Fig 1.
Fstl1-LacZ reporter mice confirmed Fstl1 expression in airway and vascular SM.
(A) Schematic representation of generation of Fstl1-LacZ reporter mice. (B) Whole-mount X-gal staining of E15.5 and E18.5 WT and Fstl1LacZ/+ lungs and X-gal staining and α-SMA staining of the tracheal longitudinal sections and lung sections of E15.5 and E18.5 embryos. X-gal staining and α-SMA staining of Fstl1LacZ/+ adjacent trachea and lung sections showed co-staining of LacZ and α-SMA in airway (arrows) and blood vessel (arrowheads). Tr, trachea, Br, Bronchi, A, airway, V, blood vessel. Scale bars, whole-mount X-gal staining, 200 μm; X-gal staining and α-SMA staining of sections, 50 μm.
Fig 2.
(A) Generation of Fstl1 exon 3 and 4 floxed mice (Fstl1flox/+) by crossing Fstl1-LacZ with ACTB-FLP mice. Fstl1flox/+ mice were crossed with EIIa-Cre transgenic mice, resulting in exon 3 and 4 deletion. (B) Western blot analysis of Fstl1 protein from E15.5 embryos. (C) Fstl1 knockout pups died of respiratory distress shortly after birth. (D) Alcian blue staining revealed impaired banding pattern of tracheal C-ring cartilage. H&E staining of E18.5 WT and Fstl1−/− embryonic trachea (E, F) and lung (G, H) sections. Scale bars, 100 μm.
Fig 3.
Loss of Fstl1 led to abnormal tracheal and bronchial SM formation in E18.5 embryos.
(A, B) α-SMA whole-mount staining revealed an extremely attenuated α-SMA signal in Fstl1−/− trachea. Trachea (C, D), proximal bronchi (E, F, the sections at the points where the tracheas split into the left and right main bronchi) and distal bronchi (G, H) sections stained for α-SMA confirmed reduced α-SMA expression (arrows). (I, J) Trachea sections of similar planes, as indicated by the common carotid artery and thymus, stained for SM22α revealed reduced SM cells in Fstl1−/− trachea. (K, L) Stitched images showed airway SM defects from proximal bronchi to distal bronchi in Fstl1−/− lung. (M) qRT-PCR analysis of the expression of Fstl1 and α-SMA in E18.5 tracheas and lungs (n = 5 per group). aa, arch of the aorta, th, thymus, ca, common carotid artery, lb, left main bronchus, rb, right main bronchus. *, P < 0.05; ****, P < 0.0001. Error bars indicate mean ± SEM. Scale bars, A, B, 200 μm; C-L, 50 μm.
Fig 4.
ASM differentiation was significantly reduced in Fstl1−/− lungs.
(A, B) α-SMA immunostaining of E10.5 trachea sections revealed rare SM cell differentiation in both WT and Fstl1−/− (arrows). Immunofluorescence staining for α-SMA of E11.5 (C, D), E12.5 (E, F), E13.5 (G, H), E15.5 (I, J) tracheas showed less SM formation and expansion in Fstl1−/− tracheas during the early development (arrows). (K) qRT-PCR of Fstl1, α-SMA, myocardin and SRF expression demonstrated a significant reduction in E11.5 Fstl1−/− lungs compared to control WT lungs (WT, n = 5, Fstl1−/−, n = 6). (L) Loss of Fstl1 inhibited the TGF-β1-induced α-SMA, SRF, and myocardin expression in MEFs. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars indicate mean ± SEM. Scale bars, 50 μm.
Fig 5.
LacZ staining and immunostaining for α-SMA on transverse sections of other organs.
(A) LacZ staining of intestine, ureter and middle cerebral artery sections. (B) Immunostaining for α-SMA on transverse sections of E18.5 esophagus, stomach, duodenum, jejunum and colon. (C) α-SMA staining of epididymis showed normal SM differentiation in ductus epididymis. (D) α-SMA immunostaining to examinate the formation of SM of ureter and bladder. Asterisks indicate the SM layer of ureter. (E) Immunostaining for α-SMA on middle cerebral artery and thoracic aorta sections. Scale bars, 100 μm.