Communication
Mol. Cells, Vol. 7, No.3, pp. 448-450
In Situ Localization of WDNMI and Ferritin Heavy Chain
Gene Expression in Mammary Gland
Myunggi Baik*, Lothar Hennighausen 1 and Yunjaie Choi 2
Department of Genetic Engineering, College of Agriculture, Institute of Biotechnology, and
Hormone Research Center, Chonnam National University, Kwangju 500-757, Korea;
lLaboratory of Biochemistry and Metabolism, National Institutes of Diabetes, Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1812, U.S.A.;
2Department of Animal Science and Technology, College of Agriculture and Life Science,
Seoul National University, Suwon 441-744, Korea
(Received on February 11, 1997)
In situ hybridization was performed with sections obtained from mammary gland at 10
days of lactation and at 1 and 3 days of involution using either digoxigenin-Iabeled antisense or sense RNA probe in order to localize expression of WDNMI and ferritin heavy
chain mRNA. The WDNMI gene was predominantly expressed in the layer of secretory
epithelial cells surrounding the lumen of mammary gland alveoli. The lower levels of
WDNMI mRNA were observed at involution day 3 compared to involution day 1. The expression of ferritin heavy chain mRNA also appears to be confined to the epithelial layer
of mammary alveoli. The lower levels of ferritin heavy chain mRNA were observed at involution day 3 compared to involution day 1.
Lactation is under multi-hormonal control, and requires the presence of prolactin, estrogen, progestrone and a variety of growth factors (Lascelles
and Lee, 1978). After completion of lactation, the
mammary gland undergoes involution, regressing to
a state resembling that of a virgin animal. This phase
of mammary gland development is characterized by
dramatic epithelial cell death and tissue remodelling.
The involution of the mammary gland is presumed
to be mediated by a decrease in serum prolactin level induced by weaning, but may also involve
changes in paracrine or autocrine growth factors.
Many questions for molecular mechanisms which regulate mammary epithelial cell death remain unan swered. Recent studies have indicated active gene expression during involution of the mammary gland.
The expression of Fas antigen (Baik et al., 1995),
sulfated glycoprotein-2 (Boudreau et al. , 1995),
stromelysin (Strange et al., 1992), interleukin-1 ~
converting enzyme (Boudreau et al., 1995), plasminogen activator (Lund et al., 1996), several genes associated with G1 cell cycle including cyclin Dl, D2,
and D3 (Marti et al., 1995) and apoptosis genes, Bax
and Bcl-x s (Heermeir et ai., 1996), was induced during involution of the mammary gland. Previously,
we have performed differential hybridization of message from the mammary gland in an attempt to find
differentially expressed messages during mammary
gland involution and reported the induction of
* To whom correspondence should be addressed.
WDNM1 (Choi et ai. , 1996) and ferritin heavy chain
(FHC) genes (Lee et aI., 1996) during involution of
the mammary gland.
During lactation, the mammary gland consists of
several types of cells with major ductular epithelial
cells and a layer of secretory alveolar epithelial cells
surrounding the lumen. The secretory alveolar epithelial cells are surrounded by a layer of elongated,
myoepithelial cells. In addition, stromal components
(adipocytes and fibroblasts) are present in the mammary gland. If the WDNMl and FHC have a functional role in the epithelial cell death of the mammary
gland, these are likely to be expressed in the epithelial cells of the mammary gland. Which cell types
express the WDNMl and FHC gene ' have not been
defined yet. In this paper, we found that both
WDNM1 and FHC genes were predominantly expressed in the epithelial cells of mammary gland alveoli.
Materials and Methods
Animals
Tissues obtained from C57BL/6 female
used in experiments. For the induction of
the young were removed at 10 days after
and the mammary tissues were obtained
dicated time after weaning.
mice were
involution,
parturition,
at the in-
The abbreviations used are: DIG, digoxigenin; FHC, ferritin
heavy chain.
©
1997 The Korean Society for Molecular Biology
Vol. 7 (1997)
449
Myunggi Baik et al.
Preparation of digoxigenin (DIG)-labeled RNA probes
For in situ hybridization, DIG-labeled RNA probes
were prepared as described below. The pBluescript
vector containing either WDNM1 or ferritin heavy
chain (FHC) cDNA was linearized with EcoRI for antisense template and with XhoI for sense template.
The WDNM1 (450 bp) and FHC cDNAs (1.0 kb)
have been previously identified by differential screening during involution of the mammary gland (Choi et
ai. , 1996; Lee et ai. , 1996). DIG-labeled antisense
RNA probes were prepared from the linearized
plasm ids using a DIG RNA labeling kit and T7 RNA
polymerase (Boehringer Mannheim, Germany). DIGlabeled sense RNA probes were prepared using the
labeling kit and T3 RNA polymerase.
In situ hybridization
Mammary gland tissues were fixed in 4% paraformaldehyde solution and embedded in paraffin. Five 11m
sections were mounted on silan-coated slides. For in
situ hybridization, sections were deparaffinized, treated with 2 ).l.g!rnl proteinase K for 10 min at room temperature, postfixed in 4% paraformaldehyde and acetylated. The sections were hybridized at 50 °C overnight with the DIG-labeled RNA probes in solution
containing 50% formamide, 5 x SSC, 0.1 M sodium
phosphate buffer pH 7.0, 1 x Denhardt' solution, 100
).l.g!ml salmon sperm DNA and 100 ).l.g!ml yeast
tRNA. The slides were washed twice at 65 °C with
50% formamide/2 x SSC for 30 min and twice at 65
°c with 25 % formamide/1 x SSC for 30 min. After
treating them with 5 ).l.g!rnl RNase A for 10 min at 37
°c, the sections were subjected to color development
using a DIG nucleic acid detection kit (Boehringer
Mannheim). Briefly, the sections were incubated with
anti-DIG alkaline phosphatase solution. The color
(purple/blue) reaction was performed overnight using
5-bromo-4-chloro-3-indolyl phosphate and nitroblue
tetrazolium. The sections were counterstained with nuclear fast red.
Results and Discussion
After completion of lactation, the mammary gland
undergoes involution, regressing to a state resembling
that of a virgin animal. This phase of mammary
gland development is characterized by dramatic epithelial cell death and tissue remodelling. Recent studies have indicated active gene expression during involution of the mammary gland. Previously, we
analyzed the levels of WDNM1 and the ferritin
heavy chain gene (FHC) mRNA by Northern hybridization and we found that expression of WDNM1
and FHC genes was induced during involution of the
mammary gland (Choi et ai., 1996; Lee et ai., 1996).
During lactation, the mammary gland consists of several cell types including duetular and secretory alveolar epithelial cells, myoepithelial cells, adipocytes
and fibroblasts. Which cell types express WDNM1
Antisense
Sense
L10
i1
i3
i1
Figure 1. III situ hybridization of WDNMI gene expression in mouse mammary gland. The tissue sections of mammary gland obtained at 10 days of lactation (LlO) and at 1
(il) and 3 days (i3) of involution were hybridized with either a digoxigenin (DIG)-labeJed antisense or sense WDNMI
RNA probe. The sections were incubated with anti-DIG alkaline phosphatase, and the color (purple/blue) reaction
was performed overnight using 5-bromo-4-chloro-3-indolyl
phosphate and nitroblue tetrazolium. The sections were
counterstained with nuclear fast red. The WDNMI gene
was predominantly expressed in the secretory epithelial
cells (arrow head) surrounding the lumen (lu). Bar, 100 ).1m
(A, B, C, D, E, F); 320 ).1m (G, H).
and the FHC gene have not been defined yet.
In order to localize expression of the WDNM1
gene, in situ hybridization was performed with sections derived from the mammary gland at 10 days of
lactation and at 1 and 3 days of involution using either a digoxigenin-Iabeled antisense WDNM1 RNA
probe or a sense RNA probe. Figure 1 demonstrated
that the WDNM1 gene was predominantly expressed
of the epithelial cells of mammary gland alveoli (Figs.
lA, 1C, and lE). Higher magnification clearly showed
that the expression of the WDNM1 gene was confined to the epithelial layer of mammary alveoli (Fig.
1G). The specificity of the signal was confirmed by
hybridizing with the corresponding sense probe. No
color was detected using a sense probe (Figs. lB, 1D,
IF, and 1H). The quantitative comparison of expression levels of the WDNM1 gene between lactation day 10 and involution day 1 was not possible
in this experiment, but lower intensities of WDNM1
expression were evident at involution day 3 com-
Localization of WDNM1 and Ferritin Heavy Chain Gene
450
Antisense
Sense
L10
i1
i3
i1
Mol. Cells
by inhibiting the action of proteases (Dear and Kefford , 1991). The functional significance of WDNM1
expression in the mammary gland remains to be studied .
FHC is a component of ferritin, an iron storage protein in eukaryotes that plays a crucial role in iron
homeostasis (Theil, 1987). In addition, ferritin functions in the detoxification of elemental iron, which is
toxic in a non-complexed form. Increased production
of ferritin in cultured endothelial cells was found to
increase cell resistance to oxidant-mediated damage
(Balla et at., 1992). Other study suggests that arsenite
exposure may lead to increased levels of oxygen radical which augmented ferritin can act to detoxify
(Guzzo et at., 1994). The functional role of FHC III
the mammary gland presently remains unclear.
Acknowledgment
This study was supported in part by the Genetic Engineering Research Program (96/97), Ministry of Education, Korea and by a grant from KOSEF through the
Hormone Research Center (HRC-97-0402).
References
Figure 2. In situ hybridization of ferritin heavy chain
(FHC) gene expression in mouse mammary gland. The tissue sections of mammary gland obtained at 10 days of lactation (UO) and at 1 (i1) and 3 days (i3) of involution
were hybridized with either a digoxigenin (DIG)-labeled antisense or sense FHC RNA probe. The sections were incubated with anti-DIG alkaline phosphatase, and the color
(purplelblue) reaction was performed overnight using 5bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium . The sections were counterstained with nuclear fast
red . The FHC gene was predominantly expressed in the secretory epithelial cells (arrow head) surrounding the lumen
(Iu). Bar, 100 11m (A, B, C, D, E, F); 320 11m (G, H).
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