LIX1 regulates YAP1 activity and controls the proliferation and differentiation of stomach mesenchymal progenitors
© McKey et al. 2016
Received: 29 January 2016
Accepted: 18 April 2016
Published: 28 April 2016
Smooth muscle cell (SMC) plasticity maintains the balance between differentiated SMCs and proliferative mesenchymal progenitors, crucial for muscular tissue homeostasis. Studies on the development of mesenchymal progenitors into SMCs have proven useful in identifying molecular mechanisms involved in digestive musculature plasticity in physiological and pathological conditions.
Here, we show that Limb Expression 1 (LIX1) molecularly defines the population of mesenchymal progenitors in the developing stomach. Using in vivo functional approaches in the chick embryo, we demonstrate that LIX1 is a key regulator of stomach SMC development. We show that LIX1 is required for stomach SMC determination to regulate the expression of the pro-proliferative gene YAP1 and mesenchymal cell proliferation. However, as stomach development proceeds, sustained LIX1 expression has a negative impact on further SMC differentiation and this is associated with a decrease in YAP1 activity.
We demonstrate that expression of LIX1 must be tightly regulated to allow fine-tuning of the transcript levels and state of activation of the pro-proliferative transcriptional coactivator YAP1 to regulate proliferation rates of stomach mesenchymal progenitors and their differentiation. Our data highlight dual roles for LIX1 and YAP1 and provide new insights into the regulation of cell density-dependent proliferation, which is essential for the development and homeostasis of organs.
The gastrointestinal (GI) tract is a vital organ, highly conserved across vertebrate species and essential for the absorption of water and nutrients. During development, the GI tract arises from a primary uniform tube composed of mesoderm and endoderm. The mesoderm gives rise to the digestive mesenchyme, which in turn differentiates into multiple tissues, such as the submucosa and the musculature, which is composed of smooth muscle cells (SMCs) and interstitial cells of Cajal [1, 2]. The process of digestive mesenchyme development into SMCs is commonly decomposed into two major steps . Mesenchymal progenitor cells first enter a determination program (that we will refer to as SMC determination), mainly characterized by the early expression of alpha smooth muscle actin (αSMA). Later during development, determined SMCs enter a more differentiated state (that we will refer to as SMC differentiation), mainly characterized by the expression of proteins involved in smooth muscle contractility, such as CALPONIN and CALDESMON.
Unlike many other mature cell types in the adult body, such as skeletal muscle cells, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate. Indeed, SMCs have the unique ability to switch between a differentiated, quiescent contractile state and a highly proliferative and migratory phenotype in response to internal or external cues [1, 4]. SMC plasticity plays crucial roles in maintaining muscular tissue homeostasis during perinatal development and postnatal stages. In humans, the disruption of this balance is a major underlying cause of disease [4, 5]. Because tissue plasticity involves the reactivation of developmental processes, developmental studies of the process regulating the differentiation of mesenchymal progenitors into SMCs have proven to be useful in identifying the molecular mechanisms involved in the regulation of digestive musculature plasticity during normal development and in pathological conditions [6, 7].
Using a microarray approach to identify candidate genes in stomach mesenchyme development , an approach that had already enabled our group to characterize the RNA-binding protein RBPMS2 as a regulator of SMC differentiation and plasticity [6, 9], we screened for genes that demonstrated higher expression at the earliest stages of stomach development. This allowed us to identify Limb Expression 1 (LIX1), a gene coding for a 281-amino acid protein. Although predictive in silico studies have shown that LIX1 has a double-stranded RNA binding domain, suggesting that it could be involved in RNA processing , no cellular function of LIX1 has yet been described. Chicken (Gallus gallus) LIX1, first identified in a gene expression screen to identify new markers of limb development, was shown to be expressed in the anterior and posterior intestinal portals, the early structures that invaginate to give rise to the primary intestinal tube . Moreover, the arthropod homolog of LIX1, lowfat, has been characterized, through its interaction with the atypical cadherins fat and dachsous, as a component of the Hippo pathway [10, 12]. The Hippo pathway has been at the centre of many studies regarding its role in maintaining tissue homeostasis through the regulation of the balance between cell proliferation and differentiation. The key downstream regulator of the Hippo pathway is Yes-Associated Protein (YAP1), a transcriptional co-activator that mainly interacts with transcription factors of the TEAD family, which are essential in mediating YAP-dependent gene expression [13–15]. Indeed, the Hippo core cascade of kinases is activated when proper cell density and organ size are reached, leading, in humans, to the inhibitory phosphorylation of YAP1 on Serine-127 [16, 17]. This leads to decreased transcription of YAP1 mitogenic targets, resulting in a decrease in cell proliferation and entry into a more differentiated state . Although LIX1 has recently been shown to stimulate progenitor proliferation during skeletal muscle regeneration in mouse , there is no evidence to date to support a role for LIX1 in regulating the activity of YAP1 in vertebrates.
In the present study, we investigated the roles of LIX1 and YAP1 during digestive SMC development. We show that LIX1 is a novel and, thus far, unique marker of stomach mesenchymal progenitors and that its expression is strong and highly dynamic during development. We demonstrate that LIX1 stimulates the expression of YAP1 transcripts and YAP1 activity and that both LIX1 and YAP1 are key regulators of the development of stomach mesenchymal progenitors. Finally, we show that YAP1 activity is required for the regulation of the proliferation and differentiation of the stomach mesenchyme.
LIX1 defines stomach mesenchymal progenitors
LIX1 silencing impairs mesenchyme determination and decreases YAP1 activity
LIX1 misexpression expands the determined SMC domain and stimulates cell proliferation and YAP1 activity
Endogenous LIX1 expression is regulated by the FGF pathway during SMC determination
Sustained LIX1 expression decreases YAP1 activity and hinders SMC differentiation
The ability of LIX1 to regulate cell proliferation is dependent on cell density
Our study first identified LIX1 as a novel and thus far unique marker of stomach mesenchymal progenitors. To our knowledge, LIX1 is the first described gene to define the population of mesenchymal progenitors and to allow discrimination between undetermined and determined SMC states in the stomach. Collectively, our in vivo gain- and loss-of-function experiments clearly demonstrate that LIX1 is a key regulator of stomach mesenchyme development, by regulating both the determination and the differentiation of SMCs. Our study further demonstrates that YAP1 is a key relay of the function of LIX1 during these developmental processes.
We first identified LIX1 as an essential regulator of stomach mesenchyme determination. We thus suspect that the expression of LIX1 must be tightly regulated in the developing mesenchyme to allow fine-tuning of the transcript levels and the state of activation of the pro-proliferative transcriptional coactivator YAP1, which in turn controls the rates of proliferation required for correct SMC determination. We further show that the FGF signalling pathway could be involved in the regulation of LIX1 expression at determination stages. Most studies published so far have identified some regulators of YAP1 at the level of its activity, through its phosphorylation, localisation and stability . Our study identifies LIX1 as a new regulator of YAP1 mRNA levels, which is a novel finding. This could result from a regulation of the transcription of YAP1 mRNA or from a regulation of its stability. Expression data were not always statistically significant for TEAD4. However, results were consistent between TEAD1 and TEAD across all experiments. We thus attribute the lack of significance in some cases of effects on TEAD4 to low statistical power rather than to absence of an effect. These functional in vivo data suggest that LIX1 also regulates the expression of the TEAD transcription factors, which are essential in mediating YAP-dependent gene expression , indicating that LIX1 is an upstream regulator of YAP signalling. Further investigations will allow us to understand by which mechanisms LIX1 regulates the level of YAP1 and TEAD transcripts. Interestingly, in silico studies have shown that LIX1 has a double-stranded RNA-binding domain, suggesting that it could be involved in mRNA or micro-RNA processing  and it has been shown that miR-506 and miR-375 regulate YAP1 expression [30, 31]. It would thus be interesting to study whether LIX1 has a direct impact on YAP1/TEAD mRNA expression and/or stability.
We then demonstrated that LIX1 is an essential regulator of SMC differentiation. Intriguingly, while the pro-proliferative activity of LIX1 presumably facilitates SMC determination, LIX1 has a negative impact on further SMC differentiation. We suspect that high proliferative activity of LIX1 led to cell contact inhibition of proliferation, revealing the presence of a negative feedback loop on the endogenous expression and activity of YAP1 within the stomach mesenchyme to compensate for aberrant cell proliferation. Accordingly, we never observed hypertrophic stomachs under LIX1 influence, suggesting that LIX1 pro-proliferation activity is limited by the overall size of the stomach. In response to high cell density, the Hippo pathway regulates YAP1 activity through inhibitory phosphorylation  and we report here that the defect in SMC differentiation is associated with an increase in inactive phosphorylated YAP1 in LIX1-misexpressing stomachs. While the Hippo pathway has already been investigated in the context of gastrointestinal epithelia [28–30, 33], our study is the first to suggest a role for this pathway in regulating the proliferation and differentiation of the gastrointestinal mesenchyme. Along these lines, the next step would be to address the possible regulation of the Hippo pathway by LIX1 in this developmental process. Lowfat, the arthropod homolog of LIX1, interacts with the atypical cadherins fat and dachsous and stabilizes FAT protein levels . Although a recent study has shown that the vertebrate ortholog of FAT does not seem to regulate the Hippo pathway , FAT signalling has been shown to decrease YAP1 activity [35, 36]. One could thus speculate that, in the context of cell contact inhibition of proliferation, LIX1 participates more directly in the inhibition of YAP1 through the stabilization of FAT levels. Further investigations should focus on uncovering the potential molecular links that tie LIX1 to the regulation of YAP1 phosphorylation and transcriptional output.
Similarly to our conclusions for LIX1, we also report that while the pro-proliferative activity of YAP1 presumably facilitates SMC determination, it is sensitive to cell contact inhibition of proliferation and has a negative impact on further SMC differentiation. Because our misexpression experiments only led to mild overexpression of YAP1 (ranging from 1.2- to 3-fold), we speculate that the native stomach mesenchyme is poised to respond to mild over-activity of YAP1 by turning on the negative feedback loop on YAP1 activity. This finding contrasts with those of previous studies where high levels of YAP1 overexpression led to sustained proliferation and overgrowth of undifferentiated cells [17, 37]. In any case, the compensatory mechanisms resulting from LIX1 or YAP1 misexpression appeared to lock the determined mesenchyme in a state where the cells were neither proliferative nor differentiated. This state could simply reflect the requirement for a dynamic proliferation event between the determination and differentiation steps. By this hypothesis, because determined LIX1/YAP1-expressing cells are in contact inhibition of proliferation, differentiation could not be initiated. Alternatively, we could speculate that a certain level of YAP1 activity is necessary to initiate SMC differentiation, and because YAP1 activity has been turned off as a consequence of aberrant cell proliferation at the determination stage, differentiation could not be initiated. This second hypothesis highlights the possibility that YAP1 plays a dual role in regulating stomach mesenchyme progenitor development, both during the proliferative phase and later on during the differentiation phase. This hypothesis concords with emerging data showing that YAP1 regulates multiple signalling pathways, such as Wnt, BMP and Notch , and that Hippo signalling regulates Notch signalling . Interestingly, all of these pathways are involved in the development of the GI tract [1, 6, 19, 40–42]. Further investigations are required to examine how YAP1 signalling is integrated in the regulation of SMC differentiation. YAP1 could be cooperating with two different transcription factors to regulate the processes of mesenchyme proliferation and SMC differentiation, similarly to what has recently been described during self-renewal of the intestinal epithelium . In that system, the authors showed that YAP1 cooperates with Klf4 in promoting differentiation of intestinal goblet cells. Klf4 has been shown to abrogate the expression of myocardin, a major regulator of SMC differentiation , and of myocardin-induced expression of SMC genes , while YAP1 has been shown to interact with myocardin and interfere with its activity .
Altogether, our results demonstrate that LIX1 is a novel and unique marker of digestive mesenchyme immaturity and a regulator of mesenchymal progenitor proliferation and differentiation through its capacity to regulate YAP1 activity and density-dependent proliferation. Additionally, we demonstrate that this activity of LIX1 is conserved in cell culture, suggesting that the mechanism of LIX1 action outlined here is not limited to the developing stomach mesenchyme. In light of these conclusions, it would be interesting to investigate whether the activity of LIX1 is conserved throughout the more general context of organ size control and tissue regeneration. Finally, we have highlighted, through a developmental approach, three properties of LIX1 that could make it essential in cancer research. LIX1 defines an immature state of stomach smooth muscle, regulates cell proliferation within this immature mesenchyme and regulates the activity of the oncogene YAP1. These three properties thus point to the interest of further studies to examine the possible function of LIX1 in tumorigenesis and tumour progression.
Chick embryonic GI tissues
Fertilized White Leghorn eggs from the Haas Farm (France) were incubated at 38 °C in humidified incubators. Embryos were staged according to Hamburger and Hamilton . Isolation of mesodermal and endodermal layers from stage 25 stomachs (referred to as E5) was performed as previously described . The efficiency of dissections was evaluated by monitoring the expression of SHH and BARX1, which are specific markers of the epithelial and mesenchymal layers, respectively.
Avian retroviral misexpression system and constructs
Chick LIX1 full-length cDNA was isolated from total mRNA extracts of E5 stomachs. The mouse YAP1, the chick full-length LIX1, the human full-length LIX1 and the Short hairpin RNA of LIX1 (Gallus target sequence: TCT TTG CAG CTG GTG ATT G, referred to as ShLIX1) associated with the mouse U6 promoter were cloned into the shuttle vector Slax13 and then subcloned into the Replication-Competent Avian Leucosis Sarcoma virus strain A (RCAS(A)) or strain B (RCAS(B)) vectors. Using RCAS vectors with two different envelopes (A and B) allows the introduction of two genes into a single cell . FGF8, sFGFR2b and GFP retroviral constructs have been previously described . RCAS(A)-shPROX1 retrovirus  served as unrelated RCAS-ShRNA retroviruses. Retroviral constructs were transfected into the chicken DF-1 fibroblast cell line (ATCC-LGC) to produce retroviruses. Retroviruses were titered using standard techniques and injected into the splanchnopleural mesoderm of E1.5 chicken embryos to target the stomach mesenchyme . Embryos were co-injected with RCAS-GFP to allow screening of correctly targeted stomachs. Eggs were then placed at 38 °C until harvested. Efficient retroviral infection was confirmed by in situ hybridization analysis on paraffin sections using ENV probes or, in LIX1 misexpression experiments only, LIX1 probes. Infection with RCAS-GFP retroviruses does not affect chick stomach development (Additional file 10: Figure S9). Stomach phenotypes from infected embryos were analysed by comparison with uninfected control embryos incubated at the same time.
Cell cultures and analysis
The chicken DF-1 fibroblast cell line was cultured as previously described . Cell growth in DF-1 cultures was assessed using the Muse Count and Viability reagent following the manufacturer’s specifications (Muse Cell Analyzer – Millipore). DF-1 cells were plated on plastic at 2000 cells/cm2 to obtain low-density cultures and 6000 cells/cm2 to obtain high-density cultures. Verteporfin (Sellekchem) was used applied to DF-1 cells for 20 hours at a final concentration of 1 μM.
In situ hybridization and immunofluorescence staining
Dissected GI tissues were fixed in 4 % paraformaldehyde at room temperature for 30 minutes, washed in PBS, gradually dehydrated in methanol and stored at −20 °C before processing for whole-mount in situ hybridization as previously described [8, 22]. For sections, GI tissues were fixed in 4 % paraformaldehyde at room temperature for 30 minutes, washed in PBS, gradually dehydrated in ethanol and embedded in paraffin. 10-μm sections were cut using a microtome and collected on poly-L-lysine-coated slides (Thermo Fisher). Partial chick YAP1, CTGF and CYR61 cDNAs were isolated from total mRNA extracts of E5 stomachs. In situ hybridization experiments on whole-mount and paraffin sections were carried out as previously described  using chick LIX1 and YAP1 probes and published SM22, BAPX1, SOX10 and ENV probes [8, 19, 24]. Immunofluorescence studies were performed on paraffin sections using polyclonal antibodies against aSMA (Abcam Cat# ab5694 RRID:AB_2223021 1:400 dilution), anti-Phospho-Histone H3-Ser10 (PH3) (Millipore Cat# 06–570 RRID:AB_310177, 1:300 dilution), cleaved CASPASE-3 (Cell Signaling Technology Cat# 9664S RRID:AB_331453, 1:400 dilution) and monoclonal antibodies against CALPONIN (Abnova Cat# MAB1512 RRID:AB_1672405, 1:500 dilution). Nuclei were labelled with Hoechst (Invitrogen). In vivo proliferation rates were assessed by counting the number of PH3-positive cells relative to the total number of nuclei in the section. Cell density was assessed on images of stomach sections by calculating the area occupied by Hoechst-stained nuclei relative to the total area of the section.
Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from stomachs or cell cultures with the HighPure RNA Isolation kit (Roche). Reverse transcription was performed using the Verso cDNA synthesis kit (Thermo Scientific) and RT-qPCR was performed using LightCycler technology (Roche Diagnostics). PCR primers (Additional file 11: Table S2) were designed using the LightCycler Probe Design 2.0 software. Each sample was analysed in three independent experiments done in triplicate. Expression levels were determined with the LightCycler analysis software (version 3.5) relative to standard curves. Data were represented as the mean level of gene expression relative to the expression of the reference genes UBIQUITIN or GAPDH. Relative mRNA expression was calculated using the 2–ΔΔCT method .
DF-1 cells and chick stomachs were re-suspended in lysis buffer (20 mM Tris pH 8, 50 mM NaCl, 1 % NP40, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)); 10 μg of total protein lysates were boiled in SDS-PAGE sample buffer, separated by 10 % SDS-PAGE and transferred to nitrocellulose membranes. Membranes probed with rabbit anti-phospho-YAP (Ser127; Cell Signaling Technology Cat# 4911S RRID:AB_2218913, 1:1000 dilution), anti-YAP/TAZ (Cell Signaling Technology Cat# 8418S RRID:AB_10950494, 1:1000 dilution) or anti-GAPDH (Sigma-Aldrich Cat# G9545 RRID:AB_796208, 1:5000 dilution) antibodies overnight. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used to confirm equal loading. All immunoblots were developed and quantified using the Odyssey infrared imaging system (LICOR Biosystems) and infrared-labelled secondary antibodies.
Data were analysed by performing two-tailed or, when appropriate, one-tailed Mann–Whitney tests using GraphPad Prism 6 software. Values of n represent the number of biological replicates. Each value used for statistical analyses is the mean of three technical replicates. Results were considered significant when P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) or P < 0.0001 (****).
Images were acquired using a Nikon Multizoom AZ100 stereomicroscope and a Carl Zeiss AxioImager microscope. Images presented in the figures are representative of the main phenotype observed in the population of infected embryos (Additional file 4: Table S1).
Availability of data and materials
Data supporting the results of this article are available in Additional file 12.
Research was supported by a Trampolin grant (N°15681) from the Association Française contre les Myopathies (AFM) to SF, a grant from AFM (N°18766) to PdSB and the French Association for CIPO patients to PdSB. JM had a studentship from the Ministère de l’Education Nationale de l’Enseignement Supérieur et de la Recherche (MENESR). The authors thank Prof EN Olson for the mouse YAP1 construct, members of the “Development of visceral smooth muscle and associated pathologies” team of the INSERM U1046 for comments, Azzouz Charrabi and Valérie Scheuermann for technical assistance, and Prof Doyle McKey for critical reading of the manuscript.
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