Hmga2is required for canonical WNT signaling during lung development
© Singh et al.; licensee BioMed Central Ltd. 2014
Received: 27 December 2013
Accepted: 10 March 2014
Published: 24 March 2014
The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2 −/− ).
We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2 −/− mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression.
Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.
KeywordsBranching morphogenesis HMGA2 GATA6 Lung development WNT signaling
The mouse lung arises from the anterior endoderm and forms during five overlapping phases of lung development: embryonic (embryonic days post coitum (E) 9 to 12.5), pseudoglandular (E12.5 to E16.5), canalicular (E16.5 to E17.5), saccular (E17.5 to post-natal day (P) 5) and alveolar (P5 to P28) [1–3]. At the end of the embryonic phase, primary and secondary lung buds formation has taken place and the embryonic lung consists of one left lobe and four right lobes. From E10.5 to E16.5, the epithelium undergoes branching morphogenesis to form the respiratory (bronchial) tree. In parallel to branching morphogenesis, the airway epithelium differentiates from a morphologically uniform cell population to different specialized cell types, thereby establishing a proximal-distal axis in the developing lung. However, most of the differentiation occurs in the canalicular and saccular phases (E16.5 to P5). The primitive lung epithelium co-expresses several lineage markers including Clara cell-specific 10 kDa protein (Scgb1a1, also CC10) and surfactant-associated protein C (Sftpc, also SP-C). Later in gestation (E16.5 onwards), Scgb1a1 is a marker for the proximal epithelium, whereas Sftpc expression defines the distal epithelium. In the adult lung these markers are characteristic of distinct cell lineages, Scgb1a1 of Clara cells and Sftpc of alveolar type II cells. Only specific progenitor cells in the adult lung, bronchioalveolar stem cells (BASCs), co-express Scgb1a1 and Sftpc.
Several evolutionarily conserved signaling pathways have been implicated in different phases of embryonic lung development. In particular, members of the fibroblast growth factor, bone morphogenetic protein, hedgehog/Gli, epidermal growth factor and wingless secreted glycoprotein (WNT) families have been implicated in lung morphogenesis and epithelial differentiation [2, 5–7]. In addition, a well-organized and balanced interplay between these signaling pathways and key transcription factors of lung development, including NK2 homeobox 1 (also known as thyroid transcription factor 1), forkhead box protein A2 (also known as hepatocyte nuclear factor 3-beta) and GATA6, is required for proper lung formation [2, 3, 7]. GATA6 is the only member of the GATA family of zinc finger transcription factors that is expressed in the distal epithelium of the developing lung [8, 9]. GATA6 is essential for branching morphogenesis and regulates differentiation of distal lung epithelium [9, 10]. Moreover, GATA6 has been implicated in blocking WNT signaling to control the balance between BASC expansion and lung epithelial differentiation required for both lung development and regeneration .
High mobility group AT-hook protein 2 (HMGA2) is a transcription regulator belonging to the family of HMG proteins. HMG proteins are the most abundant non-histone chromatin-associated proteins and regulate gene expression by altering chromatin structure and recruiting other proteins to the transcription regulatory complex . HMGA2 is present at high levels in various undifferentiated tissues during embryonic development and its levels are strongly reduced in the corresponding adult tissues [12, 13]. In addition, Hmga2 expression in adult organs has been implicated in maintaining and activating stem/progenitor cells in different tissues [14, 15]. Here, we show that Hmga2 mRNA levels are high during early stages of lung development, in which cells are undifferentiated, and become reduced and restricted to the distal airways as lung development progresses, coincident with cell differentiation. Analysis of the lung of Hmga2-knockout (KO) mice  revealed enhanced canonical WNT signaling that led to increased cell proliferation, increased number of progenitor cells and reduced differentiation of the distal airway epithelium. Using a lung explants culture system, we confirmed the causal involvement of WNT signaling mediating the effect of Hmga2-loss-of-function (LOF) and showed that Hmga2 is required for proper branching morphogenesis during the formation of the bronchial tree. Furthermore, we showed that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-KO enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium.
Hmga2is expressed in the mouse embryonic lung at the distal airways
In situ hybridization expression pattern analysis in the embryonic lung at E12.5 (Figure 1B), when branching morphogenesis of the lung bud is proceeding rapidly to establish the future bronchial tree, revealed that Hmga2 is ubiquitously expressed with higher levels of expression at the tips of the growing lung buds. Interestingly, Hmga2 expression became restricted to the distal lung endoderm at E14.5. Consistently, immunostaining on sections of the embryonic lung at E14.5 (Figure 1C) supported the presence of HMGA2 in cells of the distal lung endoderm. Co-staining with an antibody specific for the nuclear envelope protein lamin B1 (LMNB1) demonstrated the nuclear localization of HMGA2. The observed expression patterns in embryonic lung suggest a role for HMGA2 in epithelial differentiation.
Hmga2is required for proper differentiation of the distal epithelium during lung development
Hmga2knockout led to enhanced canonical WNT signaling
Enhanced canonical WNT signaling is related to cell proliferation [21–23], correlating with our histological and molecular characterization of the Hmga2-KO embryonic lung. Nevertheless, we observed increased cell proliferation in the mesenchyme of embryonic lung after Hmga2-KO, although Hmga2 expression is restricted to the distal epithelium of the embryonic lung. A plausible explanation for these two observations could be that enhanced WNT signaling after Hmga2-KO is induced in part by diffusible positive regulators of canonical WNT signaling. Indeed, our Affymetrix microarray-based expression analysis (Figure 4A) showed elevated expression of Wnt11, Wnt7b and Wnt2b in embryonic lung of Hmga2 −/− mice when compared to Hmga2 +/+ . These results were confirmed by qRT-PCR-based expression analysis (Figure 4F). Furthermore, immunostaining on sections of embryonic lung using CTNNB1-specific antibody (Figure 4G) showed increased translocation of CTNNB1 from the cytoplasm into the nucleus in cells of both the epithelium and the mesenchyme after Hmga2-KO, demonstrating elevated canonical WNT signaling in both tissues and explaining hyperproliferation and expansion of the mesenchyme after Hmga2-KO.
HMGA2 directly activates Gata6expression
To determine the causal involvement of Gata6 in Hmga2-mediated regulation of WNT signaling, we transfected MLE-12 cells with Gata6 after siRNA-mediated Hmga2- or Fzd2-depletion (Figure 7B and Additional file 3: Figure S3B). Expression analysis showed that Hmga2- and Fzd2-LOF enhanced the expression of canonical WNT targets, supporting our previous expression analysis in the embryonic lung of Hmga2-KO mice and as expected from WNT signaling antagonizing genes. Interestingly, Gata6 transfection compensated the effect of Hmga2-LOF, but not of Fzd2-LOF. Our data indicate that Gata6 acts downstream of Hmga2 and upstream of Fzd2 in negative regulation of WNT signaling (Figure 7C).
Discussion and conclusions
We showed that Hmga2 is expressed in the embryonic mouse lung at the distal airways. Hmga2 mRNA levels were high during early stages of lung development, in which cells are undifferentiated, and decreased as lung development progressed, coincident with cell differentiation. Interestingly, we detected a slight increase of Hmga2 expression at E18.5 that matches with the establishment of a bipotent progenitor cell population in the distal epithelium . Our data correlate with previous reports where HMGA2 was shown to be present at high levels in various undifferentiated tissues during embryonic development and in strongly reduced levels in the corresponding adult tissues [12–15].
Hmga2-KO induces a pygmy phenotype due to reduced expression of insulin-like growth factor 2 mRNA binding protein 2 (Igf2bp2) [15, 16, 29]. Prior to our study, the lung of Hmga2 −/− mice had not been analyzed. Detailed analysis of Hmga2 −/− mice showed that Hmga2 is required for distal epithelium differentiation during embryonic lung development. Hmga2-KO led to enhanced canonical WNT signaling due to an increase of secreted WNT glycoproteins as well as a reduction of the WNT signaling antagonizing proteins GATA6 and FZD2, thereby supporting that Hmga2 regulates WNT signaling at different points of the pathway (Figure 7C). The causal involvement of canonical WNT signaling in mediating the effect of Hmga2-LOF was demonstrated by the DKK1-induced rescue of Hmga2-LOF in embryonic lung explants (Figure 5A-E). HMGA2-mediated regulation of Gata6 seems to be a key process in fine-tuning the activity of canonical WNT signaling in airway epithelium. The sequential order of events suggested in our model (Figure 7C) in which Hmga2 acts upstream of Gata6 is strongly supported by the fact that HMGA2 directly regulates Gata6 (Figure 6A-E) as well as by the Gata6-mediated rescue experiments of Hmga2-LOF in MLE-12 cells (Figure 7B). Since HMGA2 regulates the transcription of its target genes by modulating the chromatin structure and by recruiting other proteins to the transcription regulatory complex , the scope of our future work will be to investigate in detail the mechanism of HMGA2-mediated transcriptional regulation of the Gata6 promoter.
Hmga2-KO increased cell proliferation not only in the lung epithelium, where Hmga2 is expressed, but also in the mesenchyme, suggesting a paracrine effect that could be explained by increased expression of the secreted components of WNT signaling. Since HMGA2 is known to activate transcription, the increased expression of Wnt2b, Wnt7b and Wnt11 after Hmga2-KO suggest the participation of a transcription inhibitor that could block the expression of these secreted components of WNT signaling and whose expression could be regulated by HMGA2 (Figure 7C). Identification of this unknown mediator of HMGA2 will be the scope of future studies. Interestingly, analysis of the Wnt7b promoter showed that deletion of the region between -1,005 bp and -829 bp relative to the second transcription start site significantly increased the basal transcription activity of a Wnt7b-luciferase reporter , suggesting that the binding element of a putative transcription inhibitor was deleted in this construct.
The phenotypes of Hmga2- and Gata6-LOF in embryonic lung explants are very similar (Additional file 2: Figure S2A,B) . However, the milder phenotype observed in the embryonic lung of Hmga2-KO mice when compared either with the Gata6-KO or the phenotype induced after Hmga2-KD in embryonic lung explants might be explained by redundancy in the function between Hmga2 and Hmga1, another member of the HMG protein family. Hmga1 transcript was reduced after Hmga2-LOF (Additional file 4: Figure S4C) but not affected in the Hmga2-KO mice (Additional file 1: Figure S1B). Hmga1 might compensate the Hmga2-KO, thereby avoiding lethality at early embryonic stages, as is the case after Gata6-KO [31, 32], or soon after birth due to defects in the lung, as is the case after lung epithelium-specific ablation of Gata6. In addition, the expansion of the mesenchyme in the embryonic lung after Hmga2-KO and the apparent increase of epithelium in embryonic lung explants after Hmga2-KD might be explained by the differences of both LOF systems. In the transgenic approach, Hmga2-LOF takes place soon after fertilization and affects lung development from the initial stages of lung bud formation; in the explant culture, the LOF starts at E12.5, thereby reducing the rather indirect effect on the mesenchyme and making the effect on the epithelium more dominant. Analysis of the lungs in inducible and conditional double transgenic mice (Hmga2 −/− :Hmga1 −/− ) would test these hypotheses and should be the scope of future studies.
Hmga2 expression is positively regulated by transforming growth factor beta 1 signaling . In addition, our data show that Hmga2 antagonizes canonical WNT signaling. Therefore, it will be of interest to determine a potential opposing effect between these two signaling pathways in establishing the proximal-distal axis during branching morphogenesis and lung epithelium differentiation in the developing lung. Hmga2 might play a crucial role on the interplay between these signaling pathways.
Organ regeneration requires a proper balance between self-renewal and differentiation of tissue-specific progenitor cells. Canonical WNT signaling has been implicated in different regenerative processes including zebrafish tail regeneration, zebrafish cardiac regeneration and expansion of anterior heart field progenitors in mammals [34, 35]. Moreover, canonical WNT signaling is activated upon lung epithelial regeneration, and enhanced WNT activity caused by lung epithelium-specific ablation of Gata6 led to a premature and increased number of BASCs . BASCs represent one of several regional progenitor cell populations in the adult lung and are responsible for regeneration of bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury [4, 36]. Our study suggests the possible role of Hmga2 in the adult lung controlling the balance between BASC expansion and differentiation. Our results are the starting point for future studies in which the relevance of Hmga2-mediated regulation of WNT signaling might be investigated in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells and lung regeneration during both homeostatic turnover and repair after injury. Characterization of the regulatory mechanisms controlling the proper balance between expansion and differentiation of lung stem/progenitor cells will have a profound impact on our understanding and treatment of lung disease.
Mouse work was performed in compliance with the German Law for Welfare of Laboratory Animals. The permission to perform the experiments presented in this study was obtained from the Regional Council (Regierungspräsidium in Darmstadt, Germany). The numbers of the permissions are IVMr46-53r30.03.MPP04.12.02 and IVMr46-53r30.03.MPP06.12.01. Animals were killed for scientific purposes according to the law mentioned above, which complies with national and international regulations.
C57BL/6 and Hmga2 +/− mice (stock # 002644, Jackson Laboratories)  were obtained from Charles River Laboratories (Germany) at 5 to 6 week of age. BAT-GAL transgenic reporter mice (also called beta-catenin/TCF/LEF reporter transgenic mice) were obtained as a gift from Prof. Stefan Liebner . BAT-GAL:Hmga2 −/− double transgenic embryos were obtained by crossing heterozygous BAT-GAL:Hmga2 +/− mice. Animals were housed and bred under controlled temperature and lighting (12/12-hour light/dark cycle), fed with commercial animal feed and water ad libitum. All experiments were performed with mouse embryonic lungs. Timed-pregnant C57BL/6 WT mice were killed at indicated time points; embryonic lungs were isolated according to standard methods and whole-mount in situ hybridization was performed as described  with minor modifications. Briefly, to synthesize digoxigenin-labeled RNA probes, pcDNA3-mHmga2 plasmid (gift from Prof. Peter Grouse)  was linearized, and UTP-digoxigenin (Roche) substituted antisense RNA probes were transcribed with T7 RNA polymerase. Sense RNA probes as negative control were transcribed with SP6 RNA polymerase.
Embryonic lung explants culture
Lungs of timed-pregnant C57BL/6 WT mice were dissected from the embryos at E12.5 and cultured for 72 hours till E15.5 equivalent (E15.5*) as previously reported . The explants were treated with 3 μM siRNAs against Hmga2 (Applied Biosystems, Silencer Select siRNAs, Assay ID s67600), scrambled siRNA (negative control, Ctrl) (Sigma, MISSION siRNA Universal Negative Control, SIC001) or 200 nM of mouse recombinant DKK1 (R&D Systems, 5897-DK-010) following a similar protocol as previously described [9, 41–44]. The siRNA and protein treatments were renewed every 24 hours. After 72 hours, the lungs were checked for morphological changes by standard microscopy techniques and harvested for RNA (QIAGEN RNeasy Micro Kit) and protein isolation. The images were used to determine the total number of terminal bud branches and for quantification of total branch length as described .
Cell culture transfection assays
Mouse lung epithelial cells (MLE-12, ATCC CRL-2110) were cultured following the supplier’s instructions. MLE-12 cells were transiently transfected either with 40 nM siCtrl (negative control; AM4611, Ambion), 40 nM siGata6 (L-065585-00, Dharmacon), 20 nM siFzd2 (s81164, Applied Biosystems), 20 nM siHmga2 (s67600, Applied Biosystems) and/or pcDNA 3.1(A)-Hmga2-myc/His or pCMV6-entry-Gata6-flag/myc or pBL (Ctrl) as indicated using Lipofectamine 2000 transfection reagent (Invitrogen) at a ratio of 1:2 DNA:Lipofectamine. Cells were harvested 48 hours later for further analysis.
The proximal 631 bp Gata6 promoter was amplified and cloned into the pGL4basic vector to generate pGL4-Gata6 promoter luciferase vector. Dual-luciferase reporter assays (Promega) were performed as described  following transient transfection of MLE-12 cells in 96-well plates with 20 nM effector siRNAs and a total of 100 ng DNA per well, containing 15 ng effector plasmid, 15 ng pGL4-Gata6 promoter or p3LEF-LUC luciferase reporter plasmid, 1 ng Renilla luciferase reporter plasmid and 69 ng pBluescript. Each sample was performed in triplicate. Each experiment was repeated at least three times.
Affymetrix microarrays, quantitative PCR and ChIP assays
Total RNA was isolated with RNeasy® plus mini kit (Qiagen) and quantified using a spectrophotometer. Affymetrix microarray-based transcriptome analysis of Hmga2−/− and Hmga2 +/+ embryonic lung (E18.5) was performed and analyzed as described . Kyoto Encyclopedia of Genes and Genomes pathway enrichment based analysis of dysregulated pathways in Hmga2−/− versus Hmga2 +/+ was done using DAVID software  and generation of fold change and Heat map were performed using DNAStar Arraystar 11.0. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  through accession number [GEO:GSE55340] (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55340).
The High Capacity Reverse Transcription kit (Applied Biosystems) was used for synthesis of cDNA from total RNA. Quantitative real-time PCR reactions were performed using SYBR® Green on the Step One plus Real-time PCR system (Applied Biosystems). The PCR results were normalized with respect to the housekeeping gene for tubulin alpha 1a (Tuba1a) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh).
ChIP analysis of the mouse Gata6 promoter was performed as described  with slight modifications. Briefly, MLE-12 cells were cross-linked by 1% formaldehyde for 10 minutes, lysed, and sonicated with Diagenode Biorupter to an average DNA length of 500 to 600 bp. After isolation, the soluble chromatin was immunoprecipitated with immunoglobulin G (control, Santa Cruz) or HMGA2-specific antibody (sc-30223; Santa Cruz Biotechnology). Reverse cross-linked immunoprecipitated chromatin was subjected to qPCR using the primers listed in Additional file 5: Table S1.
For paraffin-embedded mouse embryonic lung tissue, lungs were fixed overnight in 1% paraformaldehyde at 4°C, dehydrated over a graded series of alcohol, and paraffin embedded. Sections of 4 μm were prepared on a microtome (Leica). Antigen retrieval was performed by microwave heating for 8 minutes using 1 mM EDTA (pH 8) or 1 mM citrate buffer (pH 6). For cryosections of mouse lung tissue, lungs were harvested and embedded in polyfreeze tissue freezing medium (Polysciences). Sections of 10 μm were prepared on a cryostat (Leica). Sections were post-fixed in 4% paraformaldehyde for 10 minutes. Antibody staining was performed following standard procedures. All incubations and washes were performed with histobuffer containing 3% bovine serum albumin and 0.2% Triton X-100 in 1× phosphate-buffered saline, pH 7.4. Non-specific binding was blocked by incubating with donkey serum and histobuffer (1:1 (v/v) ratio) for 45 to 60 minutes. The sections were then incubated with primary and secondary antibodies for 60 minutes followed by nuclear staining. The sections were examined with a confocal microscope (Zeiss) or fluorescent microscope (Leica). Antibodies used were specific against HMGA2 (BioCheck), LMNB1 (Santa Cruz), GATA6 (R&D system), cadherin 1 (Abcam), VIM-Cy3 (Sigma), PCNA (Santa Cruz), MKi67 (Abcam), KRT (Dako and Sigma), Pro-SFTPC (Millipore), SOX9 (Santa Cruz), ABC (Millipore), CTNNB1 (Abcam), ACTA2-Cy3 (Sigma) and clCASP3 (Cell Signaling). Secondary antibodies used were Alexa 488, Alexa 633 and Alexa 555 (Invitrogen). DAPI (Invitrogen) were used as nuclear dye.
Paraformaldehyde-fixed and paraffin-embedded lung tissue sections were stained with hematoxylin and eosin and used for the lung morphology analysis. Figures were elaborated following a color scheme recommended to make them visible and easy to interpret by people with all types of color vision [51, 52].
Western blotting was performed following standard protocols and using antibodies specific for HMGA2 (Biocheck and Santa Cruz), TUBA1A (Sigma), GATA6 (R&D system), FZD2 (Abcam), active-beta-catenin (ABC, Millipore), LRP6 (Cell signaling), phosphorylated LRP6 (Cell signaling), AXIN2 (Abcam), BMP4 (Millipore), MYCN (Santa Cruz) and LMNB1 (Santa Cruz). Immunoreactive proteins were visualized with the corresponding horseradish peroxide-conjugated secondary antibodies using the Super Signal West Femto detection solutions (Thermo Scientific). Signals were detected and analyzed with Luminescent Image Analyzer (Las 4000, Fujifilm).
In total, the lungs of six Hmga2 −/− mice were analyzed using different techniques. The lungs of six Hmga2 +/+ were used as control because the heterozygote Hmga2 +/− mice presented a mild phenotype. With the exception of the Affymetrix array-based expression analysis, the experiments were performed at least three times and the samples in each experiment were analyzed in triplicates. The Affymetrix array-based expression analysis was performed one time using biological duplicates. Statistical analyses were performed using Excel Solver. All data are represented as mean ± SEM. One-way analyses of variance (ANOVA) were used to determine the levels of difference between the groups and P values for significance.
All affiliations in Germany are members of the Universities of Giessen and Marburg Lung Center (UGMLC) and the German Center of Lung Research (DZL).
bronchioalveolar stem cells
gain of function
loss of function
quantitative reverse transcription polymerase chain reaction
short interfering RNA
We thank R Bender and K Goth for technical support; S Liebner, P Grouse, PR Strauss, J Kwon for reagents and VS Nikam, R Voswinckel and N Oeztuerk for helpful discussions. This work was done according to the program of competitive growth of the Kazan Federal University and the Russian Government. GB is funded by the “LOEWE-Initiative der Landesförderung” (Wiesbaden Germany) (III L 4 – 518/15.004 2009) and the “Deutsche Forschungsgemeinschaft” (DFG, Bonn, Germany) (BA 4036/1-1).
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