Hogan Lab Projects

Overview: Molecular mechanisms underlying organ development: Mesenchymal-epithelial interactions and the branching morphogenesis of the lung

The embryonic mouse lung is a powerful model system for exploring basic principles of organogenesis. The early lung can be dissected easily from the foregut and cultured on a filter. In addition, the epithelium of the early buds can be separated from the surrounding mesenchyme and both cell populations cultured independently or in combination on a filter or in a three-dimensional gel of extracellular matrix. We can also exploit the power of mouse genetics to make conditional mutants and transgenic lines to test gene function in vivo. Genetic manipulations also allow us to make reporter mouse strains in which specific cell types are labeled with an inherited visual marker. This allows us to trace cell lineages in vivo, to sort and isolate cells using flow cytometry, and to follow the movement of cells using confocal microscopy and real time imaging.

Classical tissue recombination experiments carried out in the 1960s provided the first evidence for reciprocal inductive interactions between the epithelium and the mesoderm. It is now known that these interactions are mediated by the local production of signaling factors belonging to the Bmp/Tgfß, Wnt, Fgf, Egf and Hh families, as well as their antagonists and agonists. In particular, the tips of the growing buds act as signaling centers - integrating signals from the mesoderm, and, in turn, producing factors that influence the mesoderm (Hogan et al 1997; Bellusci et al 1996 and 1997a and b; Weaver et al 1999, 2000, and 2003). The endoderm in the distal tips has a high rate of cell proliferation and, as shown by recent lineage labeling studies by postdoctoral fellow, Emma Rawlins, is a source of multipotent progenitor cells for populating the epithelium of the entire respiratory tree. We are beginning to understand how this progenitor population is maintained and how the daughter cells become diversified into different specialized lineages (Okubo et al 2005; Eblaghie et al 2006). Postdoctoral fellow, Yun Lu, is also interested in the potential role of microRNAs in controlling progenitor cell proliferation, survival and differentiation (Lu et al 2007).

Roles for FGF signaling and extracellular guidance molecules in branching morphogenesis

Previous work from our lab and others showed that the outgrowth and branching of epithelium of the embryonic lung is controlled by FGF10, specifically localized in the distal mesenchyme, acting through FGFreceptor 2 IIIb in the epithelium. If the endoderm from an early lung bud is separated from the surrounding mesoderm and placed in Matrigel containing FGF, it quickly seals into a vesicle with the apical surfaces of the cells facing the lumen. The cells continue to proliferate and after about 10 hours numerous buds begin to appear on the surface of the vesicle (Figure 2). Over the next 24 hours these buds elongate and enlarge. If the medium contains FGF10, there are fewer buds but they grow out more than twice as far as in FGF7. Despite the simplicity of the endoderm morphogenesis in this in vitro culture system, compared with the normal embryonic lung, we know relatively little about the molecular and cellular mechanisms underlying the changes in cell shape and epithelial cell-cell interactions that initiate bud formation and drive outgrowth (Liu et al 2003; Liu et al. 2004; Eblaghie et al 2006). This problem is currently being addressed using time-lapse fluorescence and DIC microscopy, with wild type and mutant endoderm under different conditions, in collaboration with Tim Oliver, Director of the Cell Biology Imaging Facility. We are also interested in the role of signaling pathways and changes in cell behavior in the separation of the foregut into trachea and esophagus (Que et al 2007).

Role of Bmp signaling pathways in the foregut and developing lung

We and others have shown that several Bmp genes are expressed in the developing lung, either in the endoderm, mesoderm, or both. We have generated transgenic lungs in which either the gene encoding Bmp4 or its antagonist, noggin, is overexpressed in the distal endoderm (Bellusci et al. 1996; Weaver et al 1999). We have also studied the effect of these proteins on lung endoderm in vitro (Weaver et al 2000). Based on these experiments we have formulated several models for the role of Bmps in lung development (Figure 3). For example, we propose that low levels of Bmp4 in conjunction with Fgf signaling promote the proliferation and maintenance of undifferentiated distal epithelial cells, and inhibit the differentiation of proximal cell types. Higher levels of Bmp4 slow proliferation and prevent lateral bud outgrowth towards Fgf. To test these models more specifically we have generated a line of transgenic mice in which Cre recombinase is expressed in the distal endoderm from E10, using the 3.7Kb human surfactant protein C (SpC) promoter. These mice were used to inactivate Bmpreceptor1a and Bmp4 specifically in the endoderm of the developing lung (Eblaghie et al 2006). The results supported the idea that Bmps function through an auto-crine signaling pathway to maintain the proliferation and survival of the distal progenitor cells.

Figure 3 - Possible functions for Bmps in the endoderm and mesoderm of the developing mouse lung

Postdoctoral fellow, Jainwen Que, is using a variety of cellular and genetic approaches to explore precisely how this is achieved. He has also studied the role of BMPs and the BMP antagonist, noggin, in the early morphogenesis of the foregut and its separation into trachea and esophagus. He showed that about 70% of noggin null mutants have tracheal-esophageal fistula and esophogeal atresia (EA/TEF) (Que et al 2006). This finding is important because defects in the separation of the trachea and esophagus are quite common birth defects in humans.

Multipotent progenitor cells in the embryonic lung endoderm and the generation of cell diversity

As described in Figure 1, the mature lung contains several different specialized epithelial cell types - ciliated, secretory, and neuroendocrine cells in the proximal bronchi and bronchioles, and type II and type I cells in the alveoli. In the trachea there is also a population of basal cells that may represent one kind of multipotent progenitor or stem cell in the adult lung. Surprisingly little is known about how these different cell types are generated from a population of apparently uniform, undifferentiated progenitor cells. Studies in other labs have shown that Mash1 and Hes1 play important roles in the establishment of the neuroendocrine cell lineage but the function of other genes encoding bHLH and other transcription factors in the embryonic lung has not been determined. Emma Rawlins, is studying this problem, using a variety of techniques, including conditional gene targeting. She is particularly interested in the role of Id genes that encode bHLH proteins family members that lacks the DNA binding domain. Figure 4 shows one of her in situ hybridization results revealing the specific localization of Id2 transcripts to the very distal tips of lung tubules in the E11.5 and 15.5 lung.

The role of stem and progenitor cells in the response of the adult lung to injury and disease

The Hogan lab is also studying the mechanisms used in the adult lung to rapidly repair injury to epithelial cells, either in the proximal tracheal or in the distal bronchi, bronchioles and alveoli (Rawlins and Hogan, 2006). One of the major problems with studying repair is to identify the specific lung cell types that proliferate after injury and the fate of the cells that are generated. To address this problem we have generated several new lines of mice for lineage tracing and for changing the expression of potential regulatory genes in specific cell types. The Foxj1-GFP transgenic line has been used to follow the appearance of ciliated cells during development (Fig 5) while the Foxj1-CreER mouse line allows us to follow the fate of ciliated cells after injury by naphthalene and sulfur dioxide (Rawlins et al 2007). We have also made an Scgb1a1 (CC10) "knock-in" inducible Cre line for following the fate of descendants of Clara cells. As shown in Figure 5 this line shows specific expression in Clara cells throughout the lung (and trachea) after injecting Tmx into an adult Scgb1a1-CreER;Rosa26R compound mutant reporter mouse. In addition, we have recently made a K5-CreEr transgenic lines for following the fate of basal stem cells in the trachea after injury. We are particularly interested in these stem cells because the human lung, unlike the mouse lung, has basal cells right down to the peripheral bronchioles.

Discoveries and ongoing projects in the Hogan laboratory