Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis

Abstract

The development of many organs, including the lung, depends upon a process known as branching morphogenesis, in which a simple epithelial bud gives rise to a complex tree-like system of tubes specialized for the transport of gas or fluids. Previous studies on lung development have highlighted a role for fibroblast growth factors (FGFs), made by the mesodermal cells, in promoting the proliferation, budding, and chemotaxis of the epithelial endoderm. Here, by using a three-dimensional culture system, we provide evidence for a novel role for Netrins, best known as axonal guidance molecules, in modulating the morphogenetic response of lung endoderm to exogenous FGFs. This effect involves inhibition of localized changes in cell shape and phosphorylation of the intracellular mitogen-activated protein kinase(s) (ERK1/2, for extracellular signal-regulated kinase-1 and -2), elicited by exogenous FGFs. The temporal and spatial expression of netrin 1, netrin 4, and Unc5b genes and the localization of Netrin-4 protein in vivo suggest a model in which Netrins in the basal lamina locally modulate and fine-tune the outgrowth and shape of emergent epithelial buds.

Figure 1. Spatial and Temporal Expression of Genes Encoding Netrins and Their Receptors during Lung Branching Morphogenesis

(A)–(F), (H)–(J), and (M) are the results of whole mount and (G and N) radioactive section in situ hybridization. netrin 1 is expressed at E10.5 (A), continues at E11.5 (B and C), E12.5 (D and E), and E13.5 (F) but dramatically declines by E15.5 (G). Transcripts are present in the proximal epithelium and enriched in the stalks of buds but are excluded from the dilated distal tips (arrows in [B], [C], and [E]). (C) and (E) are enlargements of (B) and (D), respectively. Discrete regions of distal mesoderm in the right accessory and medial lobes also show netrin 1 expression (arrowheads in [B], [C], and [E]). (H) netrin 3 is expressed diffusely at low levels throughout the lung endoderm and mesoderm at E11.5. netrin 4 is expressed in proximal, but not distal, endoderm (arrows) at E11.5 (I) and E12.5 (J). (K and L) At E11.5, whole-mount immunohistochemistry localized DCC basally in the proximal endoderm (arrows) and apically in distal epithelium at the tips of buds (arrowhead in [L]). (K) and (L) correspond to the approximate positions of the pink and yellow squares in (B). (M and N) Unc5b transcripts are present in distal endoderm (arrows) and mesenchyme, but not in proximal endoderm (arrowhead) at E12.5 (M) and E13.5 (N). Inset in (M) shows that Unc5b is expressed in distal tip and neck region of endoderm. Scale bar = 100 µm.

Figure 2. Netrin-4 Is Expressed in the Basement Membrane of Proximal, but Not Distal, Lung Endoderm

Sections of E13.5 mouse lung were incubated with Netrin-4 and Perlecan primary antibodies. Digital images were then deconvolved. They demonstrate that Netrin-4 ([A], red) is colocalized to the basement membrane with Perlecan ([B], green) in the proximal region of the developing airway (yellow to orange in [C], overlay). In the distal bud regions (arrowheads), Netrin-4 protein is absent, while Perlecan is present. This is shown more clearly in the higher power images of the top right terminal bud in the bottom panels. Scale bar is 40 µm for upper panels and 20 µm for lower panels.

Figure 3. Morphological Response of Cultured Endodermal Epithelium to Exogenous FGF7 and Netrins

(A–F) DIC images of isolated endoderm after 40 hr culture in control Matrigel (A) or Matrigel containing 50 µg/ml full-length Netrin-4 (B), C-terminal truncated N4delC (C), Netrin-1 (D), Laminin (E), or Netrin-G1a (F). Note the presence of numerous secondary buds in (A), (E), and (F) but not in (B), (C), and (D). Arrow in (B) indicates a knob of cells inside the cyst. (G–R) Selected time-lapse images of endoderm cultured without (G–L) or with 50 µg/ml Netrin-4 (M–R). For the complete sequence see Movies in the Supplemental Data. Note the numerous secondary buds that form at the surface of control samples, while knobs of cells form inside of cyst in samples cultured with Netrin-4. As growth proceeds, the internal cells are incorporated into the surface epithelium (arrows in [Q] and [R]). (S–V) The morphological changes induced by Netrin-4 are reversible. After 40 hr culture in the presence of Netrin-4 (S and U), the endoderm is transferred to new Matrigel with FGF7, but without netrin. 24 hr later, numerous secondary buds formed in control gel (T), but not in gel containing Netrin-4 (V). All cultures are supplemented with 30 ng/ml FGF7. Scale bar = 100 µm.

Figure 4. Cell Proliferation, Unc5b Expression, and Cell Shape Change after Exogenous Netrin Treatment

(A–C) Cell proliferation in control (A) and Netrin-1-treated endoderm (B) or Netrin-4-treated (C) endoderm is estimated by double labeling with antibody to phospho-Histone H3 (red), and Alexa Fluor 488 phalloidin (green), which labels filamentous actin. (D–F) Expression of Unc5b in control (D) and Netrin-1-treated (E) or Netrin-4-treated (F) endoderm examined by whole-mount in situ hybridization. Inset in (D) shows elevated expression in secondary buds. (G–L) Cell shape visualized by double labeling with Alexa Fluor 488 phalloidin (green) and propidium iodide (red). (G–J) In control endoderm, epithelial cells in nonbudding areas are arranged in either a cuboidal ([H], corresponding to approximately the green square in [G]) or pseudostratified layer ([I], blue square in [G]). (J) In the secondary bud (pink square in [G]), cells have an elongated wedge shape and basally localized nuclei. (K and L) In Netrin-4-treated samples, cells in the internal knobs have an irregular shape (L). Scale bar = 100 µm for (A–G) and (K), 10 µm for other panels.

Figure 5. Distribution of PhosphoERK1/2 in Control and Netrin-Treated Endoderm and E11.5 Lung

ERK1/2 activity is assayed by whole mount immunohistochemistry by using antibody specific for phosphorylated forms of the protein. (A–E) endoderm cultured in Matrigel and 30 ng/ml FGF7 either without (A and D) or with 50 µg/ml Netrin-1 (B) or Netrin-4 (C and E). Note the elevated levels of phosphoERK1/2 in the secondary buds (A and D) but more uniform distribution in Netrin-treated samples (B, C, and E). The focal plane is through the lumen of the cysts in (C) and (I) and near the surface for (A, B, D–F, and H). Inset in (E) shows absence of phosphoERK1/2 in the internalized knobs in Netrin-4-treated samples. (F) Total ERK1/2 is distributed uniformly among budding and interbudding zone. (G) Western blot analysis shows that the relative level of phosphoERK1/2 is decreased in Netrin-4-treated samples. (H and I) In control samples cultured with 250 ng/ml FGF10, localized phosphoERK1/2 is restricted to the tips of elongated secondary buds (H), while Netrin-4-treated endoderm shows a uniform, low level of phosphoERK1/2 activity (I). (J–O) PhosphoERK1/2 activity in normal E11.5 lung. Note that highest ERK1/2 activity is localized to the distal tip of endodermal buds, and initiating buds shows higher phosphoERK than elongating buds (compare arrow and arrowhead in [K]). (L–O) When labeled together with TOTO-3 (blue, to visualize nuclei) and Alexa Fluor 488 phalloidin (green), a sharp boundary is seen between epithelial cells in the dilated distal tip of buds, which show high ERK1/2 activity (red), and the stalk region showing low levels. (M), (N), and (O) correspond to the blue, pink, and white squares in (L), respectively. Scale bar = 100 µm for (A)–(I), 20 µm for (J)–(L), and 10 µm for (M)–(O).

Figure 6. A Model for the Possible Roles Played by Netrins during Lung Epithelial Branching Morphogenesis

(A) Netrin-1 and -4 made by epithelial cells are deposited at the basement membrane and/or bind locally to epithelial cells at the neck region of elongating endoderm buds. Here they act through Unc5b or an unknown receptor to decrease the local ERK1/2 activity in the endoderm, thus facilitating the outgrowth of buds toward the source of FGF10 in the mesoderm, as well as preventing the generation of ectopic buds. According to this model, the basal lamina plus Netrins acts as a kind of sleeve or “corset,” restricting the morphogenesis of the emerging bud. (B) Our in vitro assays in which endoderm is surrounded by basal lamina components in the Matrigel mimic the effect of localized, ectopic Netrins at the distal tip, thus preventing bud outgrowth. In contrast, some cells accumulate at the inside of the lumen and form internal knobs.