Spry2 Regulates Signalling Dynamics and Terminal Bud Branching Behaviour During Lung Development

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 signalling 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 terminus 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 expression 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.

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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

performed on ABI7500 using TaqMan gene expression assays.

3. Results (i) 36Pub mice exhibit lung branching defects and complementation test with Spry2-BAC transgene rescue with a hypomorphic phenotype The piebald deletion mouse model is a collection of overlapping Mb-scale chromosomal deletions centred around the endothelia receptor B (Ednrb) coat colour spotting locus (O’Brien et al., 1996). In a previous study, we have used these mouse models to annotate the function of a distal 6Mb region of mouse chromosome 14. Ednrbs-36Pub (hereafter 36Pub) has been show to lose several loci essential for mammalian development: Spry2 and two other genes, Ndfip2 and Rbm26 (Roix et al., 2001; Peterson et al., 2002). Mice homozygous for Ednrbs-36Pub die from postnatal respiratory distress and craniofacial malformations (Rice et al., 2004). Spry2 has been reported to play an important role in craniofacial differentiation, and auditory and tooth development (Shim et al., 2005; Klein et al., 2006). To annotate if Spry2 is the gene causing postnatal respiratory failure, we explored the development sta- tus of 36Pub homozygous lungs. At E18·5, 36Pub mutant lung was smaller in volume and round in shape missing the sharp curved edge of each lobe (Fig. 1 (A), (C), (D) and (E)). To better characterize this phenotype we injected silicon casts into E18·5 wild- type and mutant lungs. Wild-type lungs exhibit curved edge and organized distribution of the terminal alveolar sacs, whereas 36Pub mutant lungs show ap- Fig. 1. 36Pub mutant exhibits altered lung structure and parent shape changes and characteristically dys- Spry2-BAC trangene partially rescues the defects. morphic terminal alveolar sacs (Fig. 1(a’) and (b’)). Hypomorphic rescue by Spry2-BAC transgene of lung – Additionally, cross sections show the main bronchus defects observed in 36Pub mutant mice (A C). 36Pub mutant shows a round blunt edge losing the thin sharp is larger than the wild-type lung (data not shown). transparent edge of wild-type (arrow) (C). Silicon casted Silicon casted lungs were imaged via MicroCT. The 36Pub mutant lung exhibits altered lung structure (D–E). image analysis microview of the CT data allowed E18.5 wild-type (D) show normal lung structure with the internal branch structure to be visualized two- curved edge (arrow) and surface pattern of the terminal ’ · dimensionally. Comparison of wild-type and mutant alveolar sacs (white box enlarged in a ), whereas E18 5 36Pub mutant lung loses those features (E and b’). Three lungs indicated the presence of large cystic structures dimensional reconstruction of MicroCT silicon casted lung throughout the bronchial tree of the mutant lungs. reveals the presence of cystic structure (F and G). Five Three-dimensionally reconstructed branched architec- lateral secondary bronchi are labelled with 1–5. Cystic ture from the CT images confirmed the presence of structure was also observed on a Hematoxylin and Eosin – large cystic structures within the mutant lungs stained cross section of E14.5 36Pub lung (H J). observed in the two-dimensional CT slices (Fig. 1(F) and (G)). Interestingly, we found that the cystic structure is located at the termini of the main bronchi that related to the altered branching behaviour of 36Pub define each of the five lobes of the mutant lung (data lungs. not shown). We next compared frontal sections of left The 36Pub deletion removes Spry2 and two other lobe from E14·5 embryos. Clear morphologic differ- genes (Peterson et al., 2002). As an FGF signalling an- ences of mutant lung were observed in the earlier em- tagonist, Spry2 is critical for craniofacial differen- bryonic stage with distal enlarged structure. These tiation, and auditory and tooth development (Shim results imply that the abnormal structures might be et al., 2005; Klein et al., 2006; Welsh et al., 2007).

Based on the respiratory phenotype of 36Pub and our division of the terminal bud produces a major and previous study, we prioritized Spry2 as a candidate minor bud. The major bud will elongate the main gene (Rice et al., 2004). To further investigate whether bronchus and the minor bud will grow into a lateral Spry2 was the gene, a Spry2-BAC transgene approach branch, respectively. The five lateral branches pro- was carried out. In our previous study, the transgenic duced via this process correspond to the five second- lines (Spry2-BAC-69) have a similar expression pat- ary bronchi seen in the E18·5 lung (numbered 1–5in tern to that of the endogenous locus; however, the Fig. 1 and Fig. 2). Comparison of terminal bud level of the expression was as low as 25% of wild-type branching in wild-type and mutant lungs through lung and palate levels (Welsh et al., 2007). We tested if the same developmental interval (E12.5–E13.5) dem- each transgene line could rescue the lung defects in onstrates an abnormal and progressive expansion of 36Pub deletion mutant mice. Spry2-BAC transgenic the mutant terminal bud. The expansion of the ter- mice showed rescue of the postnatal breath failure minal bud is accompanied with less branching. Over (data not shown). In addition, analysis of E18·5 the same interval the wild-type terminal bud gives lungs showed that Spry2-BAC transgenic lines par- rise to two branches whereas the mutant undergoes ex- tially rescued the lung defects associated with size, pansion (Fig. 2(e–h) and (i–l)). Besides left lobe, simi- edge and terminal alveolar sac organization, which lar cystic structure and delayed branching was also are less obvious severe phenotypes compared observed in other lobes (Fig. 2(m–r)). We were able to 36Pub homozygous lungs (Fig. 1(B) and (I)). to match the abnormalities in morphology observed However, we observed small enlarged bronchioles at in the E18.5 mutant lung to altered branching behav- the distal region of each lobe (data not shown). The iour of the terminal bud in earlier developmental phenotype of Spry2-BAC transgenic lung is hypo- stage. morphic. This might be explained by the dose of the transgene. To further evaluate if Spry2 is the candidate of the (iii) Delayed branch cycle was characterized with reduced branching event lung phenotype, a complementary cross of the 36Pub mouse with the Spry2 knockout mouse (Spry2ΔORF) Mesenchymal FGF10 signalling has been proven as a was carried out. The results showed that Spry2ΔORF dominant force in leading the epithelial bud elongation mutation fails to complement the lung branch defects during lung development (Bellusci et al., 1997 b; Park associated with the 36Pub deletion. Using a Spry2 et al., 1998;Lebecheet al., 1999). Spry2 is known to allelic series to study the effect of the dosage of function as an antagonist of the FGF signalling path- Spry2 on lung development by comparing the organ- way. Therefore, we expect to observe elevated FGF10 ization and distribution of the terminal alveolar sac, signalling in 36Pub lungs. Since FGF10 leads the we found the ranking of the phenotype to be from lung bud growth and elongation we expected to observe severe to light: Del/Del, Del/ORG, ORF/ORF, wild- more branches and larger lung bud formation. type (data not shown). The results supported that However, bud expansion with delayed growth was Spry2 contributed to the 36Pub deletion lung branch- observed in 36Pub mutant lung (Fig. 2(a) and (b)). ing phenotype and is likely the candidate gene within Quantification of branching events on whole-mount the critical interval that responds to the cystic struc- lung explants is carried out by counting the number tures. However, respiratory phenotype of Spry2 of epithelial buds around the periphery of the lung knockout is weaker compared to the phenotype of explants. Interestingly, 36Pub deletion lungs have a 36Pub mice. Spry2 knockout lungs exhibit the defect significantly lower number of branches (Fig. 3). At on the domain branching with an increased number E12.5 there are an average of seven lung edge buds in of ventral branches (Metzger et al., 2008). It is poss- wild-type lungs versus four buds in the caudal lobe of ible that in the deletion region there are other genes 36Pub lung. By E14.5, wild-type caudal lobes have 25 or functional sequences also involved in lung edge buds whereas mutant only have 16. Reduced development. branching and cystic expansion suggested FGF10 signalling was altered and an unbalanced signalling network leads to a delayed branch cycle. (ii) Cystic growth arises early in lung development The defects of 36Pub lungs were further studied in de- FGF10 and FGF10 responsive genes were altered in tail by temporally reconstructing the branching pro- (iv) the absence of Spry2 cess associated with the caudal extension of primary bronchus of the left lobe with serially staged (E12.5– The reduction in number and increase in size of the E13.5) wild-type and mutant lungs (Fig. 2). In this 36Pub lung buds reveals that without Spry2, FGF10 study we used Etv5, a protein specifically expressed signalling is changed more dynamically than simple in the branching epithelium, as an epithelium marker reduction. To further evaluate the role of Spry2 during to visualize the branching process. The asymmetric lung development we assessed the Fgf10 distribution

Fig. 2. Cystic buds appear in the early stages of lung development. (A) Bud growth was observed using in situ hybridization on the left lobe of WT and 36Pub mutant lung (from E12.5 through E13.5) for the detection of Etv5 expression. Top panel shows branching process associated with the caudal extension of primary bronchus of the left lobe (a–d). The WT left main bronchi bud undergoes a series of ﬁve asymmetric branching events (numbered 1–5). Middle panel focuses on the fourth and ﬁfth branching events and TB formation (e–h). Lower panel shows the branching process of 36Pub mutant lung, mainly focused on the expansion of the terminal bud (i–l). (B) Reduced lung buds were observed on in situ hybridization of caudal lobe of WT and 36Pub mutant lung (from E12.5 through E14.5) for the detection of Etv5 expression (m–r).

pattern and expression level by double labelled in situ deletion lung exhibits reduced FGF10 signal and hybridization. At E13.5, Fgf10 localized to a restricted loses the stereotypic lattice distribution pattern domain with an ordered lattice pattern in the mesen- (Fig. 4(B), (D), (F) and (H)). Additionally, both Shh chyme surrounding the underlying epithelial buds and Bmp4, two epithelial negative regulators of (Fig. 4(A) and (C)). A similar pattern persists in the Fgf10, were extremely elevated compared to wild-type E14.5 (Fig. 4(E) and (G)). Surprisingly, the 36Pub (Fig. 4(K) and (L)). To further evaluate the level of

Fig. 3. Cystic growth results in a reduced branch cycle. Counts of the outside edge buds from caudal lobe of WT and Spry2 mutant lung at E12.5, E13.5 and E14.5. We only count the lung buds localized to the outside edge of the lobe (data collecting from 30 WT and 31 36Pub mutant lungs).

Fgf10 and other factors we carried out quantitative analysis using RT-PCR. Surprisingly, 36Pub mutants exhibited reduced Fgf10 but elevated Shh and Bmp4. The data reﬂects a dynamic change among the signalling pathways. When there is loss of antagonism of Spry2, Fgf10 is elevated in the earlier stage of lung development (data now shown), which leads to elevated Bmp4, Shh and other factors. Stronger local inhibition from SHH and BMP4 signalling might cause a weak FGF10 signal in the later stages. An altered balance between FGF10 and other signalling factors due to the loss of Spry2 causes delayed branch cycling in the developing lung and results in cystic growth.

Fig. 4. Loss of Spry2 alters Fgf10 and other signalling 4. Discussion factors. (A–H) 36Pub lung shows reduced Fgf10 (blue) production and changed spatial distribution pattern The embryonic respiratory tree is generated by million compared with wild-type lungs (E13.5 and E14.5). Double times branching events. Each branching event is de- labelled in situ hybridization Fgf10 (blue) and NKX2.1 – pendent on restricting cellular responsiveness to sig- (pink) (A H). Altered Shh and Bmp4 expression of caudal lobe in E13.5 lungs (I–L). (M) Quantitative RT-PCR nalling. Reciprocal signalling from the epithelium analysis of Fgf10, Etv5, Shh and Bmp4 expression in and surrounding mesenchyme interact to ensure the wild-type and 36Pub mutant lung (E13.5). 36Pub mutants precise control of the branching program in both exhibit reduced expression of Fgf10 and elevated space and time during development. Signalling factors expression of Etv5, Shh and Bmp4 (error bars represent from the FGF10, BMP4 and SHH pathways are the standard error of the mean). most critical ones functioning in the branching program. In this study we have shown that Spry2 plays deletion mutant lung causes cystic structures to form an essential role in lung development. Mice homo- at the distal end of secondary bronchioles of each of zygous for 36Pub deletion exhibit postnatal respirat- the ﬁve lobes and signiﬁcantly reduces branching ory failure and the mutant lung shows abnormal events. The mouse model therefore represents a architecture of the bronchial tree and disorganized al- unique way to understand the role of epithelial veolar sac distribution. The Spry2 knockout allele fails Spry2 in the developing lung. Further analysis of mu- to complement the 36Pub deletion phenotype. A tant lungs showed cystic growth arises early in lung Spry2-BAC transgene greatly rescues the deletion development, around E12.5. Consistent with the loss lung phenotype. Absence of Spry2 in the 36Pub of SPRY2 antagonism of FGF signalling, the deletion

Fig. 5. Models of distal signalling centre regulation on branch cycle in the developing lung. Mesenchymal FGF10 signalling directs bud outgrowth and also induces the expression of Shh and Bmp4 from the epithelium. Shh, Bmp4 and Spry2 function together to antagonize the FGF10 signal and control the distribution pattern of FGF10. In the 36Pub mutant lung, absence of negative control of FGF10 signal by Spry2 results in an altered signalling balance. This leads to decreased and delayed branch cycles and cystic terminal buds. WT, Wild-type.

mice showed an altered expression pattern of Ffg10, bud in two opposite directions while the bud is and also of FGF responsive genes such as Shh and branching. Symmetric branching or asymmetric par- Bmp4. The results in this study highlight the role of titioning is determined by the relative location and Spry2 in the regulation of embryonic lung development. strength of the two newly formed distal signal centres. The accuracy of the branching program is determined by the dynamic integration of inductive and antagon- (i) Branch cycle is programmed by the mesenchyme– istic interactions among these pathways. The spatial epithelium signalling network localization of gene expression directs morphogenesis Given the complexity of the architecture of the lung is events. Many studies have suggested that disrupted remarkably stereotypic, the core developmental mech- signalling balance will result in lung malformation. anism is reused many times to build up the bronchial Defects in Fgf10 or its epithelial FGF receptor 2 tree (Metzger et al., 2008). The reiterative outgrowth (Fgf2b) cause the trachea to be terminated as a and branching of an epithelial tube into the surround- blind sac (Celli et al., 1998; Arman et al., 1999; ing mesenchymal bed by coordinating the sequential Sekine et al., 1999). Mutants of Shh and Bmp4 have steps of bud extension, tip arrest and branching, been found with severe lung branching defects which is the three-step branching cycle (Fig. 5(A)), is (Bellusci et al., 1996; Bellusci et al., 1997a; the basic simpliﬁed reused mechanism. The process Litingtung et al., 1998; Pepicelli et al., 1998; is tightly coordinated and controlled by the factors Lebeche et al., 1999; van Tuyl & Post, 2000; Zhang within the signalling centre at the distal tip of the et al., 2000 a; Zhang et al., 2000 b; Miller et al., lung bud. Mesenchymal FGF10 signalling from 2001). Spatial localization and gene expression of sig- the distal signalling centre directs the outgrowth of nalling factors are critical in directing morphogenesis the lung epithelial bud (Fig. 5(A)). In response to events. FGF10, epithelium cells at the tip of lung bud express multiple signals to antagonize FGF10, such as SHH, The signalling network is modulated by Spry2 in the BMP4 and SPRY2. Two antagonistic strengths (ii) developing lung reach certain levels and cause tip arrest. The strongest FGF10 inhibition is at the distal bud tip, and this FGF10 signalling directs epithelium layer outgrowth causes a local temporary relatively low level of and meanwhile Spry2 was found to be expressed at Fgf10 compared to the adjacent mesenchyme area. the corresponding epithelium adjacent to the distal Therefore, epithelium cell growth in this area is rela- signalling centre. Without Spry2,36Pub homozygous tively slow and a branch cleft is formed. The distal sig- mice have dramatically reduced and disorganized nalling centre splits into two separate centres. The two Fgf10 expression. Consistent with the branching newly developed buds will continue the branching cycle model (Fig. 5(A)), our data supported that dur- cycle (Fig. 5(A)). The two newly formed FGF10 sig- ing lung development the FGF10 signal is tightly nalling centres lead to the outgrowth of the primary regulated and organized. Multiple signalling pathways

function together to provide spatial and temporal re- the early stage of development attempt to overcome striction upon FGF10 signalling. The cystic structure the loss of Spry2. Lungs at E10.5 and E11.5 only of 36Pub lung is caused by the altered expression show the delayed growth. The signalling balance, and distribution of Ffg10 at the distal signalling which is the axis that drives the branching cycle, is centre. altered in the early stage because of loss of Spry2 The Spry2 gene suppresses branching during lung and also further disrupted in the later stage due to development, antagonizing Fgf10. Without the antag- altered Shh and Bmp4. In situ hybridization of E13.5 onism from Spry2, Fgf10 loses its tight control and the mutant lungs show evidence of growth of the cystic balance between bud outgrowth and branching is dis- bud. The phenotype is accumulated as the cystic struc- turbed. At an early stage, loss of Spry2 and the elev- ture locates near to L5 and D4 of the left lobe ation of the Fgf10 activated MAPK pathway results (Metzger et al., 2008). in a negative feedback loop that is activated by in- ducing it’s negative regulators such as Shh and Bmp4 from the epithelium. From our gene expression data we observed that without Spry2, Fgf10 induced (iii) Timing and spacing of branching morphogenesis is elevated expression of Bmp4 and Shh from E9.5 to coordinated by Spry2 in the developing lung E13.5. Elevated BMP4 and SHH negatively regulate 36Pub homozygous lung exhibits abnormal lung Fgf10 and lead to reduced FGF10 signalling at phenotype of cystic enlarged buds at the early stage E14.5. Continual inhibition of Fgf10 from elevated of development and cystic structures located at the ter- Shh and in the later lung development stage causes mini of the main bronchi of the five lobes with chan- the delayed branch cycle. The fewer buds of 36Pub ged alveolar sac distribution. Many previous studies homozygous lung are the evidence of delayed branch on the complexity and diversity of highly organized cycle. The data also provided evidence that Spry2 is branched organs, such as lung, kidney and mammary important for branching cleft formation (Fig. 5(B)). gland, raise the question of what is the basic mechan- Branching morphogenesis relies on signalling thresh- ism to develop these organs. One possibility is that a olds that FGF10 and anti-FGF10 signalling will branch program is encoded in the genome and drives reach. Without Spry2, there is an altered signalling growth and branching. In other words, the morpho- balance and it is relatively hard to reach the threshold logic complexity can be developed by repetitively and form the branching cleft. Therefore, the terminal using a simple branching mechanism (Metzger et al., bud continually grows and undergoes expansion. The 2008). branching cycle is delayed with cystic growth and Based on our data, without the antagonism from reduced branching. Further studies are needed to Spry2, domain branching was affected more severely test if the proliferation state of 36Pub mutant lung is than planar bifurcation and orthogonal bifurcation. altered. The prediction is that a less active prolifer- Similar to our findings, Metzger and colleagues ation state might be observed in 36Pub mutant lung. found Spry2 null lung to show the effect on domain It is interesting to note that the branching cycle is branching. However, interestingly, our data show delayed but not arrested. One possible explanation is that without Spry2 the branching events are reduced that the altered branch cycle needs a longer time to in the early stage of development and cystic growth reach the branching threshold. accumulated at the terminus of each of the five Our data also support that domain branching is not lobes, whereas they found that the Spry2 knockout equally bifurcated. We provide evidence that the cystic lung has a relatively weaker phenotype: the number structure phenotype of 36Pub is a result of accumu- of ventral branches is significantly increased in lation of delayed branch cycle. Domain branching, Spry2−/− relative to Spry2+/+ wild-type controls. which affects the proximal–distal axis of each of the Ectopic branches form earlier and proximal to normal five lobes, is different from planar and orthogonal bi- ventral secondary branch (V1). The different pheno- furcation and is not simply equal bifurcation (Metzger type of knockout and deletion lungs could be et al., 2008). The embryonic lung epithelial and mes- explained considering more function sequence is re- enchymal stem cells, which mainly give rise to second- moved in 36Pub mutant mice. However, as a mouse ary branches of the respiratory tree, have memory. In model to study lung morphogenesis, 36Pub uncovered the other words, during the domain branching L1, two the general role of Spry2 to maintain the balance of newly formed branches are not exactly the same, one the branching cycle during lung development and still remains as the elongation stem of the respiratory also the specific role that Spry2 plays in the domain tree and this feature will also remain after L2 is branching. Our results support that the branching formed. In our mouse model, as domain branching mechanism for each branch event is not exactly the continues, 36Pub deletion lung phenotype accumu- same. The core program is similar but each parameter lated in the elongation stem with relatively normal lat- will be subtly tuned to produce the morphogenetic eral branches. Several runs of the branching cycle in differences.

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