cell autonomous signaling mechanism that contributes to the mesenchymal hyperplasia that is characteristic of Type I PPB

Dicer1 regulation of lung epithelial development requires <i>Fgf9</i>.

<p>(A-D) Comparison of E12.5 whole mount lung morphology of <i>Control</i> (A), <i>Sftpc-rtTA</i>, <i>Tre-Fgf9-Ires-eGfp</i> lungs induced with doxycycline from E10.5-E12.5 (B), <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> (C) and <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i>, <i>Fgf9</i>^<i>f/+</i> (D). (E-H) H&E stained histological sections of the lungs shown above in panels A-D. (I-L) Representative immunostaining for phospho-histone H3 (pHH3) of the lungs shown in panels A-D. (M and N) Quantification of epithelial (M) and mesenchymal (N) cell proliferation of pHH3 labeled lung tissue in <i>Control</i>; <i>Sftpc-rtTA</i>, <i>Tre-Fgf9-Ires-eGfp</i> lungs induced with doxycycline from E10.5-E12.5; <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lungs; and <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i>, <i>Fgf9</i>^<i>f/+</i> lungs. For each group, at least 3 individual samples were included, 3 different slides were chosen from each sample, and for each section, three 10x fields were counted for the number of positive cells per 100 cells. (O and P) Whole mount <i>in situ</i> hybridization showing increased expression of <i>Fgf9</i> in E12.5 <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lung epithelium (P) compared to control lung (O). (Q) Quantitative RT-PCR showing increased expression of <i>Fgf9</i> in E12.5 <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lung (n = 3) epithelium compared to control lung (n = 3). *<i>P</i><0.05; **<i>P</i><0.01; ns, not significant. Scale bars: A, 200 μm; E, 100 μm; I, 50 μm; O, 200 μm. Sample numbers (n) are indicated in data bars.</p

FGF9 overexpression in Type I PPB phenocopies ectopically expressed FGF9 in mouse lung epithelium.

<p>(A, B, K, and L) Immunostaining for FGF9 showing increased expression in Type I PPB-associated lung epithelium and in doxycycline-induced <i>Sftpc-rtTA</i>, <i>Tre-Fgf9-Ires-Gfp</i> mouse lung. Non-diseased human lung and uninduced (no Dox) mouse lung were used as controls. (C, D, M, and N) Mesenchymal and epithelial proliferation in Type I PPB and induced mouse lung identified by immunostaining for Ki67. Inserts show higher magnification of the boxed regions. (E, F, O, and P) Immunostaining showing increased phosphorylated Erk1/2 (p-ERK) in Type I PPB and in induced mouse lung mesenchyme and reduced p-ERK in epithelium. Inserts show higher magnification of the boxed regions. (G, H, Q and R) Quantification of Ki67 immunostaining in C, D, M, and N above, showing increased in proliferation in both epithelial and mesenchymal tissues of Type I PPB (G and H) and <i>Fgf9</i>-induced mouse lung (Q and R). (I, J, S, and T) Quantification of p-ERK immunostaining in E, F, O and P above, showing decreased epithelial p-ERK (I and S) and increased mesenchymal p-ERK (J and T), in Type I PPB and <i>Fgf9</i>-induced mouse lung compared to control tissue. Error bars represent SD. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. (A and B) Five month-old male; (C and D) Three month-old female; (E) Six month-old female; (F) 34 month-old female. Scale bars: A and B, 20 μm; C-P, 50 μm. Sample numbers (n) are indicated on the data bars.</p

Type I PPB and induced late gestation expression of epithelial FGF9 in mice have similar histopathology and cell differentiation.

<p>(A-F) Comparison of non-diseased human lung (left) and Type I PPB (right). (G-L) Comparison of normal (no Dox) E18.5 mouse lung (left) and mouse lung from <i>Sftpc-rtTA</i>, <i>Tre-Fgf9-Ires-eGfp</i> double transgenic mice induced (+Dox) to overexpress <i>Fgf9</i> from E16.5 to E18.5 (right). (A and G) H&E stained histological sections. (B and H) Immunostaining for Nkx2.1 to identify lung epithelium. (C and I) Immunostaining for smooth muscle actin (SMA). Peri-bronchiolar SMA immunostaining (white arrow) and perivascular SMA immunoreactivity (black arrowhead) are differentially affected. (D and J) Immunostaining for Club cell secretory protein (CC10) showing reduced expression of CC10 in proximal lung epithelium (white arrow) compared to that of non-diseased human lung and uninduced mouse lung, respectively. (E and K) Immunostaining for surfactant protein C (Sftpc) showing expanded proximal expression (white arrow) in all cystic lung epithelium in human Type I PPB and <i>Fgf9</i>-induced mouse lung. In non-diseased human lung and uninduced mouse lung, Sftpc immunostaining (white arrow) was consistent with expression in Type II pneumocytes. (F and L) Immunostaining for the distal lung Type I pneumocyte marker Aquaporin 5 (Aq5, human) and T1α (mouse). In human, Aq5 was expressed in distal alveoli of non-diseased lung tissue and in Type I PPB associated epithelium. T1α was similarly expressed in distal mouse lung and throughout the epithelium of <i>Fgf9</i>-induced mouse lung (white arrow). (A) Three month-old female. (B-F) Three month-old female. Scale bar: A, 20 μm, B-L, 50 μm.</p

Epithelial Dicer1 regulation of mesenchymal Wnt/β-Catenin signaling and epithelial differentiation requires <i>Fgf9</i>.

<p>(A-C) Expression of <i>Wnt2a</i> in E12.5 lung showing increased expression in <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> distal lung mesenchyme (B) compared to the Control (A). Inactivation of one allele of <i>Fgf9</i> in <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lung epithelium (C) reduces <i>Wnt2a</i> expression to levels observed in control lungs. (D-F) The downstream target of Wnt signaling, <i>Lef1</i>, was increased in <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lung mesenchyme (E) compared to the Control (D). Inactivation of one allele of <i>Fgf9</i> in <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lung epithelium (F) reduces <i>Lef1</i> expression to levels observed in control lungs. (G-I) Expression of <i>Sftpc</i> in E12.5 lung showing decreased expression in <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> distal lung mesenchyme (H) compared to the Control (G). Inactivation of one allele of <i>Fgf9</i> in <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> lung epithelium (I) results in increased <i>Sftpc</i> expression. (J) Quantitative PCR analysis of E12.5 Control lung and <i>Shh</i>^<i>Cre/+</i>, <i>Dicer1</i>^<i>f/f</i> rescued with one or two Fgf9 floxed alleles showing increased Sftpc expression. * <i>P</i><0.04. Images shown are representative of three embryos for each genotype. Scale bars: 200 μm.</p

MiR-140 and miR-328 regulate <i>in vitro</i> lung development and <i>Fgf9</i> expression.

<p>(A-B) Validation of tiny LNA antagomers ability to block the activity of miR-140 and miR-328. Repression of the <i>Fgf9</i> 3’ UTR by 10 nM miR-140 (A) or 10 nM miR-328 (B) transfected into HEK293 cells with a luciferase reporter construct containing a wild type mouse <i>Fgf9</i> 3’ UTR was blocked by adding 10 nM of the corresponding tiny LNAs to the culture medium. The control LNA (LNA-con) contains a single mismatch in the LNA-140 sequence. (C-F) E12.5 lung explants were cultured in the presence of 100 nM tiny LNA oligonucleotides (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005242#pgen.1005242.s002" target="_blank">S2 Table</a>) for 48 hr. (C) Control LNA (100 nM), (D) LNA-328, E) LNA-140, and (F) 50 nM of LNA-140 and LNA-328 (total concentration 100 nM). Red lines indicate mesenchymal thickness. (G and H) Quantification of mesenchymal thickness (G) and the epithelial bud number (H) of lung explants in response to treatment with tiny LNA antagomers (<i>n</i> = 4–5 explants per condition). (I and J) Whole mount <i>in situ</i> hybridization showing expression of <i>Fgf9</i> in E12.5 wild type lung explants cultured in the presence of 100 nM control LNA (I) or LNA-140 (J). Images shown are representative of at least three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001. Scale bars: 200 μm.</p