Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Sep 15;23(18):3677-88.
doi: 10.1038/sj.emboj.7600371. Epub 2004 Sep 2.

Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity

John Peter McPherson  1 , Laura Tamblyn, Andrew Elia, Eva Migon, Amro Shehabeldin, Elzbieta Matysiak-Zablocki, Bénédicte Lemmers, Leonardo Salmena, Anne Hakem, Jason Fish, Farah Kassam, Jeremy Squire, Benoit G Bruneau, M Prakash Hande, Razqallah Hakem
Affiliations

Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity

John Peter McPherson et al. EMBO J. .

Abstract

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Northern analysis of Lats1 (A) and Lats2 (B) expression during embryonic development. Loading control for Northern blot shown with GADPH (C). Whole-mount in situ hybridization of Lats1 (D–G) and Lats2 (H–K) during embryonic developmental stages E8.5–10.5. Prominent Lats1 expression was detected in neuroepithelium (arrows, D, E), mesencephalon (arrows, F, G) and otic vesicles (lower arrow, F), whereas Lats2 expression was prominent in the lateral mesodermal plate (arrows, H, I) and the cardiac outflow tract (upper arrow, I). In situ hybridization (and corresponding counterstaining) of E10.5 embryonic histological sections with Lats1 (L–O) and Lats2 (P–S) showing prominent expression of Lats1 in neuroepithelium (L, M). Lats1 expression in the heart (N, O) was relatively lower compared to Lats2 expression (R, S). N, neuroepithelium; V, ventricle; A, atrium; B, first branchial arch; ot, outflow tract.
Figure 2
Figure 2
Lats2 targeted disruption and Lats2−/− embryo abnormalities. (A) Restriction maps depicting mouse Lats2 genomic fragment, targeting construct and predicted structure of targeted Lats2 allele. Exons are represented by filled rectangles. Restriction enzymes: H, HindIII; N, NotI. Digestion of genomic DNA with HindIII generates an 11.7-kb fragment from the wild-type allele and a 7-kb fragment from the mutant allele, both of which are detected using the 5′ flanking probe shown. (B) Southern analysis of genomic DNA extracted from E9.5 embryos obtained from heterozygote crosses. (C) Sequence of cDNAs corresponding to Lats2 mRNA derived from wild-type (upper) and mutant (lower) alleles. (D) Western analysis of protein extracts from wild-type and Lats2−/− MEFs. Polyclonal antibodies against Lats2 failed to detect full-length protein (upper panel) or a shorter Lats2 peptide (middle panel) in the mutant MEFs. Ku70 is shown as a loading control (lower panel). (E, F) Representative E10.5 embryos (photographed at the same magnification as at the time of dissection) depicting reduced size of Lats2−/− embryos. (G, H) Lats2−/− E11.5 embryos with distended pericardium with blood accumulation in the abdominal cavity.
Figure 3
Figure 3
Haematoxylin and eosin-stained sagittal sections of heart (A, atrium; V, ventricle) from wild-type (A, C) and mutant (B, D) embryos at E9.5 (A, B) and E10.5 (C, D) with atrial hyperplasia (arrows). (E, F) Ventricular hypoplasia in Lats2−/− embryos. Representative haematoxylin and eosin-stained transverse sections of ventricles from E10.5 wild-type (E) and Lats2−/− (F) embryos depicting trabeculation defect and thinner myocardium. Expression of cardiac actin at E9.5 (G–J) and Bmp4 at E10.5 (K–N) in wild-type (G, H, K, L) and Lats2−/− embryos (I, J, M, N) by in situ hybridization (A, atrium; V, ventricle; it, inflow tract; ot, outflow tract).
Figure 4
Figure 4
Stage-specific proliferation defect in Lats2−/− embryos. E8.5 transverse, E9.5 sagittal and E10.5 sagittal embryo sections stained for BrdU. All images for a given embryonic stage were captured at the same exposure settings and × 40 magnification, unless otherwise stated. (A, E) Left side of head fold. (B, F) Heart outflow tract. (C, G) Heart ventricle. (D, H) Neural tube. Top panels for E9.5 and E10.5 depict heart area at × 10 magnification (V, ventricle; A, atrium; ot, outflow tract; P, pericardium) with representative outflow tract (J, N, R, V) heart ventricle (K, O, S, W) and atrium (L, P, T, X) below.
Figure 5
Figure 5
Growth kinetics in Lats2−/− MEFs. (A) Growth curve representative of passage 2 MEFs derived from wild-type (▪) and two independently derived Lats2−/− MEF lines (▴ and •). (B) Representative saturation density of growth for passage 2 MEFs derived from wild-type (•) and two independently derived Lats2−/− MEF lines (▴ and ▪). (C) Plating efficiency at low seeding density (colony formation/3000 cells plated) for two wild-type MEF lines (grey bar, n=3) and two Lats2−/− MEF lines (black bar, n=3). (D) Representative determination of contact inhibition of growth. A total of 5 × 104 cells of either wild-type or Lats2−/− MEFs were plated in 6 cm dishes. Cells were maintained in culture without passage for 2 weeks and stained with crystal violet. (E, F) Morphology of wild-type MEFs (E) and foci observed in Lats2−/− MEFs (F).
Figure 6
Figure 6
Cytokinesis abnormalities, micronuclei, aneuploidy and centrosome amplification in Lats2−/− embryos and MEFs. (A, B) Lats2−/− MEFs stained with FITC-conjugated anti-α-tubulin to visualize tubulin cytoskeleton (A) with microtubule bundles (arrowhead) and counterstained with DAPI (B) to visualize DNA. (C, D) Lats2−/− MEFs stained with anti-α-actin (C) and counterstained with DAPI (D) to visualize midbody (arrowhead) and DNA, respectively. (E–G) Bridged chromatin within the midbody (E, arrowhead) of Lats2−/− MEFs could be detected following staining for phospho-histone H3 (F, arrowhead) (DAPI counterstain shown in G). (H) Graph depicting the percentage of cells exhibiting cytoplasmic bridges from three independently derived Lats2−/− MEF lines (black bar) compared to three independent wild-type or heterozygous lines of equivalent passage (grey bar). Representative photomicrographs of wild-type MEFs (I) with normal nuclear morphology (J) and a Lats2−/− MEF (K) containing micronuclei (L) are depicted. (M) Graph depicting the percentage of cells exhibiting micronuclei from three wild-type (left grey bar) and Lats2−/− (left black bar) sibling embryos and three independently derived Lats2−/− MEF lines (right black bar) compared to three independent wild-type or heterozygous lines of equivalent passage (right grey bar). MEFs (N–S) were immunostained with anti-γ-tubulin (red) and counterstained with DAPI (blue) to visualize centrosomes and DNA, respectively. Three wild-type MEFs are depicted (N, O) with single centrosomes. Lats2−/− MEFs containing three centrosomes (P, Q) and four centrosomes (R, S) are depicted. (T, U) Abnormal spindle pole formation and chromosomal segregation defects during mitosis in Lats2−/− fibroblasts. Fibroblasts were immunostained with anti-α-tubulin (green) to visualize mitotic spindles and counterstained with DAPI (blue) to visualize chromatin. (V) Graph depicting the percentage of cells exhibiting 1, 2, 3 or ⩾4 centrosomes from three wild-type (grey bar) and Lats2−/− (black bar) sibling embryos and three independently derived Lats2−/− MEF lines (black bar) compared to three independent wild-type or heterozygous lines of equivalent passage (grey bar). (W) Pooled results of karyotypic analyses of wild-type MEF lines (upper panel) compared to Lats2−/− MEF lines (lower panel) with frequencies of specific chromosomal aberrations observed.
Figure 7
Figure 7
Lats2 is a centrosomal protein that suppresses centrosome overduplication. (A–K) Subcellular localization of GFP-Lats2 at centrosomes by indirect immunofluorescence. Wild-type MEFs transfected with either GFP (A–C), GFP-Lats2 (D–K) or GFP-Lats2KD (L–N) expression constructs were analysed 24 h later for GFP fluorescence (A, D, H, L) and γ-tubulin (red) immunofluorescence (B, E, I, M), and DNA was stained with DAPI (C, F, J, N). Merged images G and K confirming colocalization of GFP-Lats2 and γ-tubulin are derived from (D–F) and (H–J), respectively. Transfected cells (GFP-positive) and untransfected cells in (D–N) are denoted as t and u respectively; arrows indicate centrosomes (B, E, I, M, P), GFP-Lats2 fluorescence (D, H), GFP-Lats2KD fluorescence (L) and merged signals (G, K). (O–Q) Centrosome overduplication in the presence of HU is suppressed by GFP-Lats2. Representative GFP vector-transfected cell (t) and untransfected (u) cell (O) demonstrating supernumerary centrosomes (arrows) as visualized by γ-tubulin immunostaining (P). DNA is visualized by DAPI stain (Q). (R) Graph depicting the percentage of GFP-positive cells exhibiting >2 centrosomes from wild-type MEFs transfected with GFP vector, GFP-Lats2 (Lats2) or GFP-LatsKD (KD) in the absence or presence of 2 mM HU. (S) Centrosome overduplication in Lats2-deficient MEFs is suppressed by GFP-Lats2. Graph depicting the percentage of GFP-positive cells exhibiting >2 centrosomes from Lats2−/− MEFs transfected with GFP (control), GFP-Lats2 (Lats2) or GFP-Lats2KD (KD).
See this image and copyright information in PMC

References

    1. Andreassen PR, Lohez OD, Lacroix FB, Margolis RL (2001) Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1. Mol Biol Cell 12: 1315–1328 - PMC - PubMed
    1. Balczon R, Bao L, Zimmer WE, Brown K, Zinkowski RP, Brinkley BR (1995) Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells. J Cell Biol 130: 105–115 - PMC - PubMed
    1. Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, Koder A, Evans RM (1999) PPARy is required for placental, cardiac, and adipose tissue development. Mol Cell 4: 585–595 - PubMed
    1. Borel F, Lohez OD, Lacroix FB, Margolis RL (2002) Multiple centrosomes arise from tetraploidy checkpoint failure and mitotic centrosome clusters in p53 and RB pocket protein-compromised cells. Proc Natl Acad Sci USA 99: 9819–9824 - PMC - PubMed
    1. Brinkley BR (2001) Managing the centrosome numbers game: from chaos to stability in cancer cell division. Trends Cell Biol 11: 18–21 - PubMed

Publication types

MeSH terms

Substances