The nuclear scaffold protein NIPP1 is essential for early embryonic development and cell proliferation

Abstract

NIPP1 (nuclear inhibitor of protein phosphatase 1) is a ubiquitously expressed nuclear scaffold protein that has been implicated in both transcription and RNA processing. Among its protein ligands are a protein kinase, a protein phosphatase, two splicing factors, and a transcriptional regulator, and the binding of these proteins to NIPP1 is tightly regulated by phosphorylation. To study the function of NIPP1 in vivo, we have used homologous recombination to generate mice that are deficient in NIPP1. NIPP1(-/+) mice developed normally. However, NIPP1(-/-) embryos showed severely retarded growth at embryonic day 6.5 (E6.5) and were resorbed by E8.5. This early embryonic lethality was not associated with increased apoptosis but correlated with impaired cell proliferation. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of NIPP1 in cultured cells also revealed an essential role for NIPP1 in cell proliferation. In further agreement with this function, no viable NIPP1(-/-) cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of NIPP1(-/+) intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of Geneticin. We conclude that NIPP1 is indispensable for early embryonic development and cell proliferation.

FIG. 1.

Targeted disruption of the NIPP1 gene. (A) Partial restriction maps of the Ppp1r8 locus (mouse NIPP1 gene), the targeting vector, and the targeted locus. Two 12.5-kb phage genomic clones, isolated from the 129SvJ mouse lambda genomic library, are shown on top. In the wild-type (wt) locus, exons 1 to 5 (E1 to E5) are shown as solid boxes and “ATG” represents the first coding exon. Restriction sites: B, BamHI (not all indicated); E, EcoRI (all indicated); P, PstI (not all indicated). Dashed lines between the wt locus and the targeting vector delineate the regions that are common to the genomic locus and the replacement vector. The neomycin resistance cassette (Neo^r) in the replacement vector is flanked by loxP (not indicated), and a thymidine kinase cassette (TK) lies downstream of flank 2 in the replacement vector. The targeted locus represents the predicted structure of the NIPP1 locus after homologous recombination with the replacement vector. A 6.6-kb fragment comprising part of the promoter region, as well as exons 1 to 2, has been replaced by the Neo cassette. (B) Southern blot genotyping of neonates derived from intercrosses of NIPP1 heterozygotes. Genomic DNA was digested with EcoRI and hybridized with probe A, yielding a 9-kb band for the wt allele and a 5.8-kb band for the mutant allele. (C) PCR genotyping of E7.5 embryos obtained from crosses of NIPP1 heterozygotes. A wt-specific nested PCR, as explained in Materials and Methods, amplifies a band of 402 bp, and a mutant-specific PCR results in a band of 696 bp. M, marker; C, control. (D) RT-PCR genotyping of blastocysts (E3.5) isolated from NIPP1 heterozygous intercrosses. The presence of the wt NIPP1 allele results in a 350-bp fragment, while the mutated NIPP1 allele leads to amplification of a 696-bp product. (E) Detection of NIPP1 by immunostaining in E6.5 embryos from intercrosses of NIPP1 heterozygotes. Serial transverse sections of paraffin-embedded embryos were made from E6.5 deciduae. Sections were stained with anti-NIPP1 antibodies (a and c) or anti-HDAC2 antibodies (b and d). The figure shows an embryo that expresses NIPP1 (a and b) and an embryo that lacks NIPP1 (c and d). The control condition in panels c and d did not contain DNA template. Bar in panel Ea, 50 μm.

FIG. 2.

Histological analysis of E6.5 NIPP1^wt and NIPP1^−/− littermates. The figure shows hematoxylin-and-eosin-stained sections of paraffin-embedded NIPP1^wt (A to C) and NIPP1^−/− (D to F) embryos at E6.5. NIPP1^wt refers to either the NIPP1^+/+ or the NIPP1^−/+ genotype. Transverse sections at the extraembryonic (B and E) and embryonic (C and F) poles as well as sagittal sections (A and D) are shown. Arrowheads in panel A indicate the junction between the embryonic and extraembryonic regions. NIPP1^−/− embryos are about half the size of their wild-type littermates. No ectoplacental cone can be distinguished. Ec, ectoplacental cone; ExEc, extraembryonic ectoderm, ExVE, extraembryonic visceral endoderm; EmEc, embryonic ectoderm; EmVE, embryonic visceral endoderm; R, Reichert membrane; PrC, proamniotic cavity. Bar in panel A, 50 μm.

FIG. 3.

Cell lineages in NIPP1^wt and NIPP1^−/− embryos. Transverse (A) and sagittal (B) sections of paraffin-embedded NIPP1^wt and NIPP1^−/− embryos (E6.5) were immunostained for NIPP1, Gata4, Cdx2, and Oct4 as indicated. Gata4 stains specifically the visceral and parietal endoderm, Cdx2 is a marker of the extraembryonic ectoderm, and Oct4 is specifically expressed in the embryonic ectoderm. All three cell lineages can be distinguished in the NIPP1^−/− embryos, but the extraembryonic and the embryonic ectoderms are not organized into a clear epithelial layer. Abbreviations are as explained in the legend to Fig. 2. VE, visceral endoderm. Bars in panels Aa and Ba, 50 μm.

FIG. 4.

Apoptosis and proliferation rates in NIPP1^wt and NIPP1^−/− embryos. (A) Paraffin-embedded transverse sections of NIPP1^wt (a and b) and NIPP1^−/− (c and d) E6.5 embryos were stained with hematoxylin and eosin (a and c) and by the TUNEL assay (b and d) for the visualization of apoptosis. No difference in apoptosis between wild-type and knockout embryos was detected. Bar in panel Aa, 50 μm. (B) Pregnant mice were injected with BrdU 1 h before sacrifice. Sections of NIPP1^wt and NIPP1^−/− embryos (E6.5) were analyzed for BrdU incorporation by immunostaining with anti-BrdU antibodies. Stained nuclei of five NIPP1^wt and six NIPP1^−/− embryos were counted and expressed as a percentage of the total number of nuclei in the section. Nearly 20% lower BrdU incorporation was found in the mutant embryos, indicating a decreased rate of cell proliferation. Results are means ± standard errors (P = 0.0074 by an unpaired t test).

FIG. 5.

Retarded outgrowth of NIPP1-deficient blastocysts. Blastocysts from intercrosses of heterozygotes were isolated, cultured, and genotyped as described in Materials and Methods. (A) Photographs of outgrowths of NIPP1^wt (a to c) and NIPP1^−/− (d to f) blastocysts following their culture in vitro for the indicated times. The inner cell mass (ICM) is surrounded by trophoblast giant cells (TG) after 3 to 5 days. After 3 days, the NIPP1^−/− blastocysts had not yet formed an outgrowth. ZP, zona pellucida. (B) Following culture for 4 days, NIPP1^wt and NIPP1^−/− blastocyst outgrowths were incubated for 16 h with BrdU. BrdU incorporation was visualized by BrdU immunofluorescence (b and e). The figure also shows staining of blastocyst outgrowths for NIPP1 by immunofluorescence (a and d) and for DNA by Hoechst staining (e and f). NIPP1^−/− blastocysts showed delayed outgrowth and severely reduced incorporation of BrdU. Ab, antibody. Bars, 20 μm.

FIG. 6.

Effects of the RNAi-mediated knockdown of NIPP1 on the proliferation of mammalian cells. (A) At 72 h after transfection of HEK293 cells with NIPP1-specific or lamin A/C-specific siRNAs or mock transfection (without siRNAs), the cells were lysed and processed for immunoblotting with anti-NIPP1 antibodies (upper panel) and with antibodies against SIPP1, a structurally unrelated nuclear scaffold protein (lower panel). (B) Levels of NIPP1 protein in mock-transfected cells (a) and in cells transfected with NIPP1-specific siRNAs (b), as determined by immunofluorescence analysis. Bar, 10 μm. (C) Number and morphology of HEK293 cells 72 h after mock transfection (a) or after transfection with NIPP1-specific siRNAs (b). The RNAi-induced knockdown of NIPP1 results in a lower number of cells, indicating slower growth. Bar, 30 μm.