ISL1 regulates lung branching morphogenesis via Shh signaling pathway

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1ShhCre lungs. There was no difference in apoptosis between the wild-type (ShhCre) and Isl1ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1 ShhCre lungs. There was no difference in apoptosis between the wild-type (Shh Cre ) and Isl1 ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1 ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.
As a respiratory organ, the main function of the lung is to realize the effective gas exchange between the blood and the external environment to maintain life activities. Lung development undergoes a series of developmental events, including branching morphogenesis and alveolar differentiation (1,2). Airways develop sequentially through early epithelial duct branching and late air sac separation. Lung branching is a highly coordinated process that generates a complex network of gas exchange units. In mice, branching morphogenesis begins with primary lung bud proliferation and continues to grow into the surrounding mesenchyme at E10.5 (3). There is an intimate crosstalk between the epithelium and the mesenchyme during branching (4)(5)(6). Lung branching relies on a complex coordination of multiple signaling pathways and transcription factors, but the underlying precise mechanisms that control lung branching remain elusive (7)(8)(9)(10).
Shh is expressed in the epithelial cells at the onset of lung growth at day E9.5. Deletion of Shh results in failure of branching and growth after the formation of the primary lung buds (11). The proliferation of mesenchymal cells is significantly decreased in the Shh knockout mice (11)(12)(13). These results highlight the importance of Shh in regulating lung branching. Genetic rescue experiments revealed that transcription factors Tbx2 and Tbx3 act mainly downstream of epithelial Shh signaling to promote mesenchymal proliferation and maintain branching morphogenesis (14). Sox9 also plays an important role in lung branching, and ablation of Sox9 in epithelial cells results in dramatic defects in lung branching (15). Sox9 promotes proper branching morphogenesis by controlling the balance between proliferation and differentiation. Iroquois homeobox (Irx) genes are reported to be involved in the regulation of proximal-distal patterning during lung development (16,17). The expression of Irx1-3 and Irx5 is restricted to branching lung epithelium, and knockdown of all lung Irx genes together significantly reduces distal branching events and increases proximal tubule dilation in vitro.
Transcription factor Isl1 has been reported to play a critical role in organ patterning (18)(19)(20)(21). Isl1 has been reported to regulate tracheo-esophageal separation and lung lobation (22). The exact molecular mechanism of Isl1 during lung branching remains to be determined. Here, we found that ablation of Isl1 in the lung epithelium resulted in defects in branching morphogenesis. Shh was identified as a target gene of ISL1 in the lung epithelium. Activation of Hedgehog signaling by Purmorphamine treatment or by ectopic Ihh expression would rescue the defects triggered by Isl1 deletion. Thus, we revealed that Isl1 regulates lung branching morphogenesis through Shh signaling.

Loss of Isl1 results in impaired lung branching morphogenesis
The expression of Isl1 in the lung was determined using Isl1 LacZ knock-in allele. Isl1 expression was detected in the lung epithelium as early as E11.5 (Fig. 1A), suggesting a role in lung development. To investigate the potential function of Isl1 in the development of lung, we generated mice with epithelial cell specific deletion of Isl1 by crossing Shh Cre with Isl1 fl mice ‡ These authors contributed equally to this work. * For correspondence: Feixue Li, lifx@hznu.edu.cn; Jianying Li, ljy241@126. com.
(Isl1 ShhCre ). The fusion of all four right lung lobes was observed in Isl1 ShhCre embryos (100% penetration) (Fig. 1C). Immunofluorescence and morphological analyses demonstrated the deletion of ISL1 (Fig. 1B) and the defect of lung branching process in Isl1 ShhCre embryos (Fig. 1, D and E). The results showed that lung branching morphogenesis was delayed in the Isl1 ShhCre mutant embryos from E11.5 to E13.5. These data suggest that Isl1 is required for lung branching morphogenesis.

Isl1 deletion reduces the proliferation of mesenchymal cells without disturbing the apoptosis
To investigate the cellular mechanisms responsible for defective lung branching morphogenesis in Isl1 mutants, we examined cell proliferation and apoptosis. E-cadherin has been characterized and used as an early-airway epithelial marker (23). BrdU was counterstained with E-cadherin to determine the proliferation of epithelial cells in the lung. The results of immunofluorescence showed that BrdU-labeled mesenchymal cells were significantly reduced in Isl1 ShhCre lungs from E11.5 to E12.5 compared with wild type (Fig. 2, A-C). Anti-Ki67 antibody (proliferation marker) was also used to detect cell proliferation, which also showed a reduction in mesenchymal cell proliferation through all detected stages (Fig. 2D). To determine whether defects in lung branch morphogenesis were due to increased abnormal cell apoptosis, we performed TUNEL assays on tissue sections from E11.5 to E12.5. There was no obvious difference in apoptosis between the control (Shh Cre ) and Isl1 ShhCre embryos (Fig. 2E). The results suggest that Isl1 is critical for cell proliferation during lunging branch morphogenesis but not for cell survival. Decreased mesenchymal cell proliferation may account for the developmental defect of the lung branching in Isl1 ShhCre embryos.

Loss of Isl1 results in differential expression of genes critical for branching morphogenesis
To explore the molecular mechanism by which Isl1 deletion caused lung branching defects, gene expression profiles were analyzed by RNA-Seq using RNA extracted from E11.5 lung tissues from mutant (Isl1 ShhCre ) and wild-type control (Shh Cre ) mice to identify the potential downstream target genes of Isl1. We uncovered dozens of protein-coding genes that were differentially expressed (≥1.5-fold, <5% false discovery rate) in the Isl1 ShhCre mutants versus the controls (Fig. 3A). Among them, the expression of multiple genes known to be critical for lung branching changed significantly. Quantitative real-time PCR using RNAs purified from independent samples confirmed the altered expression of Shh, Sox9, Irx1, Irx2, Tbx2, and Tbx3 (Fig. 3B). In situ hybridization and immunofluorescence also confirmed the downregulation of Sox9, Irx1, and Irx2 ( Fig. 3, C-F). These results indicate that the expression of all these genes is dependent on ISL1 in the lung epithelium.

Shh gene is regulated by Isl1 in lung epithelium
During branching morphogenesis, there is an intimate crosstalk between the epithelium and the mesenchyme. We hypothesized that secreted signaling molecule SHH might be the downstream target of Isl1 in the epithelium through which Isl1 controls the cellular activities of the mesenchyme. The results of in situ hybridization confirmed the downregulation of Shh in the Isl1 ShhCre mutant (Fig. 4A). Overexpression of Isl1 could significantly induce Shh expression in primary lung cells (Fig. 4B). It is known that the expression of Shh in the lung epithelium is regulated by the MACS1 (chr5:29416996-29417802) sequence. Two ISL1 consensus binding sites were found in the MACS1 sequence (Fig. 4C). The ChIP analysis showed the binding of ISL1 to the MACS1 sequence of the Shh gene (Fig. 4D). In addition, the ChIP analysis showed no binding of ISL1 to the Shh brain enhancer SBE1 (Chr5: 26858065-26858607) or Shh limb bud-specific enhancer MFCS1 (chr5: 29520611-29519698) (Fig. 4E). Our data demonstrate that Shh mediates the effects of Isl1 on lung branching development.
Tbx2 and Tbx3 are upregulated by hedgehog signaling activation In situ hybridization was used to detect the activity of Hedgehog signaling in the mesenchyme of Isl1 ShhCre mutant lungs. The expression of Ptch1, a marker gene for active Hedgehog signaling, is reduced in the mesenchyme of Isl1 ShhCre mutant lungs (Fig. 5A). The results showed that the activity of Hedgehog signaling in the mesenchyme of Isl1 ShhCre mutant was reduced. Expression of Tbx2 and Tbx3 was also decreased in the mesenchyme of Isl1 ShhCre mutant lungs (Fig. 5A). The results are consistent with reports that expression of Tbx2 and Tbx3 in the mesenchyme depends on epithelial-derived Shh signaling. Tbx2 and Tbx3 have been reported to maintain lung mesenchymal proliferation. Organ culture experiments were used to verify whether activation of Hedgehog signaling could rescue the cell proliferation defects in Isl1 ShhCre mutant lungs. The results indicated that mesenchymal cell proliferation was increased ISL1 regulates lung branching morphogenesis significantly after Purmorphamine treatment (Fig. 5, B-D). The increase of Ptch1 expression indicated that Hedgehog signaling is activated after Purmorphamine treatment (Fig. 5C).

Activation of Hedgehog signaling rescues the defects of lung branching in vivo
We conditionally overexpressed a Hedgehog ligand Ihh in the lung epithelium using a transgenic allele (Tg-pmes-Ihh).

Discussion
Lung branching morphogenesis is a complex process that generates a tree-like network consisting of proximal conducting airways and distal alveoli (1,27). In this study, we observed an obvious branching defect as early as E11.5 in the Isl1 ShhCre mutant embryos, indicating an important function of Isl1 at the beginning of branching. Shh signaling activity was downregulated in the Isl1 ShhCre mutant embryos. And activation of Hedgehog signaling could rescue the defect of lung branching.
LIM homeodomain transcription factor Isl1 serves as a marker for patterning and cell type specification in many developmental processes (20,(28)(29)(30). Isl1 highly expresses in lung epithelial cells as early as day E11.5, suggesting that it plays an important role in early lung development. Deletion of Isl1 in the lung epithelium results in defective lung branching. The fusion of all four right lung lobes was observed in Isl1 ShhCre embryos. The results showed that the proliferation of lung mesenchymal cells was significantly reduced in Isl1 ShhCre embryos, while apoptosis was not altered. Coordinated epithelial-mesenchymal interactions have been shown to be critical for lung branching morphogenesis (31). Significant downregulation of mesenchymal cell proliferation suggests a link between ISL1 and secretory signaling molecules between the epithelium and mesenchyme. It should be a secreted signaling molecule that mediates the effects of Isl1 on the mesenchymal cells.
Sox9 and Irx genes are all reported to play an important role in lung branching morphogenesis (15,16). Our results show that Sox9, Irx1, and Irx2 are all downregulated in the Isl1 ShhCre embryos, suggesting that deletion of Isl1 results in changes in the expression of multiple genes. Reciprocal communication between the epithelial layer and surrounded mesenchyme drives branching morphogenesis. During lung branching morphogenesis, many signals drive the proliferative expansion of the distal endoderm and underlying mesenchyme. Epithelial Shh is required for the formation of the lung lobation, branching, and growth (13). Shh overexpression leads to increased proliferation of mesenchymal cells (11). Therefore, we hypothesized that Shh acts downstream of Isl1 to mediate the interaction between the epithelium and the mesenchyme. Previous works have shown that ISL1 regulates the expression of Shh in the tongue and urethral epithelium (32,33). Both RNA-Seq and in situ hybridization results showed that Shh expression in the epithelium was reduced after Isl1 deletion. The Chip analysis confirmed that ISL1 could bind to the MACS1 enhancer sequence, which strictly specifies Shh expression in respiratory epithelial cells (34). The involvement of Shh signaling was confirmed by rescue experiments that activate Hedgehog signaling in vivo and in vitro. The proliferation of mesenchymal cells was rescued after activating Hedgehog signaling. The number of lung branching ends significantly increased in Isl1 ShhCre ; Tg-pmes-Ihh. Moreover, upregulation of Sox9, Irx1, Irx2, Tbx2, and Tbx3 expressions was detected in Isl1 ShhCre ; Tg-pmes-Ihh. All these data suggest that Isl1 affects branching morphogenesis by regulating Shh expression.
Decreased expression of Ptch1 confirmed a reduction in the activity of Hedgehog signaling in the lung mesenchyme of Isl1 ShhCre . Tbx2 and Tbx3 express in mesenchymal cells excluding the airway epithelium during lung development and have been identified as downstream target genes of Shh (14). Our results indicated that both Tbx2 and Tbx3 expression decreased in the Isl1 ShhCre embryos. Tbx2 and Tbx3 function to maintain lung mesenchymal proliferation by regulating the expression of Cdkn1a (14,35). The RNA-Seq data showed that cell-cycle inhibitor Cdkn1a was up-regulated in Isl1 ShhCre embryos. Downregulation of Tbx2 and Tbx3 might be responsible for the defective proliferation of mesenchymal cells. All these data suggest that decreased Hedgehog signaling activity in the lung mesenchyme leads to downregulation of Tbx2 and Tbx3, resulting in reduced mesenchymal cell proliferation.
In summary, our data indicate transcriptional factor Isl1 is important for lung branching morphogenesis. Shh expression is regulated by Isl1 in the lung epithelium. The hedgehog signaling mediates the effect of Isl1 between epithelium and mesenchyme. Activation of Hedgehog signaling rescues lung branching morphogenesis defects due to Isl1 ablation. These data suggest that Isl1 affects lung branching morphogenesis by regulating the Shh signaling pathway.

Animals
All of the animal experimental protocols were approved by Hangzhou Normal University Animal Care and Use Committee (2018036). Constructions of the Isl1 fl and Isl1 LacZ have been previously described (28). The Tg-pmes-Ihh mouse line was created by inserting full-length Ihh cDNA into a conditional transgenic expression vector as described previously (36). Mouse strains for R26R-LacZ and Shh tm1(EGFP/cre) were purchased from the Jackson Laboratory and maintained on a C57BL/6J background. The morning of observed vaginal plug was designated as day 0 (E0) of pregnancy.

X-gal staining
Whole-mount X-gal staining was performed according to the standard protocols (36). Embryos were fixed in fixing ISL1 regulates lung branching morphogenesis solution (4% PFA, 5 mM EGTA, and 2 mM MgCl2 in PBS) for 1 h at 4 C. The fixed embryos were rinsed three times in washing buffer (0.02% NP40, 0.01% sodium deoxycholate, and 2 mM MgCl2 in PBS). The embryos were then incubated in staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM Tris (pH 7.3), and 0.1% X-gal in washing buffer) 2 to 4 h in the dark at 37 C. Finally, the stained embryos were washed in PBS and post-fixed in 4% PFA. The X-gal stained sections were counterstained with Nuclear Fast Red.

Detection of cell proliferation and apoptosis
Cell proliferation activity was evaluated by 5-bromode oxyuridine (BrdU) labeling and immunofluorescence staining (37). Briefly, timed pregnant mice were injected intraperitoneally with BrdU solution (3 mg/100 g of body weight) from a BrdU labeling and detection kit (Roche) 30 min before embryo harvesting. The collected embryos were fixed in 4% paraformaldehyde solution at 4 C for 1 h and embedded in paraffin. Then embryos were processed for paraffin sectioning at 5 μm for immunohistochemical staining. BrdU-labeled cells were detected immunohistochemically in paraffin sections according to the manufacturer's instructions. Apoptosis was assayed by TUNEL staining using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's protocol. The cell proliferation rate was counted (n = 3-6 samples) and calculated as the percentage of BrdU-labeled cells among total nuclear stained cells (4 0 ,6-diamidino-2-phenylindole [DAPI] positive) within a defined arbitrary area. The proliferation rate of control (Shh Cre ) embryos was set to 1, and then the relative proliferation rate of Isl1 ShhCre mutant embryos was calculated.

Histology, immunofluorescence, and in situ hybridization
Whole-mount or section immunofluorescence staining was carried out according to the standard protocol (38). For section immunofluorescence, embryos were fixed in 4% PFA for 30 min, embedded in paraffin, and sectioned at 7 μm. After blocking with 5% BSA, samples were incubated with primary antibodies at 4 C overnight. Secondary antibodies conjugated with Alexa Fluor 488 or 594 (1:1000; Invitrogen) were applied for 30 min in the dark. Primary antibodies used were: E-cadherin (20874-1-AP; Proteintech), BrdU (ab8152, Abcam), Sox9 (mAb 82630; Cell Signaling Technology). Images were analyzed using a microscope Leica DM4 B equipped with a digital camera.
Whole-mount and section in situ hybridization was performed as previously described (39). For section in situ hybridization, Embryos were collected at the desired developmental stages and fixed in freshly made 4% paraformaldehyde (PFA) overnight at 4 C. For whole-mount in situ hybridization, samples were fixed in 4% paraformaldehyde, dehydrated into methanol, and bleached with 6% hydrogen peroxide (H2O2). Non-radioactive antisense RNA probes were generated by in vitro transcription using DIG RNA labeling kit (Roche). For histological analysis, sections were stained with Hematoxylin and Eosin according to standard protocols.
In vitro organ culture Lung rudiments were carefully dissected from E11.5 embryos and placed on Transwell permeable membranes of 0.4μm pore size in PET six-well plates supplied with Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Purmorphamine (0.5 μM) was added to activate canonical Hedgehog signaling. Cell proliferations were monitored after 2 days of culture in a humidified atmosphere of 5% CO2 at 37 C. 10 μM BrdU was administered to the organ culture medium and lungs were incubated for 1 h prior to PFA fixation. At least three sections per embryo were used for quantification.
In vitro primary cell culture Embryonic lungs were carefully dissected from E11.5 mouse embryos. The heart and esophagus were carefully removed. Lungs were incubated in dissociation buffer (Collagenase 100 U/ml, DNAse 100 U/ml and Trypsin 50 U/ml) for 15 min. Dissociated cells were cultured in 48 well plates. Isl1 overexpression vector (pCDH-Isl1) or Control vector (pCDH) was transfected with Lipofectamine3000 reagent. After culturing for 48 h in a humidified atmosphere of 5% CO2 at 37 C, the cells were used for RNA extraction and real-time PCR analysis.

Statistical analyses
All data are presented as means ± SD. Student's t test was used to test differences between two groups of data. One-way ANOVA and Tukey's test were used to analyze the difference between multiple groups. For relative analyses wild-type values were set to 1. p values of < 0.05 were regarded as statistically significant.

Data availability
All data generated or analyzed during this study are included in this published article. Materials will be made available on reasonable request.