The role of Sonic Hedgehog in postnatal mouse lung development

It is popular known that hedgehog signaling pathway plays an important role, as main regulator, in the normal development of many tissues such as lung in most animals. As the key protein of hedgehog signaling pathway, sonic hedgehog (Shh) has shown to be critical in branching morphogenesis of the lung, where it is expressed in the branching epithelium and essential for the mesenchymal-epithelial crosstalk that guides branching and epithelial tube elongation as well as smooth muscle cell/myofibroblast differentiation. The expression of Shh during embryogenesis has been found and investigated. Epithelial cell specific ablation of Shh early in lung development resulted in hypoplastic lungs with reduced bronchial branching and lung growth. Induction of Shh loss after ED13.5 did not affect fetal lung growth significantly, alveolarization was not determined (Miller et al. Dev Dyn 231:57–71, 2004). The Shh receptor ptch1 displays a peribronchial and perivascular expression pattern as well as strong expression in the primitive distal airspace regions in late fetal mouse lung, arguing for a role in mesenchymal proliferation and differentiation. Parathormone related peptide (PTHrP), induced upon fluid-regulated stretch of the acinus, may counteract Shh signaling and induces lipofibroblast differentiation (Torday, Pediatric Res 2006).

Until now the role of Shh in all tissue type of the postnatal lung is pretty quite unknown.

We hypothesize that Shh expression is critical for the regulation of the lipofibroblast and myofibroblast phenotypes, both of which are necessary elements for septation.

To address Shh expression in development lung, at first we will use western blot and reverse transcription real time PCR to detect the expression of Shh and corresponding proteins (patched-1 (ptch1), smoothened (Smo), Gli1, Gli2, desert hedgehog (Dhh), Indian hedgehog (Ihh), etc) in special time points.

To investigate the role of Shh in all tissue types of the lung we aim for global conditional Shh deletion. This will be achieved by use of global conditional cre-deleter mice (CAGGCre-ER) which express cre recombinase under the control of a chicken beta-actin promoter/CMV immediate early enhancer construct upon administration of tamoxifen (Hayashi & McMahon, Dev Biol 2002). These mice will be bred with floxed Shh-Shhlox/lox to generate CAGGCre-ER/Shhlox/lox mice. In these mice, the Shh gene will be ablated in all cells when tamoxifen is administered. This will be helpful do decipher the overall role of Shh in postnatal alveolar septation and regenerative alveolarization in adult mice, according to the models described above. We will induce Shh knock out at the second day after birth by tamoxifen injection, three days before septation occurs. In the adult pneumonectomy model, we will administer tamoxifen immediately after pneumonectomy. Moreover, in the caloric restriction model, tamoxifen will be administered either before or at the end of the starvation period. In parallel to the generation of the CAGGCre-ER/Shhlox/lox mice, we will, in collaboration with S. Bellusci, investigate the cell types which express the Shh receptor ptch1 in ptch1-lacZ mice (Goodrich, Science 1997) during postnatal and regenerative alveolarization as well as in lung regression/regeneration due to calorie restriction. Based on the effects observed with general Shh deletion and more defined expression pattern of the receptor, we aim to challenge the system in a more cell type specific way. The hypothesis to be confirmed or rejected by this approach is that Shh function is indispensable for both postnatal and adult regenerative alveolarization, with its function being mostly attributable to the proliferation and differentiation of mesenchymal myofibroblasts or lipofibroblast.

Gain of function will be achieved with doxy-inducible lung epithelial cell targeted over-expression of Shh using the tet-on system, which expresses the reverse tetracycline transactivator (rtTA) under control of the surfactant protein C promoter (SPC-rtTA). Reverse tTA binds to its respective promoter region (tetO) in the presence of doxycyclin and leads in this case to expression of Shh (SPC-rtTA/tetO-Shh; created by J. Whitsett; material transfer agreement exists). SPC-rtTA/tetO-Shh mice will be induced for Shh expression in different postnatal day to investigate whether this leads to increased septation. The effect of Shh over-expression will in addition be investigated in the lung regression/regeneration model due to calorie restriction. According to the hypothesis given above, postnatal and post-pneumectomy alveolarization is assumed to be enhanced by SPC-driven Shh over-expression, and the regression of alveolar number and surface area during caloric restriction may be partly prevented. Additionally we use CCSP (human)-rtTA mouse to overexpress the Shh in type II cells, too.

In addition, the Shh signaling pathway can be investigated by the study of down streaming protein Smo function, too. Smo is a G protein-coupled receptor protein (Chen Y, Struhl G. Cell 87 (3): 553–63, 1996) which plays an important role in the signaling pathway. We will investigate the Smo overexpression and inhibition to gain and loss the function of Shh signaling pathway.

The overexpression can be achieved by Gt(ROSA)26Sortm1(SMO/EYFP) (the Jackson Laboratory, donated by Andrew McMahon, Harvard University)/129-Gt(ROSA) 26Sortm1 (Cre/ERT) (global Cre mice from the Jackson Laboratory). The Gt(ROSA) 26Sortm1 (SMO/EYFP) mice contain an Enhanced Yellow Fluorescent Protein (EYFP)/Smoothened homolog (Drosophila) fusion gene inserted into the Gt(ROSA)26Sor locus. The mutant allele consists of a fusion product involving EYFP and the constitutively active W539L point mutation of the mouse smoothened homolog (Drosophila) gene (SmoM2). Expression of the Smo/EYFP fusion gene is blocked by a loxP-flanked STOP fragment placed between the Gt(ROSA)26Sor promoter and the Smo/EYFP sequence. When used in conjunction with a Cre recombinase-expressing strain and induced by tamoxifen, successful Cre-mediated excision results in the constitutive expression of mouse smoothened homolog (Drosophila) and unrestrained Hedgehog signaling in Cre-expressing tissues. Expression of the SmoM2 fusion protein can be monitored using EYFP-specific fluorescence protocols.

We use cyclopamine to inhibit the expression of Smo. Cyclopamine is a naturally alkaloid isolated from corn lily. It is used to treat the hedgehog overexpressed cancer because it can block the function of signaling pathway by changing the structure of Smo protein. We inject cyclopamine to postnatal mouse from the beginning of alveolarization to investigate the role of Shh signaling pathway in septation.