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Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signaling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm (PSM) during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes, however the underlying mechanisms remain obscure. Transcriptional profiling of Wnt3a−/− embryos led to the identification of the bHLH transcription factor, Mesogenin1 (Msgn1), as a direct target gene of Wnt3a. To identify Msgn1 targets, we performed genome-wide studies of Msgn1 activity in embryonic stem cells. Here we show that Msgn1 is a major transcriptional activator of a Notch signaling program, synergizing with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.
Somites are blocks of paraxial mesoderm that bud from the anterior PSM every 2 hours in the mouse embryo, and ultimately give rise to the musculoskeletal system. The timing of somitogenesis is controlled by a network of oscillating genes in the Notch, Wnt and Fgf signaling pathways1. Notch target genes such as Lunatic fringe (Lfng) are amongst the first cyclic genes to be characterized2. Lfng is first expressed in the posterior PSM and sweeps anteriorly, arresting as a stripe in the anterior PSM, before a repetitive wave of gene expression begins anew3,4,5. The oscillation frequency matches the rate of somite formation, leading to the proposal that cyclically-expressed genes constitute a segmentation clock that controls the timing of somitogenesis6. The demonstration that the Notch/Fgf pathways oscillate out of phase with the Wnt pathway1, and that the loss of activity of any one of the signaling pathways adversely affects oscillatory gene expression in all three pathways7,8,9,10, strongly suggests that these signaling pathways interact in a coordinated and reciprocal fashion. Wnt3a, signaling via ®-catenin, controls oscillatory gene expression in the Notch pathway since cyclic Lfng expression ceases in the absence of either gene 7,8,11. Although it has been proposed that Wnt signaling does so through the direct activation of the Notch ligand Dll112, Dll1 levels remain surprisingly robust in Wnt3a mutants despite the dramatic down regulation of other direct Wnt target genes such as Axin27,11. These results suggest that alternative mechanisms might account for the regulation of the Notch pathway and the segmentation clock by Wnt3a.
A posterior-anterior gradient of Wnt3a/®-catenin and Fgf activity across the PSM plays an important role in translating the rhythmic output of the segmentation clock into a periodic array of somites13,14. The Wnt/Fgf gradient positions segment boundaries by establishing a threshold of activity (termed the determination front), below which anterior PSM cells can respond to the segmentation clock by activating the boundary determination genes Mesoderm posterior 2 (Mesp2) and Ripply28,15,16,17. Reduced Wnt3a/®-catenin signaling in the anterior PSM is crucial for determination front activity since the overactivation of ®-catenin in the anterior PSM maintained the clock and blocked Mesp2/Ripply2 expression, resulting in a profound elongation of the PSM and delayed somitogenesis8,18. Thus, Wnt3a functions in both the clock and the determination front during somitogenesis, however it remains unclear how Wnt3a performs both functions.
Stimulation of the Wnt/®-catenin signaling pathway stabilizes ®-catenin, which interacts with members of the Lef/Tcf family of DNA-binding factors to activate the transcription of target genes19. We show here that the bHLH transcription factor gene Msgn1 is a target of the Wnt3a/®-catenin pathway and that the Wnt3a gradient defines the oscillatory field through the induction of Msgn1. Msgn1, in turn, directly activates the expression of Notch pathway genes, including cyclic genes. Thus, Msgn1 directly links the Wnt3a signaling gradient to the Notch signaling pathway and the segmentation clock.
Transcriptional profiling of presomite-stage wildtype and Wnt3a−/− embryos revealed that Msgn1 is downregulated in Wnt3a mutants (Fig. 1a). Msgn1 was of particular interest since the Msgn1−/− phenotype, which includes the absence of posterior somites20, is similar to the phenotypes observed in Wnt3a−/− and Ctnnb1fl (®-catenin) mutants8,21. Msgn1 is an excellent candidate target gene of Wnt3a since it is spatiotemporally coexpressed with Wnt3a in the gastrulating (Fig. 1b,c) and segmenting (Fig. 1d) embryo. The Msgn1 anterior border lies posterior to the stripes of Mesp2/Ripply2 expression22 and thus coincides with the position of the determination front. Importantly, the anteroposterior (AP) position of this border in the PSM depends upon Wnt3a and Ctnnb1 activity. Wnt3a or Ctnnb1 loss-of-function (LOF) leads to a loss of Msgn1 expression (Fig. 1e,f), while the conditional stabilization of ®-catenin in the PSM results in a remarkable anterior expansion of Msgn1 expression (Fig. 1g). To test if Wnt3a can activate Msgn1 dose-dependently, we treated pluripotent embryonic stem cells (ESC), which retain the potential to form mesoderm, with recombinant Wnt3a in serum-free conditions23. Wnt3a treatment led to a potent dose-dependent induction of Msgn1 mRNA (Fig. 1h). Taken together, the data suggest that the Wnt3a gradient spatially defines the Msgn1 expression domain in the PSM, and that the anterior border of Msgn1 may be an important component of the determination front.
Although our genetic studies, together with in vitro and transgenic analyses of the Msgn1 promoter (Fig. 2a-d), clearly demonstrate that Msgn1 is directly controlled by a Wnt3a signaling gradient, Msgn1 is not highly graded across the PSM at early somitogenesis stages (Fig.1d) suggesting that additional regulators maintain Msgn1 in more rostral domains. Previous analysis of the Msgn1 promoter showed that Msgn1 is activated by the Tbox transcription factor Tbx6, in addition to Wnt signaling24. Tbx6 is another important regulator of somitogenesis25 and is thought to be expressed in an identical domain as Msgn122. Re-examination of Tbx6 and Msgn1 coexpression demonstrates that although similar, Tbx6 expression exceeds Msgn1 (Fig. 2e,f), suggesting that Tbx6 is insufficient to define the position of the Msgn1 anterior border. To determine whether Tbx6 is required for Msgn1 expression in the anterior PSM, we examined E8.5 Tbx6−/− embryos by WISH and found that the anterior border of the Msgn1 domain was indeed shifted posteriorly, and Msgn1 mRNA was now expressed in a graded fashion in the posterior-most PSM (Fig. 2g,h). Reducing Wnt3a dosage on the Tbx6−/− background led to reductions in Msgn1 expression below levels observed in Tbx6−/− embryos (Supplementary Fig. S1), providing genetic evidence that the weak, graded Msgn1 expression observed in Tbx6 mutants depends upon Wnt3a. We conclude that Tbx6 maintains Msgn1 expression in the anterior PSM, functioning together with a Wnt3a gradient to generate an anterior boundary that defines the determination front.
The initial characterization of the segmentation phenotype in Msgn1−/− embryos was performed on E9.5 embryos, a relatively late stage when mutants already lack posterior somites and PSM and display enlarged tailbuds20. The highly abnormal tailbud complicates the interpretation of the segmentation phenotype since the lack of posterior somites could be secondary to an inability of tailbud progenitors to migrate or give rise to PSM fates. Therefore, to address the potential role that Msgn1 may play in the determination front, we examined Msgn1−/− mutants for the expression of the segment boundary determination genes Ripply2 and Mesp2, and the posterior somite marker Uncx4.1, at E8.5 when mutants are not clearly distinguished from littermates. WISH analysis revealed that all three genes were downregulated, while the primitive streak (PS) marker, Fgf8, was unaffected (Fig. 3a-d). The lack of expression of segment boundary determination genes at early somitogenesis stages demonstrates that Msgn1 is required for determination front activity and suggests that Msgn1 is a functional effector of the Wnt3a gradient.
Msgn1 may also play an important role in the segmentation clock since cycling genes in the Notch pathway were shown to be downregulated in Msgn1 mutants at E9.520. Since this reduced gene expression could arise secondarily, due to the large number of apoptotic cells reported in the E9.5 Msgn1−/− tailbud20, we reinvestigated the expression of cycling genes at the earlier E8.5 stage. We first examined mutants for the expression of cyclic Fgf and Wnt pathway genes, as interactions between all three signaling pathways are necessary for a functional segmentation clock. WISH analysis of Msgn1−/− embryos, or explanted PSMs processed by the “fix and culture” method6 to assess the dynamic expression of cycling genes, revealed that the cyclic Wnt target genes Sp5 (Fig. 4a-f) and Axin2 (not shown) were upregulated and anteriorly expanded (Fig. 4a,b) but did not oscillate in the Msgn1−/− PSM (Fig. 4c-f). The oscillating Fgf pathway genes Sprouty2 (Fig. 4g-l) and Dusp6 (not shown) were expressed at similar levels in mutants and controls (Fig. 4g,h), but cyclic expression was not observed in Msgn1 mutants either (Fig. 4i-l). Since all of the Wnt and Fgf target genes examined were expressed, we conclude that Msgn1 is not required for their activation. However, the static expression patterns in Msgn1−/− explants after culture suggests that Msgn1 is required, directly or indirectly, for the oscillatory expression of cyclic Wnt and Fgf target genes.
Although the cyclic Notch targets Lfng and Hes1 are not expressed in Msgn1 mutants at E9.5, Dll1 expression was reportedly reduced but not severely affected20, and other cyclic Notch genes such as Notch-regulated ankyrin-repeat protein (Nrarp)26,27, the Notch transcriptional coactivator Mastermind-like 3 (Maml3)28, and the periodic transcriptional repressor Hes729 were not examined. To determine if Msgn1 plays a central role in the regulation of known Notch clock genes, we examined their expression in Msgn1−/− embryos at E8.5. WISH revealed that numerous Notch pathway genes including Dll1, Dll3, Notch1, Hes7, and Lfng were significantly downregulated in the posterior PSM (Fig. 4m,n,s,t,y-d’). The Lfng expression pattern closely resembles the expression of Lfng in embryos lacking the transcriptional effector of the Notch pathway, RBP-J| 30,31, suggesting that Msgn1 and RBP-J| play equivalent roles in Lfng regulation. In contrast, Nrarp (Fig. 4e’,f’) and Maml3 (not shown) were strongly expressed in the posterior PSM of Msgn1−/− embryos. Examination of cultured PSM explants demonstrated that the stripes of Nrarp expression in the control PSM (Fig. 4o) became broader in the posterior-most PSM of the complementary half after culture (Fig. 4p). In the Msgn1−/− PSM, a large, graded, expression domain was observed (Fig. 4q), and this pattern remained unchanged after culture (Fig. 4r) indicating that Nrarp expression didn’t oscillate. Similarly, Maml3 was broadly expressed in Msgn1−/− PSM, and the pattern remained unchanged after culture (Fig. 4w, x). These results demonstrate that neither Msgn1, nor Notch signaling, is required for Nrarp and Maml3 activation, but Msgn1 is necessary for their cyclic expression. In summary, the expression of Dll1, Dll3, Notch1, Hes7 and Lfng was dramatically reduced in the posterior PSM by the absence of Msgn1, while Nrarp and Maml3 expression was qualitatively different. The lack of oscillatory expression of cyclic Notch, Wnt, and Fgf target genes indicate that Msgn1 plays a central and fundamental role in the segmentation clock.
To address the molecular mechanisms of Msgn1 function in the clock and gradient, we sought to identify the target genes of Msgn1 by genome-wide transcriptional profiling. Since early embryos provide extremely limiting amounts of tissue, ESC reagents were developed to exploit the ability of pluripotent ESC to self-renew indefinitely and to differentiate into mesoderm, including PSM, in vitro23. Recombinant ESC lines expressing a Doxycycline-inducible (Dox) FLAG epitope-tagged Msgn1 (F-Msgn1) (Supplementary Fig. S2;32), were cultured in suspension for 2 days to initiate epiblast differentiation, and then induced with Dox to prematurely express Msgn1. Transcriptional profiles of embryoid bodies (EB) +/− F-Msgn1 induction were assessed for differentially expressed genes. Examination of the cyclic Wnt (Sp5 and Axin2) and Fgf (Spry2 and Dusp6) pathway genes revealed that ectopic Msgn1 expression had little effect on their expression (Supplementary Fig. S3). Conversely, multiple genes in the Notch pathway were induced >1.5-fold by Msgn1, including Dll1, Dll3, Notch1, and Nrarp (Fig. 5a, Supplementary Table 1). Differential Lfng expression was not statistically significant due to variable expression in both treated and untreated cells. Quantitative PCR (qPCR) analysis validated the microarray data for all Notch genes examined (Fig. 5b). Notably, Maml3, which is not represented on the Affymetrix MOE430 2.0 gene chips, was also upregulated. Thus Msgn1 induced the expression of 6 different genes in the Notch signaling pathway.
The rapid activation of multiple Notch pathway genes suggests that Msgn1 could regulate their transcription directly. To determine whether Msgn1 binds to these loci, anti-Flag antibodies were used to immunoprecipitate chromatin bound to F-Msgn1, followed by sequencing (ChIP-seq) of F-Msgn1-associated DNA. Remarkably, multiple regions of the genome with significant enrichment in Msgn1-associated sequences were identified within, or upstream of, genes in the Notch pathway. Large Msgn1 peaks were found in conserved intronic regions of Notch1, Notch2, and Maml3, and in upstream regulatory regions of Dll1, Dll3, Notch1 and Lfng (Fig. 6, ,7).7). Small Msgn1 peaks were also identified upstream of Dll1 (Fig. 6a), Nrarp and Sprouty2, while none were associated with the Hes7, Sp5, Axin2, or Dusp6 loci. Sequence analysis revealed the presence of at least one consensus E box motif (CANNTG) in each Msgn1 peak (Fig. 6 and Supplementary Information). Since bHLH transcription factors bind to E boxes33, their presence is consistent with Msgn1 activating Notch pathway genes by direct binding.
Interestingly, the two Msgn1 peaks found in the Lfng locus precisely align with two previously described enhancers that drive Lfng expression in the PSM of transgenic embryos (Fig. 7a; 31,34). Peaks were also identified in the Dll1 and Dll3 loci that are distinct from, but overlapping with, previously defined regulatory elements35,36. To test these putative enhancers for Msgn1 responsiveness, peak sequences were cloned upstream of a minimal promoter driving the luciferase reporter and cotransfected with Msgn1 or control expression vectors in NIH3T3 cells. Msgn1 strongly transactivated the luciferase reporter in eight constructs (single peaks in Dll1, Dll3, Notch2, and Maml3, and 2 peaks in Notch1, and Lfng (Fig. 6b,d,f,h,j, Fig. 7c,d). Two small ChIP-seq peaks (Dll1-505 and -2313) immediately upstream of the Dll1 transcriptional start site (TSS) were not found to be significantly different from control input DNA and thus were not scored as peaks. To verify that these sequences did not contain Msgn1 responsive elements, we tested one (Dll1-2313) in reporter assays. Dll1-2313 did not respond to Msgn1 expression despite the presence of E-boxes, thereby empirically validating the ChIP-seq analysis (Fig. 6b). The small peaks observed in Nrarp and Sprouty2 upstream sequences are of similar size to Dll1-2313 and were therefore not examined further. These results demonstrate that the Msgn1 ChIP-seq peaks examined are indeed cis-acting regulatory elements, and that Msgn1 functions as a strong transcriptional activator of Notch pathway genes.
We have shown that Msgn1 can bind and activate regulatory enhancers of the cyclic genes Dll1, Maml3, and Lfng, and that Msgn1 is required for the cyclic expression of Dll1 and Lfng in vivo. These results strongly suggest that Msgn1 is an important component of the segmentation clock. To address the mechanisms of Msgn1 function in the clock, we focused on understanding the transcriptional regulation of Lfng since the regulatory elements are well-characterized in vivo31,34,37, and because Lfng is an important regulator of oscillating Notch activity37,38,39. Lfng transcription in the PSM is controlled by three separable, conserved elements termed blocks A, B, and C31 found within a 2.3 kb region upstream of the TSS31,34,37. An RBP-J| binding site (which confers Notch responsiveness) and two E-boxes identified within block A (Fringe Clock Element134), regulate cyclic Lfng expression in the posterior PSM, while E and N boxes in block B regulate expression in the anterior PSM17,31,34,37. Our genomic ChIP-seq studies showed that Msgn1 bound to both blocks A and B (Fig. 7a). Single gene ChIP-PCR assays in ESCs validated the ChIP-seq results, demonstrating that both blocks were highly enriched by F-Msgn1 ChIP (Supplementary Fig. S4). Importantly, anti-Msgn1 antibodies (Supplementary Fig. S5) detected endogenous Msgn1 bound to Lfng regulatory blocks A and B in PSM extracts but not in control extracts of the head (Fig. 7b). Electrophoretic mobility shift assays (EMSA) confirmed that Msgn1 protein indeed bound directly to E-box sequences found in the A and B block (Supplementary Fig. S6) but not to the closely-related N-boxes residing in block B. The results of the EMSA assays mirror the ChIP results, with Msgn1 generally displaying greater binding affinity for block A than for block B in both assays. Together, these results conclusively demonstrate that Lfng is a direct target gene of Msgn1, and that Msgn1 binds Lfng clock regulatory elements in vivo.
The binding of Msgn1 to block A or to block B was sufficient to activate the expression of the luciferase reporter by 5-6-fold when either block was tested in isolation, however Msgn1 alone did not strongly activate the reporter in the context of the longer 2.3 kb Lfng upstream region (Fig. 7c). This result argues that additional activators are required to generate a robust transcriptional response, and that negative factors also regulate Lfng. The 2.3 kb construct did not respond well to activated Notch (NICD) either, however the coexpression of Msgn1 and NICD resulted in a synergistic 7-15-fold induction of luciferase activity (Fig. 7c, d). This Msgn1-Notch synergism was mediated by block A since synergism was not observed with reporter constructs that lacked block A (see constructs B, C, and BC), and was very strong (50-107 fold) when Msgn1 and NICD were coexpressed with the block A enhancer alone (Fig. 7c, d). Negative regulators presumably bind to blocks B and C since the Msgn1-Notch synergism is adversely affected by the presence of these blocks. Together, the data demonstrate that Msgn1 and the Notch signaling pathway regulate Lfng by binding directly to clock elements in block A to cooperatively activate transcription.
Msgn1 activates the expression of multiple genes in the Notch pathway, including cyclic genes, however Msgn1 is not expressed periodically. How then are some Msgn1 target genes cyclically expressed, while others are not? In the case of the Msgn1-Notch target gene Lfng, oscillatory expression presumably arises from the dynamic activity of the Notch pathway. The Hairy enhancer-of-split (Hes) gene, Hes7, is a periodically expressed Notch target gene and bHLH transcription factor that is required for somitogenesis and cyclic gene expression40,41. Hes7 has been proposed to function as the segmentation clock pacemaker as it operates in an autoinhibitory feedback loop and periodically represses Lfng transcription29. If Msgn1 is a major driver of clock gene expression and Hes7 is the pacemaker, then Hes7 should repress the activating activity of Msgn1 on Lfng. Indeed Hes7 completely repressed the activation of the full-length 2.3 kb Lfng enhancer by Msgn1 alone, or Msgn1 and Notch together (Fig. 7d). Examination of Hes7 activity on the isolated A or B blocks revealed that Hes7 repressed the activation of the block B enhancer by Msgn1 alone, but had no effect on the Msgn1-dependent activation of the block A cyclic enhancer. Since Hes7-binding N-boxes are only found in block B, these results argue that Hes7 represses Lfng by binding to N-boxes. On the other hand, Hes7 does repress the combined activity of Msgn1 and Notch on the A block by 47% suggesting that at least some Hes7 repressor activity is N-box independent. Nevertheless, Msgn1 and Notch still function as powerful, synergistic activators of the Lfng block A clock enhancer, activating it 57-fold despite the presence of Hes7.
The original clock and wavefront model first proposed that the sequential and repetitive formation of somites arises from a cellular oscillator (the clock) periodically interacting with a wavefront of rapid cell change that moves progressively down the embryonic AP axis42. The discovery of oscillating genes in the Notch, Wnt3a and Fgf signaling pathways, and the demonstration that Wnt3a/Fgf signaling gradients define the traveling wavefront or determination front, suggests that Wnt3a functions in both the clock and the wavefront during somitogenesis. In this study, we have shown that Wnt3a controls the clock and the wavefront through the activation of the direct target gene Msgn1 and that Msgn1 plays a pivotal role in the segmentation clock by functioning to coordinate the Wnt and Notch pathways.
The expression of Wnt3a in the PS and tailbud13,21 establishes a descending posterior-to-anterior nuclear gradient in the PSM of the transcriptional coactivator ®-catenin18. This ®-catenin gradient is reflected in the graded expression of some direct Wnt3a/®-catenin target genes such as Axin213. Axin2 appears to be universally responsive to Wnt/β-catenin signaling since it is expressed at multiple sites of Wnt activity in the embryo and adult, as well as in Wnt/β-catenin-dependent tumors and cancer cell lines43,44,45,46. In contrast, Msgn1 expression is restricted to the embryonic PSM and is less graded than Axin2 during early somitogenesis stages, displaying a more defined border in the anterior PSM. The absence of Msgn1 expression at other sites of Wnt activity argues that additional pathways are required for the activation of Msgn1 in the PSM. This is supported by the demonstration that Tbx6, which is coexpressed with Msgn1 in the PSM, cooperates with the Wnt3a/β-catenin pathway to directly regulate the Msgn1 promoter (this work; 24). The broad expression of Msgn1 throughout the posterior PSM, collapses into a weak gradient in the absence of Tbx6, suggesting that a major function of Tbx6 is to convert the “slope” of the Wnt3a gradient into a “step” of Msgn1 expression that displays a defined anterior border.
The anterior Msgn1 border lies immediately caudal to the segmental Mesp2 stripe and therefore marks the position of the determination front. Recent genetic studies have shown that the Wnt3a/β-catenin gradient regulates somitogenesis, at least in part, by positioning the determination front in the anterior PSM8,18. Our present work demonstrates that these same genetic manipulations of Wnt signaling that alter the determination front position, also correspondingly alter the position of the anterior boundary of Msgn1 expression. This boundary posteriorly regresses at the rate of one somite length during one period of somite formation47, indicating that the posterior-ward movement of the Msgn1 expression domain parallels the regression of the wavefront. Importantly, activation of Mesp2 and Ripply2 does not occur in Msgn1−/− mutants (Fig. 3;20), suggesting that Msgn1 is required for posterior PSM progenitors to transition to anterior PSM fates and establish a segmental pre-pattern. Taken together, the data strongly argue that the Wnt3a/β-catenin gradient controls determination front activity through Msgn1, and that the anterior border of Msgn1 plays a key role in defining this activity.
Recent work has affirmed the essential role that the Notch pathway plays in the mammalian segmentation clock9. Wnt3a functions upstream of Notch to control cyclic gene expression in both the Wnt and Notch pathways, leading to the hypothesis that Wnt/β-catenin signaling drives the segmentation clock13. Studies of β-catenin mutants support this contention however the demonstration that Lfng continues to oscillate in the presence of a stabilized, non-cyclical form of β-catenin argues that β-catenin is not the clock pacemaker8,18. How then does the Wnt3a/β-catenin pathway regulate Notch signaling and the segmentation clock? One way is through the direct activation of Dll112,48. We show here that Wnt3a, acting via Msgn1, has a much broader role in Notch regulation, activating almost the entire Notch signaling pathway including cyclic target genes. Thus Msgn1 is a master regulator of the Notch pathway.
Although Msgn1 is required for Notch target gene expression, the Wnt target genes Sp5 and Axin2 were upregulated in Msgn1 mutants. This expression is similar to the enhanced expression previously reported20 for another Wnt3a target gene T/Brachyury49, suggesting that Msgn1 directly, or indirectly, represses some Wnt targets. The divergent response of Wnt and Notch targets suggest that Msgn1 functions to coordinate these pathways in the segmentation clock. Despite our proposal that it plays a central role in the clock, Msgn1 is unlikely to function as the pacemaker since it is not expressed periodically. Our demonstration that Hes7 blocks the synergistic Msgn1-Notch activation of Lfng is consistent with previous suggestions that Hes7 is the pacemaker29. We suggest that cyclic gene expression in the Notch pathway requires two primary regulatory components: an activator, arising from the combined activity of Msgn1 and the Notch signaling pathway, and a short-lived periodic repressor, Hes7, which functions in an autoinhibitory feedback loop to periodically repress Hes7 and Lfng (Fig. 8). Although it is well known that the Fgf pathway is also required for cyclic gene expression and for the proper expression of Msgn110, the precise mechanisms remain unclear.
Interactions between the Notch and Wnt pathways are known to regulate the maintenance of stem cells, including hematopoietic and gastrointestinal stem cells50,51,52. This raises the intriguing possibility that a similar relationship between the Wnt and Notch pathways could maintain mesodermal stem cells in the segmenting embryo. Msgn1 regulates the maturation of these mesodermal progenitors since they accumulate as an undifferentiated mass in the tailbud of Msgn1−/− embryos20 and display impaired anterior-ward migration and epithelial-mesenchymal transitions (EMT). Since Wnt3a regulates the same processes, then it is possible that Wnt3a regulates mesodermal stem cell homeostasis, migration, EMT, and positional information through Msgn1. The identification of the interacting proteins, and the genomic targets, of Msgn1 will greatly assist in our understanding of how Wnt signals regulate such numerous and diverse processes.
Ctnnb1tm2Kem mice were purchased from the Jackson Labs. The Wnt3a 21, Mesogenin1 20, Ctnnb1lox(ex3) 53, Tbx625 and T-cre mice54 were obtained from the originating labs. Transgenic mice were generated in the Transgenic Core Facility, NCI-Frederick, by pronuclear injection following standard procedures. All animal experiments were performed in accordance with the guidelines established by the NCI-Frederick Animal Care and Use Committee.
Single and double whole-mount in situ hybridization (WISH) and section ISH were performed as described15. Unless indicated otherwise, at least 4 mutant embryos were examined for each probe, and all yielded similar results.
Serum independent GFP-Bry ES cells were maintained and differentiated into mesoderm as described23. Recombinant Wnt3a protein (R&D Systems) was added to embryoid bodies on D2 of differentiation and cells were harvested for RNA extraction 48 hours later.
The Msgn1 ORF with N-terminal 1× or 3× FLAG tags was cloned into p2lox targeting vector and site-specific recombination was accomplished by inducing A2lox.cre ESCs32 with Doxycycline (Dox) 24 hours prior to electroporation with the P2lox-FLAG-Msgn1 vector. For EB differentiation, ESC were plated on 10cm Petri dishes at a density of 2.5 million cells per plate in 15ml of DMEM, 10% FBS, NEAA, Pen/Strep, 2-Mercaptoethanol and 50ug/ml ascorbic acid (Sigma). After 2 days of differentiation, EBs were then transferred to 6 well ultra low attachment dishes (Corning) and expression of FLAG-Msgn1 was induced using 1ug/ml Dox. Samples were collected at 12h interval up to 48h. The same EB differentiation protocols were used for transcriptional profiling and ChIP-seq assays.
Gene expression profiling on microdissected node and primitive streak regions of E7.75-E8 wildtype and Wnt3a−/− embryos was performed as described8.For ESC expression profiling, total RNA was isolated using RNeasy mini kit (Qiagen) from EBs treated with and without doxycycline at 12h, 24h and 48h time intervals according to manufacturer’s instructions. Protocols for synthesis of cDNA and cRNA were performed using the Affymetrix Genechip 3′ IVT Express kit (Affymetrix, Santa Clara, CA) according to manufacturer’s recommendations. Statistical analysis was performed on probe-intensity level data (CEL files) using Genespring GX10 (Agilent) and BRB ArrayTools v.3.2 software. Initial filtering and preprocessing, including background correction, quantile normalization and summarization, was performed using the robust multi-array analysis (RMA) algorithm. Statistical analyses were performed using an unpaired T-test at p≤0.05 on genes displaying a fold change of 1.5 or greater. The mean-normalized intensity values (log2 values) and standard deviation for each gene were calculated and plotted.
Polyclonal mouse antibodies against Msgn1 protein were made by A&G Precision antibody (Columbia, MD) using a cocktail of peptides CWKSRARPLELVQESP, CDLLNSSGREPRPQSV, CSHEAAGLVELDYS conjugated to KLH antigen.
ChIP of EBs: 36 hours after Dox induction, EBs were fixed in 1% formaldehyde solution for 15 min and quenched with 0.125M Glycine. Cells were lysed with lysis buffer and chromatin was sheared to an average length of 300-500bp as described55. Protein DNA complexes were immunoprecipitated using anti-FlagM5 antibody or corresponding isotype. Crosslinks were reversed and DNA was extracted with phenol:chloroform and purified by ethanol precipitation.
The Chromatin immunoprecipitation-sequencing (ChIP-Seq) approach was performed by Genpathway (San Diego, California)56. Briefly EBs were fixed and quenched as described above, and flash frozen. The nuclei were extracted, sonicated and precleared with Staph A cells (Pansorbin, Calbiochem, San Diego, CA) before incubation with anti-Flag M2 antibody (Sigma). Samples were incubated with prepared Staph A cells to extract antibody-chromatin complexes, followed by washing and elution. Precipitated DNA fragments were released and purified. ChIP DNA fragments associated with FLAG-Msgn1 were amplified using the Illumina ChIP-Seq DNA sample prep kit (Illumina, San Diego, CA). DNA libraries were sequenced on a Genome Analyzer II by Illumina Sequencing Services. 35-nt sequence reads were mapped to the genome using the Eland algorithm. The 35bp sequences were extended in silico at the 3′ end up to 110bp, which is the average DNA fragment length after shearing. The resulting histograms were stored in BAR (Binary Analysis Results) files. For each BAR file, intervals were calculated and compiled into BED files (Browser Extensible Data) for viewing in the UCSC genome browser. Enriched intervals, referred to as peak values, were determined by applying a threshold p-value cutoff of 10e-5 and a false discovery rate (FDR) of 2.35%. BAR files were uploaded to the Integrated Genome Browser (IGB-Affymetrix) for extensive analysis.
ChIP of embryo tissues: About 60 PSMs and 25 heads from E9.5 stage embryos were collected and fixed overnight in 4% PFA at 4°C and dehydrated in methanol series57. On the day of the CHIP assay, PSM and head tissues were rehydrated, cross-linked with 1% formaldehyde for 15min at room temperature and quenched with 0.125M glycine. Tissues were washed 2x with ice-cold PBS, resuspended in lysis buffer LB155 and disrupted by passing through 26 3/8 gauge needle. Pellets in lysis buffer LB3 were sonicated using Branson Sonifer450 and lysates were pre-cleared for 15min at 4°C with 20ul proteinG dynal beads (Invitrogen) pre-blocked with 0.5% BSA in PBS and 1mg/ml sonicated salmon sperm DNA (Ambion). Equal amounts of lysates were immunoprecipitated with anti-Msgn1 ascites or control ascites (Clone NS-1; Sigma).
Half-embryo explant cultures were done as described8. PSM explants of wild type and Msgn1−/− embryos were dissected at E8.25-E8.5 and manually bisected along the midline. The left half of the embryo was immediately fixed in 4% PFA, while the right half was cultured at 37C for 60 or 90 minutes in DMEM with 10% FBS supplemented with 10ng/ml bFGF (R and D Systems) before fixation.
Reporter constructs of 1 kb Msgn1 promoter and mutant version of 3 TCF/LEF binding sites were generated in pGL4.10(luc2) vector (Promega). For generating transgenic mice, the hsp68 minimal promoter was replaced with the wildtype and TCF/LEF mutated 1kb Msgn1 promoter followed by lacZ and SV40polyA signal in pASShsp68-lacZ-pA vector. The luciferase reporter construct containing the 2.3kb Lunatic-fringe cyclic enhancer31, Lfng enhancer deletion constructs, and positively identified CHIP seq peaks were generated in the pGL4.23 [minp-luc2] vector (Promega). HEK293T cells or NIH 3T3 cells were seeded at 0.25 × 10^5 cells per well in 24 well plates and cultured for 18 hours. A total of 500ng of DNA containing the reporter plasmids (200ng) and empty luciferase vectors were co-transfected with or without expression vectors pSG5-T-VP16 (50ng), pcDNA-Tbx6-VP16 (50ng), ΔN-ßcatenin-myc (50ng), pCS2-Myc-Msgn1 (100ng), CMV-3xFlag-Mesp2 (100ng), and pCS2-NICD-Venus (50ng), CMV-3xFlag-Hes7 (100ng) using Lipofectamine 2000 (Invitrogen). Equivalent amounts of DNA were achieved with pcDNA3 or pCS2 vector. Cells were lysed 40 hours after transfection and luciferase activity was measured using the Dual Luciferase Assay Kit (Promega) as per manufacturers recommendations. In each condition, 5ng of pGL4.74 [hRluc/TK] Vector (Promega) was used as an internal control to normalize for transfection efficiency. Fold change was calculated as a ratio of the luciferase vector containing Msgn1 or Lfng regulatory elements relative to empty luciferase vector for identical experimental conditions, normalized to a control condition minus expression vectors. The reported values are from one experiment but are representative of at least three independent experiments.
Lef1 and Myc-Msgn1 proteins were made using the TNT Reticulocyte Lysate System (Promega) in vitro. Double stranded DNA oligonucleotides were end labeled with DIG-ddUTP and protein-DNA complexes were analyzed using the DIG Gel Shift Assay Kit (Roche) according to manufacturer’s instructions.
1ug of RNA was Dnase1 treated and first-strand cDNA was synthesized using oligo dT and superscript III reverse transcriptase (Invitrogen) or iScript cDNA synthesis kit (Roche) according to manufacturers instructions. qPCR analysis was used to quantitate cDNAs using CFX96 Real-Time PCR Detection System (Bio-Rad) and Fast Start Universal SYBR Green Master (Roche). For the qPCR results shown in Fig.1h, Taqman gene expression assays were performed (Mm00490407_S1) using Taqman universal PCR Master Mix (Applied Biosystems) according to manufacturer’s recommendations. Gapdh expression was used to normalize Msgn1 expression values. The specificity of all primers was monitored by electrophoresis of the amplicons on agarose gels. The mean expression values obtained for each gene were normalized to GAPDH (ΔΔC (t) method) and to the expression in undifferentiated ES cells. For normalization of CHIP-qPCR analysis, first percent input method was used to calculate from the obtained Ct values and was further normalized to the values obtained in the control ascites to obtain the fold change values. All CHIP and qPCR experiments were done in triplicates.
See Supplementary Table S2 for oligonucleotide sequences used in EMSA and qPCR assays.
We thank Y. Saga, B. Herrmann, and R. Kageyama for providing reagents. We are grateful to S. Mackem, A. Perantoni, M. Lewandoski, M. Anderson, N. Adler, and M. Kennedy for providing comments on versions of this manuscript. We are particularly indebted to J. Greear and R. Wolfe (SAIC-Frederick) for excellent animal husbandry. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Author Contributions: R.B.C., W.C.D. and T.P.Y. conceived the experiments. R.B.C., W.C.D. and K.K.B. predominantly performed the work with the help of R.A. and A.B. M.I. and M.K. developed the ESC reagents, L.F. generated transgenic animals, D.L.C. and J.K.Y. provided embryos and mice. T.P.Y. supervised the project and wrote the paper.
Competing Financial Interests Statement: The authors declare no competing financial interests.