Preparing for the first breath: genetic and cellular mechanisms in lung development

Abstract

The mammalian respiratory system--the trachea and the lungs--arises from the anterior foregut through a sequence of morphogenetic events involving reciprocal endodermal-mesodermal interactions. The lung itself consists of two highly branched, tree-like systems--the airways and the vasculature--that develop in a coordinated way from the primary bud stage to the generation of millions of alveolar gas exchange units. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as branching morphogenesis, vascular development, and the differentiation of multipotent progenitor populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding respiratory disorders, congenital defects in human neonates, and how the disruption of morphogenetic programs early in lung development can lead to deficiencies that persist throughout life.

Figure 1. Early Patterning and Morphogenesis of the Anterior Foregut

For references other than those cited, see Cardoso and Lü (2006). (A) The anterior foregut begins as a single epithelial tube surrounded by mesoderm. Early specification of the future trachea and lungs (here marked by Nkx2-1, blue) and other endodermal organs is regulated by the dose and timing of Wnt and FGF signals from the mesoderm (Goss et al., 2009; Serls et al., 2005). Shh in the endoderm regulates Bmp4 in the mesoderm in part through the transcription factors Foxf1 and Gli1 and Gli3. The localized expression of Fgf10, which drives lung bud outgrowth, requires retinoic acid (RA) signaling and involves inhibition of Tgfβ (Chen et al., 2007). In the chick, Fgf10 is downstream of Tbx4. Embryos lacking Hoxa5 have defects in tracheal cartilage development, but roles for other Hox genes have not been determined. (B) Schematic section through undivided foregut at the level of the dotted box in (A) showing the highest Sox2 levels dorsally, and Nkx2-1, ventrally. The action of Bmp4 (and earlier, Bmp7) is antagonized by noggin (Li et al., 2008b; Que et al., 2006). Asterisks mark the site of separation of foregut into two tubes. Deficiencies in Shh, Gli1/Gli3, RARs, Fgfr2, Fgf10, Foxf1, Sox2, Nkx2-1, Bmp4, noggin, and Wnts lead to defects in foregut development including tracheoesophageal fistula and esophageal atresia (EA/TEF). (C) Around E9.5, the primary lung buds appear with the right bud larger than the left. This asymmetry is downstream of the genetically regulated L/R patterning of the whole-body axis. Separation of the foregut tube into trachea and esophagus begins just anterior of the lung buds (asterisk). The domain of separation extends over time. There is also extensive elongation of the two tubes (green arrows) as well as outgrowth of primary lung buds. (D) Whole-mount in situ hybridization for Shh showing expression in the endoderm with highest levels in distal tips. Domain branching gives rise to the primordial buds of the right cranial (Cr), medial (Md), caudal (Cd), and accessory (Ac) lobes.

Figure 2. Schematized Depiction of Distal Buds at the Pseudoglandular Stage

(A) Fgf10 in distal mesoderm acts on the epithelium through the Fgfr2IIIb isoform to promote cell proliferation and bud morphogenesis, possibly through different downstream pathways. Signaling through Tgfβr2 in the mesoderm inhibits Fgf10 expression (Li et al., 2008a). RA expressed in a gradient in the mesoderm, with highest levels proximally, also inhibits Fgf10. Fgf9 made in the mesothelium and epithelium promotes the proliferation of Fgf10-express-ing mesenchymal cells that express Fgfr2IIIc, and inhibits the expression of Bmp4 and noggin in response to Shh. (B) Epithelial Shh promotes proliferation and expression of Bmp4 in the mesenchyme, as well as inhibits Fgf10 expression and upregulates hedgehog-interacting inhibitory protein (Hhip). The phenotype of Shh null lungs is partially rescued by deleting Gli3, suggesting that the repressor form of Gli3 (Gli3R) functions in the mesenchyme to inhibit proliferation and to regulate Foxf1. (C) Absence of Wnt7b results in lungs that are small but almost normally proportioned. Wnt7b regulates the expression of Bmp4 and Idb2 in the epithelium through independent pathways and promotes proliferation of the mesoderm through Axin2 and Lef1 (Rajagopal et al., 2008; Shu et al., 2002). Loss of Wnt2 results in significant lung hypoplasia due to decreased proliferation in both epithelial and mesenchymal compartments (Goss et al., 2009). Wnt5a is expressed in both endoderm and mesoderm. (D) Bmp4 is transcribed in distal epithelium and in the mesoderm with highest levels around bud stalks. Bmp5 is expressed in mesoderm. Bmpr1a is expressed in both the epithelium and mesenchyme, while Bmpr1b is restricted to the proximal epithelium. The level of Bmp4^lacZ in the epithelium increases as the bud elongates, suggesting a role in limiting this process (Weaver et al., 2000). Moreover, addition of Bmp4 to buds in culture inhibits their directed outgrowth toward a source of Fgf10 (Weaver et al., 2000). Conversely, deletion of Bmpr1a in the epithelium reduces proliferation in vivo and inhibits the ability of epithelium to bud in response to Fgf in vitro. The precise response of cells to Bmps likely depends on the level of ligand, the activity of other signaling pathways, and cell type. Bmps likely play additional roles in later lung development and postnatally (Alejandre-Alcázar et al., 2007).

Figure 3. Bud Elongation and Bifurcation

(A) Movement of isolated lung endoderm in Matrigel from an E11.5 lung toward a bead soaked in recombinant Fgf10. Images are from a continuous series over 36 hr. To date there have been no detailed studies on dynamic cell rearrangements in these buds as with ureter bud (Costantini, 2006) and mammary gland epithelium (Ewald et al., 2008). (B) Schematic representation of the spatial expression in the distal lung of some semaphorins, plexins, and neuropilins first described as “axonal guidance” factors. Based on in vitro studies, netrin 4 may help to restrict expansion of the stalk and lateral budding while sema3a may help to restrict outgrowth of the bud (Hinck, 2004). (B–D) Schematic representation of (B) bud elongation, (C) cessation of outgrowth, and (D) bifurcation. The basal lamina (dashed line) surrounds the epithelial cells in the stalk and extends into the tip but is less dense in this region, meaning that the basal surface of the tip epithelium can be in contact with mesenchyme (Bluemink et al., 1976). There is also evidence for differential sulfation of the matrix associated with budding (Izvolsky et al., 2003). As the bud elongates (arrow in A) toward Fgf10 in the distal mesenchyme (red), expression of the Bmp4^lacZ knockin reporter (blue) increases in the tip (blue) (Weaver et al., 2000). PhosphERK1/2 expression is confined to cells at the tip (Liu et al., 2004). There is deposition of new matrix adjacent to the epithelium in the cleft formed by the bifurcation process; this may be promoted by Tgfβ signaling (Goss et al., 2009).

Figure 4. Schematic of Alveolarization

(A) Alveolar development begins in late gestation as the endothelial plexus becomes tightly associated with the distal epithelial saccules. Bottom panel shows terminal bronchioles with saccules attached while top panel is a higher magnification after the saccules have been removed to reveal the lumen. (B) The epithelium of the developing saccules/primary septae differentiates into several important cell types found in the mature alveolus. These include AEC1 cells, which are very flat and thin walled and when mature characteristically express aquaporin 5 and T1alpha (podoplanin), and the much larger, cuboidal AEC2 cells. These cell types are closely juxtaposed to each other and AEC1 cells form intimate interactions with the underlying vascular endothelium. (C) Mature AEC2 cells secrete abundant surfactant proteins and lipids that are trafficked to the cell surface in organelles called lamellar bodies marked by the ACBC3 transporter protein. Mature AEC2 cells express high levels of the gene encoding surfactant protein C (Sftpc). Maturation of the alveolar compartment is accompanied by generation of secondary septae, which involves growth of alveolar crests. Crest formation requires elastin deposition and Pdgfa for myofibroblast development (Boström et al., 1996). (D) Hand Estained histological sections demonstrating the change in distal lung morphology from the canalicular/saccular stages at approximately E17.5 through the adult where mature alveoli are found with secondary septae (arrows).

Figure 5. Schematic Representation of Mesoderm Development at the Pseudoglandular Stage

Undifferentiated multipotential progenitors in the distal tip express Twist2, Foxf1, and Tbx4. As shown above the dashed line, they include precursors of bronchiolar smooth muscle (Pdgfra+) that translocate proximally and associate with the endoderm. As shown below the dashed line, other progenitors give rise to pericytes (Pdgfrb+) that migrate around blood vessels. Some pericytes and interstitial cells may arise from the mesothelium (yellow). Blood vessels (BV) likely arise by sprouting angiogenesis and remodeling of the vascular plexus (VP) surrounding the distal tip that is in continuity with the embryonic circulation. There is some evidence for distal vasculogenesis (see text). The growth and differentiation of BVs and smooth muscle is regulated by signals from the endoderm (Shh, Wnt7b, Vegf, and Fgf9) and mesothelium (Fgf9). It has been proposed that early mesoderm is subdivided into submesothelial and subepithelial domains that differ in their response to signaling factors (White et al., 2007).

Figure 6. Schematic of Epithelial Cell Lineages in the Lung

(A) At the pseudoglandular stage (E11–16.5), progenitor cells (P1, red) within the distal tip epithelium both self-renew and generate descendents (green) that exit the tip and populate the conducting airways. At least up to E13.5 the P1 cells are able to generate all of the different airway epithelial cell types, strongly suggesting multipotency. The pool is maintained by multiple factors (Figure 2 and text). P1 cells persist to the canalicular stage when they generate cells (P2, orange) that populate the future alveoli. It is not clear if this switch from the production of bronchiolar to alveolar descendents is intrinsic or imposed by extrinsic factors. However, there is evidence that Wnt and Notch signaling regulate this change in behavior. (B) Putative airway progenitors (green) express Sox2. One model for their differentiation is that the first bronchiolar lineage decision is mediated by Notch signaling via Hes1 and is between an NE (blue) and putative non-NE fate (light green). Mash1 expression is required for NE cell commitment and differentiation. The non-NE cells subsequently undergo a second Notch-mediated decision to commit to the secretory cell (yellow, Scgb1a1^hi) or ciliated cell (purple, FoxJ1⁺, β-tubulin IV⁺) lineages. However, other models are equally possible, including all three lineages sorting out at once. There is good lineage tracing evidence that once Scgb1a1+ cells are formed, they are able, as a population, to self-renew over the long term and to give rise to ciliated cells in the postnatal lung (Rawlins et al., 2009b). Mucus-producing cells can be derived from Clara cells directly (Chen et al., 2009), but other origins are possible. (C) Similarly to the bronchioles, there is no direct evidence that a common alveolar epithelial progenitor exists. However, the evidence is consistent either with an alveolar progenitor giving rise to AEC1 (Aqp5⁺, T1alpha⁺) and AEC2 (Sftpc⁺) cells, or AEC2 cells themselves generating AEC1. It is not clear what genetic mechanisms regulate these decisions.

Figure 7. Schematic of Epithelial Cell Lineages in the Trachea

In the embryonic trachea all the progenitors express Sox2, p63, and Nkx2-1. They give rise to basal cells, Scgb1a1+ secretory cells, ciliated cells, and NE cells, but the precise mechanism is not known. Postnatally, at least some basal cells function as long term self-renewing progenitors, although there is likely to be heterogeneity. Lineage-labeled Scgb1a1+ cells can divide and give rise to ciliated cells but they do not self-renew over the long term. Basal cells can give rise to Scgb1a1+ and ciliated cells but the precise mechanism is not yet known.