Figure 1.
Miz1 expression in the mammary gland epithelium at different developmental stages.
(A) Immunocytochemistry revealed a nuclear expression of Miz1 in the virgin gland as well as during pregnancy and involution. During lactation also a cytoplasmic staining was observed in addition to the nuclear stain. (B) Western blot analysis of Miz1 expression exhibited a strong Miz1 expression during lactation while the protein could only occasionally be detected in virgin gland. Scale bar in A: 50 µm.
Figure 2.
Female mice with a Miz1ΔPOZ mammary gland exhibit a lactation defect.
(A) Time course of the body weight of the offspring from control (black, n = 6) and Miz1ΔPOZ mothers (blue, n = 6). The number of pups per mother was set to 6 at birth. (B) Size differences in 24-day-old pups did not depend on their gender. (C) Mammary glands from mothers of lactation day 6 were investigated by histology with H & E sections and glands from mothers of lactation day 1 were analysed in whole mounts (D). Morphometric analysis of the adipose tissue content from H & E sections (Lactation day 6) are shown in (E). Note that the difference in the ratio of glandular to adipose tissue is similar during the first and second pregnancies. Scale bar in C: 500 µm.
Figure 3.
Miz1 function on proliferation and apoptosis of mammary gland epithelial cells in vitro and in vivo.
(A) Ki67 immunostaining in control and Miz1ΔPOZ lactation day 6 mammary glands. (B) Ki67 labeling index (up) and quantitative RT-PCR (down) obtained from lactation day 6 samples. (C) The expression of potential proliferation regulators like Cdkn1a and Myc was measured by qRT-PCR. (D) The TUNEL assay was performed on tissue from lactation day 1 and lactation day 6, respectively. Quantifications obtained from 20x pictures are shown (at least n = 3 per genotype and time-point). (E) Representative confocal microscopy pictures of acinar structures formed 6, 8 and 10 days after seeding shscr and shMiz1 transfected HC11 cells onto Cultrex-coated coverslips. Nuclei were stained with Hoechst (blue), actin filaments with Phalloidin-TRITC (red) and apoptotic cells by cleaved Caspase-3 immunostaining (green). (F) Western Blot showing the knock-down of Miz1 in shMiz1 HC11 cells. Numbers indicated are fold changes of band intensities obtained by densitometry (see Materials and Methods). Acinar cell apoptosis and proliferation were quantified by analysis of cleaved caspase-3 (G) and Ki67 (H) positivity in double immunofluorescence confocal microscopy pictures. (I) Quantification of the number of nuclei per acinus in shscr and shMiz1 transfected HC11 cells. Data from two independent experiments were merged and the total number of acini analysed is indicated. See Materials and Methods for experimental details. Scale Bar in A: 50 µm; E: 25 µm.
Figure 4.
Differentiation of mammary gland epithelial cells in control (Ctr) and Miz1ΔPOZ tissue and in HC11 cells with reduced levels of Miz1.
(A) Gene expression of the milk proteins α-casein, ß-casein and whey acidic protein (Wap), measured by quantitative RT-PCR, in control and Miz1ΔPOZ mammary gland tissue (ΔPOZ). (B) Immunoblot analysis for ß-casein from mice with the indicated phenotype. Each lane represents an individual animal. (C) Immunostaining with an antibody against milk proteins in the acini from control and Miz1ΔPOZ mammary glands. (D) Sudan III staining was performed on cryosections from lactating mammary glands of Ctr and MizΔPOZ mice (n = 4 per genotype). Data from A to D was obtained from lactation day 6 samples. (E) Time course of growth and differentiation as performed in the experiments with HC11 cells. EGF: epidermal growth factor; DIP: differentiation media containing dexamethasone, insulin and prolactin. Time points indicated correlate to the time points in (G) and (H). (F) HC11 cells were stably transfected with scrambled short hairpin (sh) RNA (shscr), a Miz1 shRNA and a vector expressing Miz1 as a positive control. The film was exposed 1 and 5 minutes, respectively. (G) PCR and (H) Western blots revealed a down-regulation of ß-casein when Miz1 concentration is decreased. Numbers indicated in (B) and (H) are fold changes of band intensities obtained by densitometry (see Materials and Methods). Scale Bar in C: 50 µm; D: 100 µm.
Figure 5.
Stat5 phosphorylation is reduced in Miz1ΔPOZ mammary glands and in shMiz1 HC11 cells.
(A) Analysis of Stat5a/b mRNA expression in Miz1ΔPOZ (ΔPOZ) and control mammary gland tissue (Ctr). (B) Immunoblot analysis of Stat5 in wildtype and Miz1ΔPOZ mammary glands. (C) Immunohistochemical staining of pStat5 in the mammary gland tissue from wildtype and Miz1ΔPOZ animals. (D) Western blots from HC11 cells stably transfected with a scrambled short hairpin (sh) RNA (shscr) or a Miz1 shRNA (see also Fig. 4E and F). Numbers indicated are fold changes of band intensities obtained by densitometry (see Materials and Methods). Quantitative RT-PCR for the prolactin receptor (E; Prlr), the Supressors of cytokine signalling (Socs) 1, 2 and 3 and caveolin-1 (F; Cav1), and for ErbB4 (G). Lactation day 6 samples were used in all in vivo experiments. Scale bar in C: 50 µm.
Figure 6.
Genes related to vesicular transport processes are bound by Miz1 and down-regulated in Miz1ΔPOZ mammary glands.
(A) GSEA analysis comparing the gene expression of wildtype versus Miz1ΔPOZ mammary glands. The 100 Miz1 target promoters with the highest tag number were used as the gene set in this analysis. (B) Browser pictures of Miz1 ChIP-Seq profiles at the Miz1 target genes Exoc2, Vamp4 and Lrp12. (C) Quantitative RT-PCRs testing the expression of genes indicated in Table 1. (D) Electron microscopy showing vesicles with (arrows) and without (asterisks) casein micelles in tissue from control and Miz1ΔPOZ animals. (E) Percentage of the two vacuole types in mammary gland epithelial cells of control and Miz1ΔPOZ animals from lactation day 10. Data obtained from 4 animals per genotype. Total number of vacuoles counted: ctr, 309–379; ΔPOZ, 339–404. Scale bar in D: 1 µm.
Figure 7.
Protein expression and localization of Prlr and ErbB4.
PrlR (A) and ErbB4 (B) immunofluorescence from control and Miz1ΔPOZ mammary gland tissue (lactation day 6). (C) Hypothetical model about the function of Miz1 in the mouse lactating mammary gland. Vesicular transport processes are impaired in Miz1ΔPOZ mice due to a decreased gene expression of Miz1 target genes which are involved in the vesicular transport. This causes a reduction of the PrlR and ErbB4 exposure to the plasma membrane, hampering the autoamplifying expression of PrlR. Reduction of PrlR and ErbB4 expression and their diminished availability at the cell surface leads to a decreased amount of phosphorylated Stat5, which is the key regulator during lactation. In consequence, reduced levels of phosphorylated Stat5 dimers (represented by smaller symbol size) cannot adequately activate the transcription of proliferation and differentiation genes in Miz1ΔPOZ glands [50], [53]. Scale bars in A and B: 50 µm.
Table 1.
GSEA analysis.