Research ArticleDevelopmental Biology

Gain-of-function mutations in the gene encoding the tyrosine phosphatase SHP2 induce hydrocephalus in a catalytically dependent manner

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Science Signaling  20 Mar 2018:
Vol. 11, Issue 522, eaao1591
DOI: 10.1126/scisignal.aao1591
  • Fig. 1 Neural cell–specific expression of Ptpn11E76K induces hydrocephalus and brain developmental defects.

    (A) Brain tissues dissected from 1-month-old mice (n = 3 mice per genotype) were lysed, and SH2 domain–containing phosphatase 2 (SHP2) catalytic activities in the lysates were assessed by the immunocomplex phosphatase assay. SHP2 abundance in the lysates was examined by immunoblotting. (B) Kaplan-Meier survival curves of mice of the indicated genotypes. (C) Representative images of 1-month-old mice and their brains. (D) Sagittal brain sections were processed for hematoxylin and eosin (H&E) staining. Analyses in all panels were performed in three independent experiments, n = 4 mice per genotype, and representative images are shown. Data are means ± SD of biological replicates. Cx, cortex; Hp, hippocampus; LV, lateral ventricles. Scale bars, 2 cm (C, mice), 5 mm (C, brain), and 500 μm (D).

  • Fig. 2 Ptpn11E76K decreases self-renewal in NSPCs.

    (A and B) Brain sections prepared from 1-month-old mice (n = 4 mice per genotype) were processed for immunofluorescence staining of neuronal nuclei (NeuN) (neurons) and glial fibrillary acidic protein (GFAP) (astrocytes) in the cortex (A) and hippocampus (B). DAPI, 4′,6-diamidino-2-phenylindole. (C) Brain sections (n = 3 mice per genotype) were processed for immunohistochemistry staining for the migrating neuroblast marker specificity protein 8 (Sp8). SVZ, subventricular zone. (D to F) Cerebral cortices dissected from embryonic day 14.5 (E14.5) embryos (n = 4 mice per genotype) were assessed by neurosphere assays. (D) Quantification of the size distribution of neurospheres formed by tissue from the indicated genotypes in culture. (E) Total cell numbers at the indicated time points after dissociation of neurospheres into single cells and culture in medium containing basic fibroblast growth factor (bFGF). (F) Cell cycle profiles of the dissociated neurosphere cells as determined by Ki67 and Hoechst 33342 staining followed by fluorescence-activated cell sorting analyses. Analyses in all panels were performed in three to four independent experiments. Data are means ± SD of biological replicates. Representative images are shown. Scale bars, 200 μm (A and B, top, and D) and 100 μm (A and B, bottom, and C).

  • Fig. 3 Aberrant development of ependymal cells and cilia in Ptpn11E76K mice.

    (A) Brains dissected from 1-month-old mice (n = 3 mice per genotype) were processed for scanning electron microscopic analyses. Ependymal cilia on the lateral ventricular walls were examined. Scale bars, 5 μm (top) and 2 μm (bottom). (B and C) Brain sections prepared from postnatal day 5 (P5) pups (n = 3 mice per genotype) were processed for immunofluorescence staining for the ependymal cell marker FoxJ1 (B) and the cilium marker acetyl α-tubulin (C). The images show the ependymal cells and ependymal cilia on the walls of the lateral ventricles in brain sections. FoxJ1+ cells were quantified. Scale bars, 50 μm (B) and 20 µm (C). (D) Lateral ventricular walls dissected from newborn pups (n = 3 mice per genotype) were processed for ependymal cell differentiation assays. Differentiated cells were immunostained for acetyl α-tubulin. Scale bar, 50 μm. Analyses in all panels were performed in three independent experiments. Data are means ± SD of biological replicates. Representative images are shown.

  • Fig. 4 Ptpn11E76K decreases STAT3 activity and enhances ERK and AKT activity.

    (A) Immunohistochemical staining for phosphorylated extracellular signal–regulated kinase (p-ERK) in the cortex and hippocampus of brain sections prepared from 1-month-old Ptpn11+/+/Nestin-Cre+ and Ptpn11E76K/+/Nestin-Cre+ mice (n = 3 mice per genotype). Scale bars, 100 μm (top) and 200 μm (bottom). (B to D) Immunoblot showing p-ERK and p-AKT (B and C) or signal transducer and activator of transcription 3 (STAT3) phosphorylated at Tyr705 (p-STAT3; D) in whole-cell lysates of neurospheres that were generated from Ptpn11+/+/Nestin-Cre+ and Ptpn11E76K/+/Nestin-Cre+ mice (n = 3 mice per genotype), dissociated into single cells, and stimulated with bFGF or ciliary neurotrophic factor (CNTF) as indicated. Densitometric data of phosphoproteins (normalized to the total abundance of the respective proteins) are summarized in fig. S6 (B to D). (E) Immunofluorescence staining for FoxJ1, acetyl α-tubulin, and p-STAT3 in brain sections prepared from 1-month-old Ptpn11+/+/Nestin-Cre+ and Ptpn11E76K/+/Nestin-Cre+ mice (n = 3 mice per genotype). Scale bar, 50 μm. Double-positive FoxJ1+p-STAT3+ cells were quantified. Analyses in all panels were performed in three independent experiments. Data are means ± SD of biological replicates. Representative images are shown.

  • Fig. 5 Depletion of STAT3 in neural cells results in developmental defects in ependymal cells and cilia.

    Immunohistochemical and immunofluroescence staining showing (A) STAT3 and (B) acetyl α-tubulin and FoxJ1 in ependymal cells on the walls of the lateral ventricles in brain sections of newborn Stat3+/+/Nestin-Cre+ and Stat3fl/fl/Nestin-Cre+ pups (n = 3 mice per genotype). FoxJ1+ cells were quantified (B). Scale bars, 500 μm (A, top), 50 μm (A, bottom, and B, bottom), and 20 μm (B, top). (C) Acetyl α-tubulin in cells isolated from lateral ventricular walls dissected from newborn Stat3+/+/Nestin-Cre+ and Stat3fl/fl/Nestin-Cre+ pups (n = 3 mice per genotype). Scale bar, 50 μm. (D) Quantification of the number of primary neurospheres (1° NS) and secondary neurospheres (2° NS) derived from cerebral cortices dissected from E14.5 embryos (n = 3 mice per genotype) of the indicated genotypes. (E) Quantification of the proliferation of cells in primary neurospheres derived from the indicated genotypes. (F) Immunofluorescence staining of brain sections from newborn Stat3+/+/Nestin-Cre+ and Stat3fl/fl/Nestin-Cre+ pups (n = 3 mice per genotype) showing the neuron marker NeuN and the astrocyte marker GFAP. Scale bars, 100 μm. Analyses in all panels were performed in three independent experiments. Data are means ± SD of biological replicates. Representative images are shown.

  • Fig. 6 Inhibition of SHP2 but not ERK or AKT rescues ependymal cell differentiation of Ptpn11E76K/+ NSPCs.

    (A to D) Lateral ventricular walls dissected from newborn pups (n = 3 mice per genotype) were processed for ependymal cell differentiation assays in the presence of the mitogen-activated protein kinase kinase 1 inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, the SHP2 inhibitor #220-324, or vehicle. Inhibitor-treated cells were immunostained for acetyl α-tubulin to mark cilia (A and C) and examined by immunoblotting for p-AKT, p-ERK, and p-STAT3 (B and D). Analyses in all panels were performed in three independent experiments. Representative images are shown. Scale bars, 50 μm.

  • Fig. 7 Pathological effects of Ptpn11E76K/+ on NSPCs and brain development depend on SHP2 catalytic activity.

    (A and B) Cerebral cortices dissected from E14.5 embryos (n = 4 mice per genotype) were assessed by neurosphere assays. Quantification of total numbers of primary (1° NS), secondary (2° NS), and tertiary (3° NS) neurospheres (A) and proliferation of cells in primary neurospheres (B). (C) Lateral ventricular walls dissected from newborn pups (n = 4 mice per genotype) were processed for ependymal cell differentiation assays and stained with acetyl α-tubulin to mark cilia. Scale bar, 50 μm. (D) Scanning electron microscopy showing ependymal cells in brains dissected from 1- to 2-month-old mice (n = 3 mice per genotype) of the indicated genotypes. Scale bar, 10 μm. (E) Immunoblot showing p-AKT, p-ERK, and p-STAT3 in neurosphere cells generated from mice of the indicated genotypes (n = 3 mice per genotype) and treated with CNTF. Densitometric data of phosphoproteins (normalized to the respective total proteins) are summarized in fig. S8D. Assays in all panels were performed in three independent experiments. Data are means ± SD of biological replicates. Representative images are shown.

  • Table 1 Ependymal cilia defects are proportionate to the catalytic activities of various mutant forms of SHP2.

    Brain sections prepared from 12-month-old mice (n = 3 mice per genotype) were immunofluorescently stained for acetyl α-tubulin to mark cilia, and the ependymal cilia on the walls of ventricles were examined. NS, Noonan syndrome; NSML, NS with multiple lentigines.

    MiceCatalytic activity of
    mutant SHP2
    Ependymal cilia
    Ptpn11E76K/+/
    Nestin-Cre+
    Substantially
    enhanced
    Severely abnormal
    Ptpn11D61G/+
    (NS mice)
    EnhancedAbnormal in some
    areas*
    Ptpn11Y279C/+
    (NSML mice)
    No catalytic activityNormal

    *Ependymal cilia of the third ventricle but not the lateral ventricles were abnormal.

    • Table 2 The increased catalytic activity of SHP2E76K is required for its detrimental effect on ependymal cell development.

      Ptpn11E76K,C459S/+ double-mutation knockin and Ptpn11E76K/+ single-mutation knockin mice were monitored for 15 months. The incidences of frank hydrocephalus in the euthanized animals were documented.

      Mice*Incidence of frank
      hydrocephalus
      Ptpn11E76K/+/Nestin-Cre+26/56
      Ptpn11+/+/Nestin-Cre+0/58
      Ptpn11E76K,C459S/+0/56
      Ptpn11+/+0/44

      *Mice were monitored for 15 months.

      Supplementary Materials

      • www.sciencesignaling.org/cgi/content/full/11/522/eaao1591/DC1

        Fig. S1. The Ptpn11E76K mutation induces brain developmental defects with aberrant behaviors.

        Fig. S2. Neurons are reduced, and astrocytes are increased in the cerebral cortex and hippocampus of adult Ptpn11E76K/+/Nestin-Cre+ mice.

        Fig. S3. Total number and survival of NSPCs in the developing brain of Ptpn11E76K/+/Nestin-Cre+ mice are not significantly changed.

        Fig. S4. Neuron and ependymal cell differentiation is decreased, whereas astrocyte differentiation is increased in Ptpn11E76K/+/Nestin-Cre+ NSPCs.

        Fig. S5. ERK activity in the developing brain of Ptpn11E76K/+/Nestin-Cre+ mice is enhanced, but cell proliferation does not change.

        Fig. S6. Similar abundance of bFGF and CNTF receptors in Ptpn11E76K/+/Nestin-Cre+ and Ptpn11+/+/Nestin-Cre+ NSPCs.

        Fig. S7. Defects of ependymal cilia are proportionate to the catalytic activity of various mutant forms of SHP2.

        Fig. S8. Generation and characterization of Ptpn11E76K,C459S/+ mice.

      • Supplementary Materials for:

        Gain-of-function mutations in the gene encoding the tyrosine phosphatase SHP2 induce hydrocephalus in a catalytically dependent manner

        Hong Zheng, Wen-Mei Yu, Ronald R. Waclaw, Maria I. Kontaridis, Benjamin G. Neel, Cheng-Kui Qu*

        *Corresponding author. Email: cheng-kui.qu{at}emory.edu

        This PDF file includes:

        • Fig. S1. The Ptpn11E76K mutation induces brain developmental defects with aberrant behaviors.
        • Fig. S2. Neurons are reduced, and astrocytes are increased in the cerebral cortex and hippocampus of adult Ptpn11E76K/+/Nestin-Cre+ mice.
        • Fig. S3. Total number and survival of NSPCs in the developing brain of Ptpn11E76K/+/Nestin-Cre+ mice are not significantly changed.
        • Fig. S4. Neuron and ependymal cell differentiation is decreased, whereas astrocyte differentiation is increased in Ptpn11E76K/+/Nestin-Cre+ NSPCs.
        • Fig. S5. ERK activity in the developing brain of Ptpn11E76K/+/Nestin-Cre+ mice is enhanced, but cell proliferation does not change.
        • Fig. S6. Similar abundance of bFGF and CNTF receptors in Ptpn11E76K/+/Nestin-Cre+ and Ptpn11+/+/Nestin-Cre+ NSPCs.
        • Fig. S7. Defects of ependymal cilia are proportionate to the catalytic activity of various mutant forms of SHP2.
        • Fig. S8. Generation and characterization of Ptpn11E76K,C459S/+ mice.

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        © 2018 American Association for the Advancement of Science

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