The control of branching morphogenesis involves determining when and where a branch will occur, how long the tube grows before branching again, and at what angle the branch will form. The development of different organs (such as salivary gland, mammary gland, kidney, and lung) creates branching patterns easily distinguished from each other. Moreover, during the development of a particular organ, the form of branching often changes, depending on the place or time when the branching occurs.
The branching development of the lung depends upon the interactions between the endodermal foregut diverticulum and the splanchnic mesenchyme by which it is surrounded. Mesenchyme from the tracheal region will inhibit branching when apposed to distal lung epithelium, while distal mesenchyme (from the region of lung bud formation) will induce ectopic branches when grafted adjacent to isolated tracheal epithelium (Alsecio and Cassini, 1962; Wessels, 1970; Goldin and Wessels, 1974; Shannon, 1994). Moreover, the regional cytodifferentiation of the respiratory epithelium is also specified by the mesenchyme (Shannon et al., 1998).
Taderera (1967) showed that the effect of the respiratory mesenchyme was transmitted across porous filters, thereby implicating soluble molecules in the mesenchymal regulation of lung development. Recent experiments have demonstrated that paracrine factors such as sonic hedgehog, Wnt proteins, Bone morphogenetic protein 4, scatter factor, and Fibroblast growth factor 10 each play important roles in the lateral branching of the mouse lung bud (Hogan et al., 1997, 1999; Warburton et al., 2000). Genes encoding Bone morphogenetic protein 4 (Bmp4), Wnt2, and Sonic hedgehog (Shh) are expressed at high levels in the bud-forming distal epithelium, while genes encoding Fibroblast growth factor 10 (Fgf10) and the Shh receptor Patched (Ptc) are expressed in the distal mesenchyme (Levay-Young and Navre, 1992; Bellusci et al., 1996, 1997a, 1997b; Urase et al., 1996).
In the embryonic mouse lung, Fgf10 regulates the placement and expansion of the lung bud (Bellusci et al., 1997b). Mice homozygous for loss-of-function mutations of Fgf10 lack limbs and lungs, while endodermal expression of a dominant negative Fgf receptor (Fgfr2IIIb) causes mice to lack terminal buds in their lungs (Peters et al., 1994; Min et al., 1998; Sekine et al., 1999). Moreover, the addition of Fgf10 to 11.5d embryonic mouse lung rudiments in Matrigel causes extensive budding (Bellusci et al., 1997b). FGF10 is seen both in the mesenchyme around both the terminal and lateral branches.
The regulation of FGF10 appears to be controlled, at least in part, by Sonic hedgehog and BMP4 (Lebeche et al., 1999). Shh is expressed throughout the respiratory epithelium, with the highest expression being in the terminal buds (Bellusci et al., 1997a). In lung rudiments where Shh is overexpressed, Fgf10 transcription is reduced significantly (Bellusci et al., 1997b). In normal mouse lung development, the lateral buds become surrounded with Shh-expressing mesenchyme after they form (Figure 1).
One possible scenario is envisioned in Figure 1. (A) During bud outgrowth, Shh and Wnt7b from the epithelium induce FGF10 and cell proliferation of both the epithelium and mesenchyme cells. (B, C) As outgrowth progresses, the levels of BMP rise in the distal tip, and it reaches a level where it can inhibit FGF10. FGF10 expression is then seen more laterally, where it initiates the formation of new buds. (D) At the most distal region, a cleft appears, and extracellular matrix molecules stabilize this cleft.
The molecular bases of lung branching morphogenesis is also being mathematically modeled on computers. This new field has generated four dimensional structures that look very much like developmental lung branching patterns (Metger et al 2008; Miura 2008).
Extracellular matrix molecules may play several roles in lung branching, and they interact with the paracrine factors. First, they probably play a critical role in maintaining the epithelial integrity once branching has occurred. TGF-b proteins may be involved in stabilizing the extracellular matrix and in keeping the epithelial cells mitotically arrested. Extracellular matrices are strengthened along the non-budding areas (Serra et al., 1994). Conversely, in the areas where buds form, there is usually metalloproteinase activity and scatter factor activity (see Shiratori et al., 1995; Ohmichi et al., 1998). These proteins dissolve extracellular matrices and allow budding and cell proliferation to occur.
Second, extracellular matrix molecules, especially collagens, may be important in forming the cleft where the alveoli form. This hypothesis was originally proposed by Grobstein (1967) and has received substantial support from the work of Nakanishi and colleagues (1988).
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