Role of FGF10 in Early Mouse Mammary Gland Development

Institution: Childrens Hospital, Los Angeles
Investigator(s): Saverio Bellusci, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9PB-0082 Award: $446,400
Award Type: Request for Applications
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2003)
Significant development of the breast takes place before birth in humans and mice. Early breast development is dependent on secreted molecules that control the migration of epithelial cells. Mis-regulation of this normal process occurs during cancer metastasis. The genes involved in normal breast cell migration are largely unknown, but we have found that a gene encoding a secreted molecule called Fibroblast Growth factor 10 (FGF10) controls the early steps of breast development in a mouse model deficient for the gene. The mechanism of FGF10 action in this process is unknown. In this proposal, we are going to determine the role of FGF10 and its potential interaction with another important family of growth factors, called WNTs (known to be involved in normal breast development as well as in the process of tumor progression) in the process of epithelial cell migration. This proposal should allow a better understanding of the important process of epithelial cell migration.

Our hypothesis is that FGF10 in association with members of the WNT family controls the migration of breast cells during embryonic development. Our long term goal is to apply the knowledge that we will have gained in studying normal breast development to understand the process of migration during metastasis of primary breast tumors.

We are going to use both in vivo and in vitro techniques to test the central hypothesis. State of the art imaging techniques will be used to follow the migration of the breast cells as the breast develops. We will analyze the timing of when key genes potentially controlled by FGF10 during the process of the breast development are activated.

Our very innovative proposal will allow us to identify the function of key genes in controlling migration during normal development of the breast. Understanding how cell migration is controlled during normal breast development is critical to design strategies to fight against cancer metastasis, which is characterized by active migration of a subpopulation of very aggressive cells and represents the major complication of the evolution of a tumor.

Final Report (2006)
Migration of epithelial cells at the surface of the skin is a critical event to form the future breast. The migration of the epithelial cells (which are this stage should be considered as epithelial stem cells) is associated with the formation of the five pairs of mammary gland rudiment in the embryo and our previous work indicated that Fibroblast Growth factor 10 (Fgf 10) was potentially controlling either the very early event of the formation of the breast or the maintenance of the breast rudiments.

During this third and final year of funding, we have dedicated our efforts towards determining the mechanism of action of FGF10 on either the maintenance of the breast rudiment or on its formation per se.

The results generated during the total funding period have been published recently in a seminal article in Development. Our work demonstrates that FGF10 controls the expression of Wnt10b in the putative mammary line located on each flank of the mouse embryo and from which the five pairs of breast rudiments (also called placodes) are forming. Using reporter mice called Topgal mice (to follow in vivo the activation of canonical Wnt signaling and the formation of the mammary line) in the Fgt10 null background, we found that the absence of placodes is preceded by a lack of mammary line formation. Our previous work also suggested a role for Fg110 expressed in the somites in the induction of mammary placode formation in the mouse embryo. During this funding period, we have redefined the spatial-temporal expression of Fgf10 in the somites by whole mount in situ hybridization. From E10 to E10.25, Fgt10 is transiently expressed in the fore- and hindlimbs. From E10.5 onwards, Fg110 is expressed in the somites, first in the ventral area and later extending to the medial and dorsal areas. Notably somitic Fgr10 is expressed in the anterior somites of the interlimb area only (where placode 3 will form), and does not extend to the low abdominal/inguinal region where the breast rudiments 4 and 5 would emerge from within the epithelial cells. We demonstrate that Pax3 null mutants, lacking the ventral bud of the somites in the interlimb area, lack the induction of mammary placode 3 only, preceded by a delayed formation of the mammary line in the interlimb region. This suggests that Fg110 expression in the somites is not required for the formation of the mammary line, whilst it is required for the formation of the placode 3 from this line. Embryos exhibiting a decrease in Fg110 expression compared to the wild type embryos form a mammary line, but not mammary placode 3, suggesting different sensitivity levels of Fgt10 expression for the mammary line and placode 3 or redundancy for Fgf 10 in the formation of the mammary line, but not in the formation of placode 3 from this line.

This work shows that FGF10 from the somites controls Wnt signaling which is critical for the formation of the mammary line. These two pathways are likely to be critical throughout breast development both during the embryonic and post-natal phases for the proper maintenance, proliferation and differentiation of the breast epithelial stem cells, These two pathways have already been shown to be involved in breast cancer progression and the knowledge accumulated during these early phases of development about the exact function of these two signaling pathways will be instrumental in designing strategies to interfere with cancer progression.

Symposium Abstract (2005)
This project focuses on the role of FGF10 in breast development, taking the 5 pairs of mouse breasts as a model for human breast development. In humans, breasts develop from so-called milk-lines, thickenings of the skin spanning from thorax to groin, both on the left and right side of the body. These lines regress completely except at one spot on the thorax, where the breasts develop. However, in 1-5% of all humans, additional breasts develop on other positions along these milk-lines, most often near the normal breast, but sometimes remote, in the groin. Normal breasts alike, additional breasts are susceptible to pathology, including breast cancer.

In the mouse, we identified each milk-line to exist of at least three independent mammary streaks. One streak appears in the axilla, giving rise to gland #1. Another streak appears on the flank between the forelimb and hind limb, half-way in between giving rise to gland #3. The third streak appears in the groin, giving rise to gland #5. Glands #2 and #4 appear on the intersections of the axillary streak with the streak on the flank, and of the inguinal streak with the streak on the flank respectively. These different origins of the mammary glands suggest that the onset of the formation of the different glands is regulated by different molecular mechanisms. This is supported by our observation that in mice lacking the function of FGF10, the mammary streaks do not appear, except at the position of the prospective gland #4.

Additionally, our analysis of the expression of FGF10 in normal mice suggests that glands #1, 2, 3, and 5 use different sources of FGF10 to start their formation. We identified the somites as one of the sources. Somites are transiently existing structures giving rise to vertebrae and ribs, skeletal muscles, and importantly, the dermis. The dermis is in contact with the epidermis (the top layer of the skin) and instructs it to form a mammary gland. We conclude that in the absence of FGF10 in the somites, the dermis has lost the capacity to properly communicate these instructions to the epidermis.

We furthermore identified that the complete loss of FGF10 in specific parts of the somites only, restores the formation of glands #1, 2, and 5, and allows for a delayed but impaired formation of gland #3. By contrast, a partial reduction of FGF10 throughout the entire somite, likewise restores the formation of glands #1, 2, and 5, but does not allow the formation of gland #3. This adds another level of sensitivity to FGF10 for mammary gland formation.

Deregulation of genes that regulate normal development of a particular organ is often a cause of cancer. Therefore, our data suggest that each pair of mammary glands, whether it is the normal or an additional pair in humans, may also have a different genetic 'barcode' for the development of breast cancer. This difference may be relevant for studies using the mouse breast as a model for human breast cancer.

Identification of the mammary line in mouse by Wnt10b expression.
Periodical:Developmental Dynamics
Index Medicus: Dev Dyn
Authors: Veltmaat JM, Van Veelen W, Thiery JP, Bellusci S.
Yr: 2004 Vol: 229 Nbr: 2 Abs: Pg:349-356

Fgf10 Expression Identifies Parabronchial Smooth Muscle Cell Progenitors and is Required for their Entry into the Smooth Muscle Cell Lineage
Index Medicus:
Authors: Mailleux, A. A., Kelly, R.G., Veltmaat, J.M., ...and Bellusci, S.
Yr: 0 Vol: 132 Nbr: Abs: Pg:2157-2166

Gli3-mediated somatic Fgf10 Expression Gradients are Required for the Induction and Patterning of Mammary Epithelium along the Embryonic Axes.
Index Medicus:
Authors: Veltmaat, J.M., Relaix, F., Le. L.T., Kratochwil, K., Sala, F.G....and Bellusci, S.
Yr: 0 Vol: 133 Nbr: Abs: Pg:2325-2335