Key stages in mammary gland development: the mammary end bud as a motile organ

Abstract

In the rodent, epithelial end buds define the tips of elongating mammary ducts. These highly motile structures undergo repeated dichotomous branching as they aggressively advance through fatty stroma and, turning to avoid other ducts, they finally cease growth leaving behind the open, tree-like framework on which secretory alveoli develop during pregnancy. This review identifies the motility of end buds as a unique developmental marker that represents the successful integration of systemic and local mammotrophic influences, and covers relevant advances in ductal growth regulation, extracellular matrix (ECM) remodeling, and cell adhesion in the inner end bud. An unexpected growth-promoting synergy between insulin-like growth factor-1 and progesterone, in which ducts elongate without forming new end buds, is described as well as evidence strongly supporting self-inhibition of ductal elongation by end-bud-secreted transforming growth factor-beta acting on stromal targets. The influence of the matrix metalloproteinase ECM-remodeling enzymes, notably matrix metalloproteinase-2, on end bud growth is discussed in the broader context of enzymes that regulate the polysaccharide-rich glycosaminoglycan elements of the ECM. Finally, a critical, motility-enabling role for the cellular architecture of the end bud is identified and the contribution of cadherins, the netrin/neogenin system, and ErbB2 to the structure and motility of end buds is discussed.

Figure 1

Photomicrographs illustrating motility and histoarchitecture of end buds. (a) Natural and experimentally induced motility 'behavior' of end buds in the mammary ductal system of a 5-week-old nulliparous mouse. The 'open' ductal architecture of the mammary tree leaves 80% or more of the gland epithelium-free. Large terminal end buds identify the most actively growing region of the gland (top arrows), and progressively smaller lateral end buds extend to each side of the center, indicating slowed forward growth as the end bud encounters a thinning fat pad. End buds may also reverse direction to grow back into accommodating stroma (side arrow). Bifurcating end buds (top arrows) are arrayed along the growth front. Original magnification approx. ×12. (b) Cross-section through end bud with accompanying diagram. End buds are bilayered structures; an outer layer of myoepithelial progenitor cells (cap cells) overlays a multilayered mass of luminal cells fated to form the walls of the ductal lumen (L). Stained with hematoxylin and eosin. Original magnification approx. ×300.

Figure 2

Photomicrograph of a longitudinal section through an end bud and its subtending duct. A lateral end bud stained to reveal sulfated glycosaminoglycans and mitotic cells with the cap cell layer is indicated by the dashed line. Constriction of the end bud to ductal dimensions coincides with induction of a collagenous extracellular matrix along the end bud flank. Note that this sheath is continuous with the subtending duct as well as the duct of origin (dotted lines). The basal lamina along the end bud flank (large arrows) as well as in the cleft of a bifurcation (triangle) stained deeply for sulfated glycosaminoglycans (Alcian blue stain). This contrasts with weakly stained basal lamina around the end bud tip indicative of non-sulfated hyaluronate (short arrows). Silver grains (dark spots) are from tritiated thymidine autoradiography and mark mitotic cells in the end bud and subjacent ducts. Original magnification approx. ×300.

Figure 3

Loss of Ntn1 disorganizes end buds. The Ntn1^+/+ end bud (top) displays normal CDH3 staining of the cap cell layer at the tip of the end bud (delineated by the dashed line). In contrast the Ntn1^-/- end bud (bottom) displays a loss of adhesion between the cap and luminal cell layers, with a large space forming under the cap cell layer (delineated by the dashed line). This space fills with dissociated cap cells (arrows show three examples) that either die by apoptosis or migrate inappropriately into the body of the end bud. Original magnification approx. ×300.