Genet. Res., Camb. (2015), vol. 97, e5. © Cambridge University Press 2015 1 doi:10.1017/S0016672315000026 Spry2 regulates signalling dynamics and terminal bud branching behaviour during lung development 1,2 2 YINGYING ZHAO AND TIMOTHY P. O’BRIEN * 1Shenzhen University Diabetes Center, AstraZeneca-Shenzhen University Joint Institute of Nephrology, Department of Physiology, Shenzhen University Health Science Center, Shenzhen 518060, China 2Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA (Received 7 November 2014; revised 12 January 2015; accepted 3 February 2015) Summary Development of mammalian lung involves reiterative outgrowth and branching of an epithelial tube into the surrounding mesenchymal bed. Each coordinated growth and branching cycle is driven by reciprocal signal- ling between epithelial and adjacent mesenchymal cells. This signalling network includes FGF, SHH, BMP4 and other pathways. We have characterized lung defects in 36Pub mice carrying a deletion that removes an antagonist of FGF signalling, Spry2. Spry2 deficient mice show an enlarged cystic structure located in the ter- minus of each lobes. Our study shows that Spry2 deficient lungs have reduced lung branching and the cystic structure forms in the early lung development stage. Furthermore, mice carrying a targeted disruption of Spry2 fail to complement the lung phenotype characterized in 36Pub mice. A Spry2-BAC transgene rescues the defect. Interestingly, cystic structure growth is accompanied by the reduced and spatially disorganized ex- pression of Fgf10 and elevated expression of Shh and Bmp4. Altered signalling balance due to the loss of Spry2 causes a delayed branch cycle and cystic growth. Our data underscores the importance of restricting cellular responsiveness to signalling and highlights the interplay between morphogenesis events and spatial localization of gene expression. 1. Introduction The airway goes through million times branching and fi fi Branching morphogenesis is characteristic of the de- nally lls the pleural cavity. Three simple branching velopment of many organs. Mammalian lung, kidney, modes are adopted including domain branching, pla- mammary gland, pancreas and vasculature share com- nar bifurcation and orthogonal bifurcation (Metzger fi mon processes in organ formation. The process et al., 2008). The respiratory system is nally gener- includes reiterative steps of duct outgrowth, bifur- ated with conducting airways and a maximized gas cation and formation of new branches (Hogan, exchange surface two days after birth. 1999; Metzger & Krasnow, 1999; Hogan & The elaborate branching mechanism is genetically Kolodziej, 2002; Affolter et al., 2003). At embryonic programmed. Each branching cycle has three sequen- day (E) 9·5, mouse lung starts to develop as a diver- tial steps including bud outgrowth, tip arrest and ticulum from the ventral side of the foregut. It has branching. Common genes and signalling pathways, “ ” two layers: outside mesenchyme and underlying epi- known as the branching module , are activated thelium. The epithelial layer evaginates into surround- (Davies, 2002). Embryonic lung development is regu- ing mesenchyme and elongates rapidly giving rise to lated by several intercellular signalling pathways, such fi the trachea. At the very tip it splits into two buds, as the broblast growth factors (FGFs), bone mor- and they will emerge into the primary bronchus. The phogenetic protein (BMP) and Sonic hedgehog primary bronchi buds undergo outgrowth, extension (SHH) (Bellusci et al., 1997 a; Bellusci et al., 1997 b; and branching to give rise to the secondary bronchus. Celli et al., 1998; Arman et al., 1999; Lebeche et al., 1999; Weaver et al., 2000; Mailleux et al., 2001). These signalling pathways interact with each other * Corresponding author: Tel: (607) 253-4326. and form a network that controls the programming E-mail: [email protected] and accuracy of the reiterated branching cycle. Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.42, on 26 Sep 2021 at 12:16:58, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672315000026 Y. Zhao and T. P. O’Brien 2 Mesenchymal Fgf10 plays the dominant role to guide The periodic outgrowth, tip arrest and branching of the epithelial layer outgrowth and branching lung buds is precisely controlled by signalling net- (Sutherland et al., 1996; Bellusci et al., 1997 b;Park works from both mesenchyme and epithelium. The et al., 1998;Lebecheet al., 1999). Moreover, other sig- role of Spry2 within the lung branching signalling net- nal pathways, such as SHH and BMP4, are important work remains unclear. In this study, we report that to regulate lung morphogenesis (Bitgood & lung branching morphogenesis is disrupted in mice McMahon, 1995; Bellusci et al., 1996; Bellusci et al., that have lost the Spry2 locus. Abnormal lung pheno- 1997 b;Lebecheet al., 1999; Miller et al., 2001). Shh type with reduced branching and enlarged terminal is expressed at the highest level at the distal tip of the cystic structures was observed. We demonstrate that epithelium bud, and regulates lung mesenchymal cell without Spry2, Fgf10 spatial distribution was dis- proliferation. Remarkably, over expression of Shh rupted and expression levels of Fgf10 and other signal- caused an increase in the ratio of interstitial mesen- ling factors were altered. Furthermore, this finding chyme to epithelial tubules (Bellusci et al., 1997 a; provides novel evidence that a reiteratively formed sig- Lebeche et al., 1999). Another key player, Bmp4,is nalling centre guides the development of embryonic localized at high levels in the distal tips of the epithelium lung and the signalling balance is critical to coordinate and at lower levels in the adjacent mesenchyme each branching cycle during respiratory airway (Bitgood & McMahon, 1995). Moreover, Bmp4 func- formation. tions upstream to Shh by regulating Shh expression in developing mouse lung and tooth germ (Bellusci et al., 1996; Zhang et al., 2000 a). These results suggest 2. Materials and methods that reciprocal signalling between mesenchyme and the underlying epithelium is critical for lung development. (i) Mice and production of Spry2-BAC transgene Sprouty (spry), the first identified FGF signalling an- 36Pub deletion mice were maintained on a C57BL/6J tagonist, functions as an intracellular inhibitor of the genetic background. 36Pub mice were genotyped in a receptor tyrosine kinase pathway in Drosophila PCR assay using the deletion flanking markers (Hacohen et al., 1998). Interestingly, Sprouty2 is an in- D14Mit265 and D14Mit177 as previously described tracellular protein localized to the cell membrane and (Roix et al., 2001; Welsh et al., 2007). Spry2ΔORF binds Gap1 and Drk to inhibit Ras activation (Casci mice were genotyped as described (Shim et al., et al., 1999). Upon growth factor stimulation, Spry2 2005). Spry2 BAC transgenic mice were genotyped translocates to the plasma membrane and becomes and maintained as described (Welsh et al., 2007). phosphorylated, which induces binding to the adaptor protein Grb2 to inhibit the recruitment of the Grb2– Sos complex to either the FGF receptor (FGFR) dock- (ii) Silicon cast injection and MicroCT data analysis ing adaptor protein FRS2 or to Shp2. Membrane Silicon casts were injected into E18·5 wild-type and translocation of Spry is necessary for its phosphoryla- mutant lungs. The lungs were imaged via MicroCT. tion, which is essential for its inhibitor activity The image analysis microview of the CT data allowed (Hanafusa et al., 2002). Many studies have demon- the internal branch structure to be visualized two- strated that Sprouty genes play an important role in dimensionally. Three-dimensional reconstructs of the early organ formation, such as kidney development, branched architecture were made from the CT images auditory epithelium differentiation, regulation of odon- using MATLAB software. togenesis, and limb and craniofacial development (Minowada et al., 1999; Hansen et al., 2003;Basson et al., 2005;Shimet al., 2005;Kleinet al., 2006; (iii) Histology and whole mount in situ hybridization Welsh et al., 2007). As a common antagonist of FGF and EGF signalling pathways, Spry2 has been demon- Embryonic lung tissue was dissected in PBS (4 °C) and fi strated to regulate airway branching in Drosophila xed overnight in 4% paraformaldehyde and strained (Kramer et al., 1999). Mouse Spry2 is expressed in a with Hematoxylin and Eosin following standard pro- domain adjacent to Fgf10 and is down regulated in tocols. Whole mount in situ hybridization was per- the clefts between new lung branches (Tefft et al., formed as standard protocol and a detailed protocol 1999). FGF10 beads can up regulate the expression is available upon request. of mSpry2 in adjacent epithelium (Mailleux et al., 2001). Inhibition of mSpry2 using antisense oligonu- cleotides stimulates branching and increases epithelial (iv) Quantitative RT-PCR analysis proliferation (Tefft et al., 1999). In addition, over ex- E14.5 lung tissue was dissected and frozen in RNAlater pression of mSpry2 leads to less branching (Mailleux (Ambion) and stored at −80 °C. Following the et al., 2001). These results suggest that Spry2 might Invitrogen TRIzol instruction, total RNA was extracted play important role during mouse lung formation. using TRIzol. Quantitative RT-PCR analysis was Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.42, on 26 Sep