Developmental programs of lung epithelial progenitors: a balanced progenitor model

Marker discovery using Eurexpress. Left panel: an example in situ hybridization image from Eurexpress (Cldn7, euxassay_002232) showing that major organs can be recognized at E14.5. Right panels: our screen to identify markers for the progenitors (top: Sox9, euxassay_018446; Lin7a, euxassay_011082; Lama3, euxassay_011044) and conducting airways (bottom: Sox2, euxassay_019525; Mycl1, euxassay_019486; Acsl1 (Acyl‐CoA synthetase long chain family member 1, euxassay_001151). The expression pattern is identified based on the presence (solid line) and absence (dashed line) of in situ signals in the branching (green) and nonbranching (red) regions.

A complete developmental history of lung epithelial progenitors. (a) OPT (top panels, scale: 250 µm) and confocal (bottom panels, scale: 20 µm) projection images of mouse lungs at indicated embryonic stages immunostained for E‐Cadherin and SOX9. The confocal images show the growing edges of the lungs, allowing comparison over developmental time. The E‐Cadherin staining is shown as a sectional view in grey scale. The branching program expands progenitors (filled arrowhead) and leaves behind differentiating descendants (open arrowhead) for the conducting airways (top panels) or the gas exchange region (bottom panels). Branches with a bud‐stalk structure form as late as E18.5 (dashed line in bottom panels), after which progenitors cluster around an expanded lumen [postnatal day (P)2, P7] and eventually are depleted (∼P14) and replaced by juxtaposed AT1 and AT2 cells tiling the gas exchange surface. Alveolar walls can form between (early) or within (late) branches. The accessory lobe grows from the right main bronchus to the left side (dashed line in top panels). SOX9 is also expressed in the cartilages (square bracket). (b) Two modes of branch formation: bifurcation via splitting an existing branch bud, and lateral branching via de novo bud outgrowth from an existing branch stalk. (c) Types of abnormal branching: overgrown with cystic branches and hypoplastic with or without cystic branches.

OPT imaging of foregut specification. (a) OPT images of an E9.5 mouse embryo immunostained for E‐Cadherin (a pan‐epithelium marker), SOX2 (foregut and future conducting airway marker), and SOX9 (lung progenitor marker) showing the formation of the lung (Lu), liver (Li), dorsal and ventral pancreas (dP and vP). The left (filled arrowhead) and right (open arrowhead) lung buds are viewed from different angles (0°, 45°, and 90°). Cross sections at four levels of the anterior foregut (I, II, III, and IV) show that SOX9‐expressing lung buds (cross section III) occupy a subregion of SOX2 low‐expressing foregut (cross sections II and III), which is presumably also NKX2.1 positive. Scale: 100 µm. (b) Schematics showing the specification of the trachea and lung from the ventral anterior foregut between E9.5 and E10.5. The side view is drawn based on (a). The contribution of SOX9 expressing cells at E9.5 to the trachea and lung is unknown (question mark). A cross section of the trachea region at E9.5 and a longitudinal section of the lung bud at E10.5 are shown in the lower panel to illustrate the similarity with respect to epithelial gene expression and mesenchymal signals.

Balanced progenitor model in the mouse lung. (a) Lung epithelial progenitors (SOX9, green nucleus) balance among three developmental programs: branching (green arrow), which expands progenitors and builds a tree‐like structure; airway differentiation (red arrow), which activates SOX2 (red nucleus) and then forms specialized airway cells (e.g., club and ciliated cells); and alveolar differentiation (blue arrow), which forms AT1 and AT2 cells and the former needs to fold extensively. (b) This balance shifts over time: early in development, progenitors branch/expand and undergo airway differentiation; late in development, progenitors continue to branch/expand and undergo alveolar differentiation; in perinatal periods, progenitors stop branching, are depleted (dashed green arrow) via alveolar differentiation (thicker blue arrow) and transition from development to homeostasis. (c) Regulated deployment of the three developmental programs following one branch. The left side is color‐coded for the progenitors (green line) and their early (red line, conducting airways) versus late (blue line, gas exchange region; wavy line, deformation of tubular structures by air space expansion, see also Figures (a) and ) descendants, and the developmental programs (arrows, see (b) for details). The right side shows the morphology and is labeled for the three generations of conducting airways. (d) Three possible outcomes of a cystic branch resulting from an early branching defect. The left side is colored coded as in (c). Depending on whether a cystic branch bud becomes a normal‐looking branch stalk (corrected) and whether it undergoes alveolar or airway differentiation, the location of the cyst and ratio between the gas exchange and airway compartments differ. Therefore caution should be taken in interpreting phenotypes beyond early branching.

A model for acinus morphogenesis in the mouse lung. Branching continues beyond the terminal bronchiole to generate tubes that will become the alveolar ducts (duct). Eventually, there is insufficient number of progenitors to support further branching and the remaining branch buds expand to form alveolar sacs (sac), which is subsequently divided into alveoli ‘a’ via secondary septation. Air space expansion is accompanied and possibly driven by AT1 cell flattening. Primary and secondary septa may correspond to early and late forming alveolar walls (Figure (a)). Side top panel: overlay of the left diagrams showing transformation of a tube to a clefted bubble. Note that cleft formation may involve inward growth and/or expansion of neighboring epithelia. Side bottom panel: the alveolar ducts are surrounded by alveolar sacs/alveoli possibly derived from short tubular branches perpendicular to the ducts and therefore may appear to have outpouchings on sections.

Sox2 promoter analysis. (a) Top: a putative Sox2 promoter contains a positive regulatory element (binding site) for the airway differentiation program and two negative regulatory elements for branching and alveolar differentiation. For simplicity, an alternative scenario is not illustrated but does not affect the discussion: each developmental program may not have its own element, but instead interfere with alternative elements. For example, the branching program may compete with and block the positive element for airway differentiation, therefore does not require its own regulatory element. Bottom: predicted Sox2 expression patterns in early and late development when each element is individually disrupted. See Figure (c) for color scheme. Without the airway differentiation element, Sox2 expression is lost and Sox2‐independent mechanisms may prevent expansion of the alternative programs (dashed line). Without the branching element, Sox2 expression expands into the branching region and such expansion inhibits branching and likely interferes with subsequent alveolar differentiation. Without the alveolar differentiation element, Sox2 expression is normal in early development but continues to expand in late development such that all progenitors' descendants express Sox2. (b) Stereomicroscope images of an E14.5 Sox2‐BAC (bacterial artificial chromosome, RP23‐4D1) transgenic embryo expressing a membrane Tomato fluorescent protein and its littermate control (Chen J. and Krasnow M.A.). The promoter is active in the neural tube but not the lung, suggesting that the airway differentiation element (a) is at a distant site from the coding region.

Branching versus alveolar differentiation in evolution. (a) Top: OPT images of E11.5 control and epithelial Sox9 mutant mouse lungs and a stage 42 Xenopus embryo immunostained for SOX9. Bottom: Surfactant protein B (Sftpb) whole‐mount in situ hybridization of E10.5 control and epithelial Sox9 mutant mouse lungs and a stage 42 Xenopus embryo. When Sox9 is absence in the lung buds (dashed line) in the mouse mutant and Xenopus, Sftpb is expressed. (Reprinted with permission from Ref . Copyright 2013 the National Academy of Sciences) (b) The branching program may be an evolutionary addition that temporarily delays alveolar differentiation to allow an increase in lung complexity by expanding the progenitors and building the conducting airways. Genes promoting branching (e.g., Sox9) have a second function to suppress (blunt arrow) the ancestral program that starts alveolar differentiation immediately after lung specification (gray dashed line in Xenopus).