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. 2009 Aug;238(8):1999-2013.
doi: 10.1002/dvdy.22032.

Conditional Gene Inactivation Reveals Roles for Fgf10 and Fgfr2 in Establishing a Normal Pattern of Epithelial Branching in the Mouse Lung

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Free PMC article

Conditional Gene Inactivation Reveals Roles for Fgf10 and Fgfr2 in Establishing a Normal Pattern of Epithelial Branching in the Mouse Lung

Lisa L Abler et al. Dev Dyn. .
Free PMC article

Abstract

Fibroblast growth factor 10 (FGF10) signaling through FGF receptor 2 (FGFR2) is required for lung initiation. While studies indicate that Fgf10 and Fgfr2 are also important at later stages of lung development, their roles in early branching events remain unclear. We addressed this question through conditional inactivation of both genes in mouse subsequent to lung initiation. Inactivation of Fgf10 in lung mesenchyme resulted in smaller lobes with a reduced number of branches. Inactivation of Fgfr2 in lung epithelium resulted in disruption of lobes and small epithelial outgrowths that arose arbitrarily along the main bronchi. In both mutants, there was an increase in cell death. Also, the expression patterns of key signaling molecules implicated in branching morphogenesis were altered and a proximal lung marker was expanded distally. Our results indicate that both Fgf10 and Fgfr2 are required for a normal branching program and for proper proximal-distal patterning of the lung.

Figures

Figure 1
Figure 1
Dermo1cre/+ activity and Fgf10 inactivation. (A,B) Cre activity of the Dermo1cre/+ allele as assayed by β–gal staining of Dermo1- cre;R26R embryos at stages indicated. Dotted line in A frames caudal periphery of lung. Dotted lines in B outline each of the lobes: L, left; Ro, rostral; Me, medial; Ac, accessory and Ca, caudal. (C) Amount of intact Fgf10 transcript remaining per lobe of Dermo1-cre;Fgf10 mutant lung as determined by qRT-PCR. Full length (exon 2-containing) Fgf10 transcript was undetectable in the mutant rostral lobe. (D-G) FGF signaling as assayed by RNA in situ hybridization using a Spry2 probe as a readout of FGF signaling at stages indicated. Arrowheads indicate comparable regions of the lung to highlight differences in Spry2 expression. Arrows indicate normal position of the rostral lobe (solid arrow) to illustrate its absence in the mutant (open arrow). Ventral views of lungs shown in all figures unless otherwise indicated.
Figure 2
Figure 2
Sftpc-cre activity and Fgfr2 inactivation. (A-D) Cre activity of the Sftpc-cre transgene as assayed by β–gal staining of Sftpc-cre;R26R embryos at stages indicated. B is a lateral view of the right side of the lung shown in A. Dotted lines in A, B frame caudal periphery of lung. D is a magnified view of the bracketed region indicated in panel C. Uneven β-gal signal indicates variations in the efficiency of recombination. (E-I) An RNA in situ hybridization probe targeted to floxed exons of Fgfr2 (Fgfr2 exon8,10) was used to detect intact receptor in lungs of indicated genotypes and stages. (J-N) FGF signaling as assayed using a Spry2 in situ probe. Arrowheads in J,K indicate comparable regions of the lung to illustrate differences in Spry2 expression. I, N are magnified views of bracketed regions in H, M, respectively. Arrowheads indicate regions of residual Fgfr2 exon8,10 (I) or Spry2 (N) expression. Esophagus is included in this figure and others as an internal standard for equivalence of staining between control and mutant samples.
Figure 3
Figure 3
Morphology of Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutant lungs. (A-E) Epithelial branching pattern of Dermo1-cre;Fgf10 lungs. (A-D) An Fgfr2 TK probe was used to outline the epithelium in lungs of indicated genotypes and stages. Solid arrows in A, C and dotted line in C indicate normal position of the rostral lobe and open arrows in B and D indicate absence of the rostral lobe in the mutant. (E) Samples shown in whole-mount at E17.5. (F-L) Epithelial branching pattern of Sftpc-cre;Fgfr2 lungs as outlined by Fgfr2 TK in situ at stages indicated. Arrow in K indicates a dilated sac. Bracketed region in K is magnified in inset; arrowheads indicate small nodules. L is a magnified view of the right lung shown in K; arrow indicates outgrowth of lobe mesenchyme without outgrowth of epithelium.
Figure 4
Figure 4
Cell death analysis of Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants. (A-H) LysoTracker staining of apoptotic cells in lungs of indicated genotypes and stages. For all samples, dotted lines outline lobes of lungs and arrows indicate caudal extent of cell death in epithelium of primary bronchi. Arrowheads in C, D indicate cell death in epithelium of medial and accessory lobes. Asterisks in D, H indicate aberrant mesenchymal death. (I-M) Immunofluorescent staining on paraffin sections of lungs of indicated genotypes and stages. Cleaved Caspase 3 antibody staining labels dying cells within the trachea (I, K, M). E-Cadherin antibody staining on adjacent serial sections outlines the tracheal epithelium (J, L, N). Arrows in I, K, M indicate cell death within the tracheal epithelium.
Figure 5
Figure 5
Shh and Ptch1 expression in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutant lungs. (A-P) Gene expression in lungs of indicated genotypes and stages. Arrowheads in A,B indicate comparable regions of the lung to illustrate differences in Shh expression. Bracketed regions in C-D and K-L are magnified in insets. Shh is not upregulated in the distal epithelium of the Dermo1-cre;Fgf10 mutant but remains upregulated in the Sftpc-cre;Fgfr2 mutant, albeit in smaller domains. Dotted line in D and L indicates outgrowth of lobe mesenchyme without outgrowth of epithelium. (Q-T) Gene expression in wild type lungs cultured with protein-soaked beads (dashed circles). Arrows in R, T indicate domains of upregulated expression in tissue immediately adjacent to the FGF10-soaked beads.
Figure 6
Figure 6
Bmp4 and Fgf10 expression in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants. (A-T) Gene expression in lungs of indicated genotypes and stages. Arrow in H indicates upregulated Fgf10 expression in the residual rostral lobe mesenchyme of the mutant. M and N are magnified views of bracketed regions in L; arrowheads indicate isolated domains of Bmp4 upregulation. S is a frontal section of the sample shown in R; T is a transverse section of a mutant littermate.
Figure 7
Figure 7
Expression of P-D lung patterning markers in Dermo1-cre;Fgf10 and Sftpc-cre;Fgfr2 mutants. (A-R) Expression of P-D markers in lungs of indicated genotypes and stages. Arrowhead in A indicates Sox9 expression in the proximal mesenchyme where cartilaginous tracheal rings will form. M,R are magnified views of bracketed regions indicated in L,Q, respectively. Arrows in G,H and P-R indicate caudal limit of Sox2 expression. Asterisks in G,H and P,Q serve as reference points indicating the position where the main bronchi split. Sox2 is expressed in a longer region of the main bronchi (distance between asterisk and arrow) in the mutant lungs shown in H and Q compared to their respective controls. Arrowheads in M, O and Q indicate isolated domains of expression.

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