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First published online 3 September 2003
doi: 10.1242/dev.00715

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Impaired differentiation and lactational failure of Erbb4-deficient mammary glands identify ERBB4 as an obligate mediator of STAT5

Weiwen Long1, Kay-Uwe Wagner2, K. C. Kent Lloyd3, Nadine Binart4, Jonathan M. Shillingford5, Lothar Hennighausen5 and Frank E. Jones1,*

1 Department of Structural and Cellular Biology, Tulane Cancer Center, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112-2699, USA
2 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA
3 Center for Comparative Medicine, School of Veterinary Medicine, University of California, Davis, California 95616, USA
4 Faculté de Médecine Necker, Paris 75730, France
5 Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA



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  		Fig. 1. Conditional deletion of Erbb4 in the mammary gland. (A) Schematic representation of the wild-type ERBB4 locus, targeting vector, recombinant allele and CRE-deleted allele. The targeting vector replaces the coding region of exon 2 (black box) with a loxP-flanked exon 2. A frt-flanked PGK neomycin-resistance (neo) cassette (gray box) was included for positive selection, and a PGK thymidine kinase (tk) cassette (white box) was included for negative selection. The PGK-neo cassette was not removed in the CRE-deleted allele. An external genomic probe and an internal neo probe were used for screening embryonic stem cells. The position of PCR primers (1 and 2) are indicated by arrowheads. (B) Southern-based analysis of genomic DNA from embryonic stem cells. The 9 kb and 7 kb BamH1-digested fragments correspond to the targeted and wild-type allele, respectively. (C) PCR analysis of DNA extracted from tails of mice derived from matings between mice heterozygous for the wild-type (wt) allele and the recombinant (floxed) allele, showing the 350 bp wild-type band and the 400 bp floxed band. (D) Southern blot analysis showing CRE-mediated excision of Erbb4 exon 2 in mammary glands from 8-week-old nulliparous (N8wk) mice and two L12 Erbb4Flox/FloxWap-Cre mice. The 9 kb and 6 kb BamH1-digested fragments were detected using an exon 2 probe and the internal control exon 10 probe, respectively.

 


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  		Fig. 2. Immunohistochemical localization of ERBB4 expression. Paraffin wax-embedded number 4 inguinal mammary glands from Erbb4+/+Wap-Cre (A,C,E) and Erbb4Flox/FloxWap-Cre (B,D,F) mice, at L1 (A,B), L14 (C,D) and P18 of the second pregnancy (E,F), were stained for ERBB4 expression by IHC. Nuclear ERBB4 protein can be detected in Erbb4+/+Wap-Cre mammary glands and Erbb4Flox/FloxWap-Cre mammary glands at L1. Membrane staining of ERBB4 at L14 is indicated by the arrowhead in C and stromal expression is indicated by the arrow in D. Scale bar: 50 µm.

 


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  		Fig. 7. STAT5 activation is abolished in Erbb4Flox/FloxWap-Cre mammary glands. Immunohistochemical analysis of biparous control Erbb4+/+Wap-Cre (A,C,E,G) and Erbb4Flox/FloxWap-Cre (B,D,F,H) paraffin wax-embedded mammary glands, at P13.5 (A,B), P17.5 (C,D) and L1 (E,F), for STAT5 activation using an affinity-purified antibody directed against STAT5 phosphorylated at the regulatory amino acid Y694 (A-F; P-STAT5). A STAT5 antibody was used in immunohistochemistry of paraffin wax-embedded mammary glands to identify both phosphorylated and inactive STAT5 populations (G,H; STAT5). The inset in B is a higher magnification view of positive P-STAT staining. Arrowheads and arrows indicate positive nuclear and cytoplasmic staining, respectively. Scale bar: 50 µm. (I) Expression of inactive STAT5 in Erbb4Flox/FloxWap-Cre mammary glands was confirmed by western blot analysis of mammary gland protein lysates. STAT5 and ERBB4 were immunoprecipitated from Erbb4+/+Wap-Cre control and Erbb4Flox/FloxWap-Cre mammary gland protein lysates prepared from biparous mice at L1. ERBB4 immune complexes were probed with an ERBB4 antibody (I; ERBB4/ERBB4). Phosphorylation of immunoprecipitated STAT5 protein was determined by western blot analysis using a phosphotyrosine antibody (I; STAT5/P-tyr) and an antibody specific for STAT5 phosphorylated at Y694 (I; STAT5/P-STAT5). The relative epithelial cell population was determined by probing 50 µg of total mammary gland lysate with an antibody specific for keratin 18 (I; NA/K18).

 


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  		Fig. 3. Impaired mammary gland development in biparous Erbb4Flox/FloxWap-Cre mice. Wholemounts of carmine stained mammary glands from biparous Erbb4+/+Wap-Cre (A,C,E) and Erbb4Flox/FloxWap-Cre (B,D,F) mice at P13.5 (A,B), P18.5 (C,D) and L1 (E,F). Alveolar clusters are indicated by arrows and normal distention of mammary ducts is indicated by arrowheads. Scale bar: 500 µm.

 


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  		Fig. 4. Condensed alveolar structures in biparous Erbb4Flox/FloxWap-Cre mice. Histological analysis of Hematoxylin and Eosin stained paraffin wax-embedded biparous Erbb4+/+Wap-Cre (A,C,E) and Erbb4Flox/FloxWap-Cre (B,D,F) mammary glands at P13.5 (A,B), P18.5 (C,D) and L1 (E,F). Abnormal secretory activity is indicated by the accumulation of lumenal lipids in Erbb4Flox/FloxWap-Cre mammary glands at parturition. Arrowheads indicate alveolar lumens. Scale bar: 50 µm.

 


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  		Fig. 5. Erbb4Flox/FloxWap-Cre mice exhibit defects in mammary epithelial proliferation. Immunohistochemical detection of in situ BrdU incorporation in biparous Erbb4+/+Wap-Cre control (A) and Erbb4Flox/FloxWap-Cre (B) mice at L1. Arrowheads indicate BrdU-labeled nuclei. (C) A significant reduction in the percentage of BrdU-labeled cells was observed in Erbb4Flox/FloxWap-Cre mammary glands at P13 (t[8]=4.41, P<0.01) and L1 (t[8]=9.11, P<0.001), but not at P18 (t[13]=1.71, P<0.20). Significant differences within each data set are represented by asterisks. Scale bar: 50 µm.

 


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  		Fig. 6. Erbb4Flox/FloxWap-Cre secretory epithelium fails to express the differentiation marker NPT2B. Immunohistochemical staining of NKCC1 (red) and SMA (green) in mammary glands of biparous Erbb4+/+Wap-Cre and Erbb4Flox/FloxWap-Cre mice, at P13 (A,B), P18 (C,D) and L1 (E,F). Immunohistochemical staining of NPT2B (red) and ß-catenin (green) in mammary glands of biparous Erbb4+/+Wap-Cre and Erbb4Flox/FloxWap-Cre mice, at P13 (G,H), P18 (I,J) and L1 (K,L). Lack of NPT2B staining in Erbb4Flox/FloxWap-Cre mammary glands at L1 indicates a defect in epithelial differentiation. Arrowheads (A,B,E) indicate NKCC1 staining and arrowheads (I,K) indicate NPT2B staining of the apical surface of secretory epithelium.

 


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  		Fig. 8. Reduced milk-gene expression in Erbb4Flox/FloxWap-Cre mice. Northern blot analysis of milk gene expression in biparous control Erbb4+/+Wap-Cre (lanes 1,2) and Erbb4Flox/FloxWap-Cre (lanes 3,4) mice at L1. Total mammary gland RNA was isolated at L1 and subjected to northern blot analysis using probes specific for the milk genes ß-casein, Wap and {alpha}-lactalbumin. A GAPDH probe served as a control for RNA loading.

 


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  		Fig. 9. PRLR signaling in mammary glands at late pregnancy. Erbb4Flox/FloxWap-Cre mice at P18 were either mock injected or injected with PRL at 5 µg/g body weight for 15 minutes. Mammary gland lysates were analyzed by western blot for phospho-STAT5 (A, top panel) and total STAT5 protein (A, bottom panel). In addition, paraffin wax-embedded mammary glands from mock- (B) or PRL (C)-injected Erbb4Flox/FloxWap-Cre mice at P18 were stained for phospho-STAT5 by immunohistochemistry. Detection of PRL-stimulated STAT5 activation in mammary glands from biparous Erbb4Flox/FloxWap-Cre mice at P18 (A,C) indicates intact PRLR signaling. Pregnancy was rescued in Prlr-/- mice by the administration of progesterone. Mammary glands from progesterone-treated Erbb4Flox/FloxWap-Cre control mice (D) and progesterone-rescued Prlr-deficient mice (E) at P18 were embedded in paraffin wax and stained for phospho-STAT5 by immunohistochemistry. Phospho-STAT5 immunohistochemical staining of progesterone-rescued Prlr-null mammary glands indicates PRLR-independent STAT5 activation at late pregnancy. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2003