The development of organs such as the lung, pancreas, and intestine proceeds through distinct stages, each coordinated by sets of conserved intercellular signaling pathways. Initially, an organ primordium is established within a larger embryonic field. This is followed by the proliferation of progenitor cells, their diversification into different lineages, cell differentiation, and the sequestration of organ-specific stem cells in distinct niches. In the adult, these stem cells give rise to new progenitors that normally differentiate along the same tissue-specific lineage pathways. Occasionally, however, a process known as metaplasia can occur, usually in response to local inflammation or injury. Under these conditions, cell types specific for a different organ arise in situ. A well-known example in humans is Barrett's esophagus, in which epithelial cell types characteristic of the small intestine differentiate ectopically in the lower esophagus [1, 2]. Despite the medical relevance of this and other metaplastic conditions, little is known about the underlying mechanisms and whether they involve changes in the lineage specification of progenitors and/or stem cells, a process known as transdetermination. Insight is likely to come from greater knowledge of the pathways regulating normal lineage commitment and differentiation in embryonic epithelia, including the esophagus, intestine, pancreas, and lung, all of which are derived from foregut endoderm [3, 4].
One intercellular signaling pathway that is involved at multiple stages in organ development in both vertebrates and invertebrates is the canonical Wnt signaling pathway. Initiated by the interaction between extracellular Wnt ligands and their receptors, this pathway culminates in the stabilization of β-catenin, which then interacts with nuclear T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors to modulate the activity of target genes . In Drosophila development, depending on the cellular context, the Wnt homolog Wingless (Wg) can regulate cell proliferation, embryonic patterning, and/or differentiation. Of particular relevance to the findings of this article, Wg can drive transdetermination of third instar larval imaginal disc cells (reviewed in ). For example, ectopic expression of Wg in leg imaginal discs induces, in a subset of proliferating cells that co-express other signaling pathway components and competency factors, the expression of selector genes specific for wing imaginal disc progenitors. The descendants of these cells subsequently differentiate into wing cell types.
Studies in the vertebrate embryo have identified multiple roles for components of the canonical Wnt pathway in organ development. For example, in the small intestine, Tcf4 is required for the rapid proliferation of the embryonic intervillus epithelium that gives rise to the crypts . These contain the stem cells of the adult intestine, which generate the progenitors of the major epithelial cell types. Lineage choice among these progenitors is thought to involve signaling via the Notch/Delta pathway and the expression of so-called neurogenic basic helix-loop-helix (bHLH) genes. Cells transcribing high levels of Notch and Hes1 give rise to enterocytes, while descendants of cells that express high levels of Delta and the bHLH gene Atoh1 (Math1) keep their options open and undergo further rounds of lineage restriction to generate secretory cell lineages (Paneth, goblet, and neuroendocrine cells) . Blocking Wnt signaling in the intestine inhibits both cell proliferation and the generation of secretory cells [7, 9]. This abnormal phenotype is accompanied by the down-regulation of Atoh1 (Math1), consistent with the phenotype of Atoh1-null mice, which also lack all secretory cell lineages in the intestine .
Much less is known about either Wnt signaling or lineage diversification in the embryonic lung. This organ arises in the ventral wall of the foregut tube between the thymus and the stomach. The trachea and primary bronchi develop by separation from the future esophagus, while the remaining respiratory tree develops from two small ventrolateral buds (for reviews see [10, 11]). These buds proliferate rapidly and undergo reiterative branching to generate an arborization of epithelial tubes of decreasing diameter. The epithelium in the larger, more proximal tubes differentiates into several specialized cell types (ciliated cells, the various subsets of secretory Clara cells, and the pulmonary neuroendocrine cells). The epithelium of the smaller, peripheral tubes that appear towards the end of gestation gives rise to the distal alveolar cell types - the type I and type II cells. Genetic studies have shed some light on mechanisms underlying lung lineage diversification. For example, as in the intestine, the bHLH gene Ascl1 (Mash1) is required for the development of lung neuroendocrine cells, while Hes1 apparently promotes non-neuroendocrine lineages [12, 13]. However, Atoh1 (Math1) is not expressed during lung development ( and our unpublished observations) and it is not known what regulates the generation of ciliated, Clara and mucus-producing cells.
With respect to the Wnt signaling system, a number of Wnt ligands and receptors are expressed dynamically during lung development . For example, Wnt7b is transcribed in the distal endoderm during branching morphogenesis, while Wnt2 is expressed in the adjacent mesoderm ( and our unpublished observations). Transcription factors of the TCF/LEF family are also expressed in the developing lung, both in the endoderm and mesoderm . Although the submucosal glands that arise from epithelial cells in the trachea and main bronchi are absent from Lef1
-/- mice, the respiratory portion of the lung develops normally, suggesting that other factors can compensate for the absence of Lef1 . Recently, an inducible transgenic system was used to delete β-catenin in the epithelium at different times during lung development . Although the β-catenin protein persisted for some time, its eventual depletion resulted in a dramatic down-regulation of the number of differentiated distal alveolar epithelial cells in the lung before birth, and an increase in the relative proportion of proximal ciliated and Clara cells.
The experiments described here were initially designed to explore Wnt function in lung development using the complementary approach of pathway overexpression. We used the same lung epithelial cell-specific promoter as Mucenski et al. ; it is active from the time the primary buds first appear. We employed an activated β-catenin-Lef1 fusion protein that had previously been used to rescue embryonic expression in Wnt3a-null mouse mutants . We found that transgenic lungs looked grossly normal but contained rapidly proliferating epithelium and a relative paucity of fully differentiated pulmonary cell types. Unexpectedly, use of Affymetrix array analysis to study gene expression revealed very high levels of expression of multiple genes normally characteristic of intestinal epithelial secretory cell types (small intestine, duodenum, and stomach). This finding was confirmed by in situ hybridization. In addition, transgenic epithelium ectopically expressed genes such as Cdx1, which regulates gut development, and Atoh1, which is required for the determination of the secretory lineage in the intestine. These results provide strong evidence that the developmental fate of early lung progenitor cells can be switched in vivo to that of gut/intestine by elevated and/or prolonged Wnt signaling. We discuss this finding in the light of previous examples of transdetermination in response to abnormal Wnt signaling and its relevance to the pathological condition of intestinal metaplasia in humans.