ErbB3 is required for ductal morphogenesis in the mouse mammary gland - PubMed (original) (raw)

ErbB3 is required for ductal morphogenesis in the mouse mammary gland

Amy J Jackson-Fisher et al. Breast Cancer Res. 2008.

Abstract

Introduction: The receptor ErbB3/HER3 is often over-expressed in human breast cancers, frequently in conjunction with over-expression of the proto-oncogene ERBB2/HER2/NEU. Although the prognostic/predictive value of ErbB3 expression in breast cancer is unclear, ErbB3 is known to contribute to therapeutic resistance. Understanding ErbB3 functions in the normal mammary gland will help to explain its role in cancer etiology and as a modulator of signaling responses to the mammary oncogene ERBB2.

Methods: To investigate the roles of ErbB3 in mouse mammary gland development, we transplanted mammary buds from ErbB3-/- embryos into the cleared mammary fat pads of wild-type immunocompromised mice. Effects on ductal outgrowth were analyzed at 4 weeks, 7 weeks and 20 weeks after transplantation for total ductal outgrowth, branch density, and number and area of terminal end buds. Sections of glands containing terminal end buds were analyzed for number and epithelial area of terminal end buds. Terminal end buds were also analyzed for presence of mitotic figures, apoptotic figures, BrdU incorporation, and expression of E-cadherin, P-cadherin, alpha-smooth muscle actin, and cleaved caspase-3.

Results: The mammary ductal trees developed from ErbB3-/- buds only partly filled the mammary fat pad. In contrast to similar experiments with ErbB2-/- mammary buds, this phenotype was maintained through adulthood, pregnancy, and parturition. In addition, and in contrast to similar work with ErbB4-/- mammary buds, lobuloalveolar development of ErbB3-/- transplanted glands was normal. The ErbB3-/- mammary outgrowth defect was associated with a decrease in the size of the terminal end buds, and with increases in branch density, in the number of terminal end buds, and in the number of luminal spaces. Proliferation rates were not affected by the lack of ErbB3, but there was an increase in apoptosis in ErbB3-/- terminal end buds.

Conclusions: Endogenous ErbB3 regulates morphogenesis of mammary epithelium.

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Figures

Figure 1

Figure 1

Whole mount analysis of ductal morphogenesis in transplanted glands. Mammary buds from ErbB3+/+ or ErbB3+/- and _ErbB3_-/- day 12.5 embryos were transplanted into contralateral cleared mammary fat pads of 3-week-old _Rag1_-/- females. The glands were harvested at (a, b) 4 weeks, (c, d) 7 weeks, (e, f) 20 weeks after transplantation, or (g, h) 1 day postpartum. Shown are paired contralateral transplanted glands from the same recipient mouse. The 20-week glands were from a mouse that was mated but did not deliver a litter and was not pregnant at time of sacrifice. The 1 day postpartum (1D PP) mouse was mated about 8 weeks after transplantation and a litter was delivered about 12 weeks after transplantation. The glands were harvested 1D PP. Inserts in panels g and h are hematoxylin and eosin stained sections of +/+ (panel g) and -/- (panel h) paired transplants at 1D PP.

Figure 2

Figure 2

Analysis of epithelial outgrowth. Diamonds represent the average end to end length of total ductal outgrowth measured in arbitrary units in a transplanted gland at (a) 4 weeks and (b) 7 weeks for the null and wild-type or heterozygous (Wt/het) donor genotypes. The average numbers of branchpoints within a unit area are shown for (c) 4 weeks and (d) 7 weeks after transplantation. (e) Ductal outgrowths for 20 weeks after transplantation, mid-pregnancy, and 1 day postpartum (1D PP) are shown together. In all panels, solid lines connect the null with the Wt/het pair of contralateral transplants from the same recipient mouse. Dotted lines connect the mean of the outgrowths from contralateral glands in complete null-Wt/het pairs, and the mean lengths are listed at the termini of these lines. Asterisks represent the mean from all recipients of a given genotype, with mean lengths presented in italics in panels b and d.

Figure 3

Figure 3

Whole mount analysis of TEBs. (a) High-magnification TEBs in carmine alum stained whole mounts. Large bulbous TEBs are present at the end of the advancing duct in +/+,+/- glands at 4 weeks after transplantation (top left), but have regressed by 7 weeks (middle left) and 20 weeks (bottom left) after transplantation. The TEBs in the -/- glands are present at 4 weeks (top right), they persist at 7 weeks (middle right), and have regressed by 20 weeks (bottom right). Whole mounts at a given time point are contralateral glands from the same recipient mouse at the same magnification. (b) Number and area of TEBs after transplantation. Shown are the number of TEBs in a carmine alum (CA)-stained whole mount (top left) and the average epithelial area of the TEBs (top right) at 4 weeks after transplantation in three mice with paired contralateral outgrowth; and the number of TEBs (bottom left) and the epithelial area of the TEB in stained paraffin-embedded sections (bottom right) at 4 weeks aftertransplantation in six _ErbB3_-/- glands and six _ErbB3_+ glands, two of which are paired contralateral outgrowths. Solid lines connect the paired null and wild-type or heterozygous (wt/het) contralateral transplants from the same recipient mouse. Dotted lines connect the mean of the outgrowths from contralateral glands in the complete null-wt/het pairs, and the means are listed at the termini of these lines. Asterisks represent the mean from all recipients of a given genotype, with means listed in italics. Circles mark the TEBs shown in panel c. (c) Representative TEBs in paraffin-embedded sections. An _ErbB3_+ TEB (bottom right) in an hematoxylin and eosin stained paraffin-embedded section compared with a _ErbB3_-/- TEB (bottom left) taken at the same magnification. Both TEBs were near average size for the respective genotype. Circles in panel b, lower right, mark the corresponding diamonds. TEB, terminal end bud.

Figure 4

Figure 4

Immunohistochemical analysis of a TEB in serial sections. (a-d) TEBs from serial sections of an ErbB3+/+ gland 4 weeks after transplantation (panels a-d) have a typical single layer cap cell/myoepithelial cell outer layer (panels b and d) and a multilayered body cell section (panel c). (e-i, j-n, and o-s) Serial sections of three representative -/- TEBs at 4 weeks after transplantation. The three TEBs vary in the cap cell layer (panels h, l, and q), in the layers in the body cell layer (panels g, m, and d), and in the size of the spaces between the cap and body cell layers (panels i, j, and o). (t-w) The multilayered cap cell compartment has large spaces and the body cell layer is a single layer in the serial section of an _ErbB3_-/- TEB from a 7-week post-transplantation gland. cc3, cleaved caspase 3; H&E, hematoxylin and eosin; IgG, negative control antibody; E-cad, E-cadherin; P-cad, P-cadherin; SMA, smooth muscle actin; TEB, terminal end bud.

Figure 5

Figure 5

Apoptosis in TEBs. H&E stained TEBs in +/+ transplanted glands (top left) have few apoptotic nuclei and one cleaved caspase 3 positive (brown stain) cell (middle left). H&E stained TEBs in -/- transplanted glands (top right) have numerous apoptotic nuclei and cleaved caspase 3 positive cells (middle right). BrdU-positive cells (brown stain) are present in both the +/+ transplanted glands (bottom left) and in the -/- transplanted glands (bottom right). BrdU, 5-bromo-2-deoxyuridine; cc3, cleaved caspase 3; H&E, hematoxylin and eosin; TEB, terminal end bud.

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