EGF-R ligands are required for development and mucosal maintenance in mouse small intestine. Both maternal and neonatal sources of growth factors are required for optimal pup growth.

Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the TGFalpha-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless, pups born to triple null dams frequently died or were runted, suggesting a mammary gland defect. Comparison of individual and combinatorial knockouts established that specific loss of AR severely stunted ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal developing ducts. Surprisingly, loss of all three ligands did not significantly affect cellular proliferation, apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females, but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFalpha in mammopoiesis and lactogenesis. In triple null glands, alveoli were poorly organized and differentiated, and milk protein gene expression was decreased. Additionally, Stat5a activation was frequently reduced in AR single and combinatorial nulls in association with impaired lactation. Collectively, our results provide genetic confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of growth factors from pups nursed by triple null dams further worsened their survival and growth, establishing functions for both maternal- and neonatal-derived growth factors.

The four ERBB receptors and their multiple polypeptide ligands are differentially expressed during development of the mouse mammary gland. Profiles suggest that ERBB1/EGF receptor (EGFR)4 and ERBB2/Neu are required during ductal morphogenesis, whereas the Neuregulin (NRG) receptors, ERBB3 and ERBB4, are preferentially expressed through alveolar morphogenesis and lactation. Consistent with these profiles, recent gene knockouts established that EGFR and its ligand, Amphiregulin (AR), are essential for ductal morphogenesis in the adolescent mouse and likely provide the required epithelial-stromal signal. In contrast, the phenotypes of transgenic mice expressing dominant negative ERBB2 and ERBB4 proteins suggest that these receptors differentially act to promote or maintain alveolar differentiation. This view of ERBB action provides a conceptual framework for future testing using more sophisticated conditional knockout models. New or existing transgenic mice are also being used to better understand the contributions of ERBB receptors and ligands to mammary tumorigenesis, as well as to more closely mimic the human disease. Recent studies have focused on defining molecular events in neoplastic progression, and in the case of ERBB2/Neu, the requirement for ERBB heterodimerization partners as well as the relative importance of gene amplification versus gene mutation. Collectively, these recent studies establish that normal development and homeostasis of the mammary gland is critically dependent on regulated ERBB signaling. They also illustrate the value of animal models in deciphering roles for the complex ERBB network in this dynamic tissue.

We previously reported that multiparous WAP-TGFa transgenic mice develop mammary gland carcinomas with complete incidence. TGFa-induced tumors appear stochastically and with relatively long latency, indicating an additional requirement for other genetic alterations. To identify genes that cooperate with TGFa in mammary tumorigenesis, we used a retroviral insertion approach featuring a cloned and infectious hybrid MMTV (C3H/ Mtv-1; (Shackleford and Varmus, 1988)). Tumor latency was decreased approximately 30% in MMTV-infected WAP-TGFa transgenic animals compared to noninfected transgenic controls, and 430% of the corresponding tumors displayed evidence of integrated C3H/ Mtv-1 DNA. PCR-based analyses of DNAs from two virus-infected, transgenic tumors revealed integration of hybrid MMTV in 3' untranslated exons of the Wnt-1 or Wnt-3 oncogenes. Moreover, Northern blots con®rmed dramatic induction of Wnt-1 or Wnt-3 transcripts in the respective tumors, indicating that MMTV integration resulted in activated expression of these genes. Semiquantitative RT ± PCR analyses showed that overexpression of Wnt-1 or Wnt-3 was a common occurrence in MMTV-infected WAP-TGFa tumors, and some noninfected WAP-TGFa tumors also showed evidence of elevated Wnt-3 transcripts. Collectively, these results reveal cooperative induction of mammary gland tumorigenesis by simultaneous deregulation of EGF-like (TGFa) and Wnt growth factors. Oncogene (2000) 19, 3193 ± 3199.

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