Wenduo Ye

Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation

Significance Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia in the human population. It is critical to elucidate the molecular mechanisms underlying AF, given that the prevalence of AF is expected to dramatically increase as the human population ages. We identified a microRNA (miR)-regulated genetic pathway that delimits sinoatrial node development and inhibits AF. To our knowledge, our data are the first genetic evidence showing that miR deletion results in AF predisposition. Moreover, to our knowledge, our data are the first demonstration that sinoatrial node regulatory genes are regulated by miRs. Our findings suggest attractive therapeutic targets to treat AF given that miR-based therapeutics are feasible using miR antagonists and mimics. The molecular mechanisms underlying atrial fibrillation, the most common sustained cardiac arrhythmia, remain poorly understood. Genome-wide association studies uncovered a major atrial fibrillation susceptibility locus on human chromosome 4q25 in close proximity to the paired-like homeodomain transcription factor 2 (Pitx2) homeobox gene. Pitx2, a target of the left-sided Nodal signaling pathway that initiates early in development, represses the sinoatrial node program and pacemaker activity on the left side. To address the mechanisms underlying this repressive activity, we hypothesized that Pitx2 regulates microRNAs (miRs) to repress the sinoatrial node genetic program. MiRs are small noncoding RNAs that regulate gene expression posttranscriptionally. Using an integrated genomic approach, we discovered that Pitx2 positively regulates miR-17-92 and miR-106b-25. Intracardiac electrical stimulation revealed that both miR-17-92 and miR-106b-25 deficient mice exhibit pacing-induced atrial fibrillation. Furthermore electrocardiogram telemetry revealed that mice with miR-17-92 cardiac-specific inactivation develop prolonged PR intervals whereas mice with miR-17-92 cardiac-specific inactivation and miR-106b-25 heterozygosity develop sinoatrial node dysfunction. Both arrhythmias are risk factors for atrial fibrillation in humans. Importantly, miR-17-92 and miR-106b-25 directly repress genes, such as Shox2 and Tbx3, that are required for sinoatrial node development. Together, to our knowledge, these findings provide the first genetic evidence for an miR loss-of-function that increases atrial fibrillation susceptibility.

A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node.

In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5+ domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5+ domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2+ cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2− cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome-wide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development. Summary: Antagonism between Shox2 and Nkx2-5 in the cardiac venous pole of mouse embryos regulates cell fate, morphogenesis and the distinction between pacemaker cells and working myocardium.

FGF signaling sustains the odontogenic fate of dental mesenchyme by suppressing β-catenin signaling.

Odontoblasts and osteoblasts develop from multipotent craniofacial neural crest cells during tooth and jawbone development, but the mechanisms that specify and sustain their respective fates remain largely unknown. In this study we used early mouse molar and incisor tooth germs that possess distinct tooth-forming capability after dissociation and reaggregation in vitro to investigate the mechanism that sustains odontogenic fate of dental mesenchyme during tooth development. We found that after dissociation and reaggregation, incisor, but not molar, mesenchyme exhibits a strong osteogenic potency associated with robustly elevated β-catenin signaling activity in a cell-autonomous manner, leading to failed tooth formation in the reaggregates. Application of FGF3 to incisor reaggregates inhibits β-catenin signaling activity and rescues tooth formation. The lack of FGF retention on the cell surface of incisor mesenchyme appears to account for the differential osteogenic potency between incisor and molar, which can be further attributed to the differential expression of syndecan 1 and NDST genes. We further demonstrate that FGF signaling inhibits intracellular β-catenin signaling by activating the PI3K/Akt pathway to regulate the subcellular localization of active GSK3β in dental mesenchymal cells. Our results reveal a novel function for FGF signaling in ensuring the proper fate of dental mesenchyme by regulating β-catenin signaling activity during tooth development.

BMPRIA mediated signaling is essential for temporomandibular joint development in mice

The central importance of BMP signaling in the development and homeostasis of synovial joint of appendicular skeleton has been well documented, but its role in the development of temporomandibular joint (TMJ), also classified as a synovial joint, remains completely unknown. In this study, we investigated the function of BMPRIA mediated signaling in TMJ development in mice by transgenic loss-of- and gain-of-function approaches. We found that BMPRIA is expressed in the cranial neural crest (CNC)-derived developing condyle and glenoid fossa, major components of TMJ, as well as the interzone mesenchymal cells. Wnt1-Cre mediated tissue specific inactivation of BmprIa in CNC lineage led to defective TMJ development, including failure of articular disc separation from a hypoplastic condyle, persistence of interzone cells, and failed formation of a functional fibrocartilage layer on the articular surface of the glenoid fossa and condyle, which could be at least partially attributed to the down-regulation of Ihh in the developing condyle and inhibition of apoptosis in the interzone. On the other hand, augmented BMPRIA signaling by Wnt1-Cre driven expression of a constitutively active form of BmprIa (caBmprIa) inhibited osteogenesis of the glenoid fossa and converted the condylar primordium from secondary cartilage to primary cartilage associated with ectopic activation of Smad-dependent pathway but inhibition of JNK pathway, leading to TMJ agenesis. Our results present unambiguous evidence for an essential role of finely tuned BMPRIA mediated signaling in TMJ development.

Altered FGF Signaling Pathways Impair Cell Proliferation and Elevation of Palate Shelves.

In palatogenesis, palatal shelves are patterned along the mediolateral axis as well as the anteroposterior axis before the onset of palatal fusion. Fgf10 specifically expressed in lateral mesenchyme of palate maintains Shh transcription in lateral epithelium, while Fgf7 activated in medial mesenchyme by Dlx5, suppressed the expansion of Shh expression to medial epithelium. How FGF signaling pathways regulate the cell behaviors of developing palate remains elusive. In our study, we found that when Fgf8 is ectopically expressed in the embryonic palatal mesenchyme, the elevation of palatal shelves is impaired and the posterior palatal shelves are enlarged, especially in the medial side. The palatal deformity results from the drastic increase of cell proliferation in posterior mesenchyme and decrease of cell proliferation in epithelium. The expression of mesenchymal Fgf10 and epithelial Shh in the lateral palate, as well as the Dlx5 and Fgf7 transcription in the medial mesenchyme are all interrupted, indicating that the epithelial-mesenchymal interactions during palatogenesis are disrupted by the ectopic activation of mesenchymal Fgf8. Besides the altered Fgf7, Fgf10, Dlx5 and Shh expression pattern, the reduced Osr2 expression domain in the lateral mesenchyme also suggests an impaired mediolateral patterning of posterior palate. Moreover, the ectopic Fgf8 expression up-regulates pJak1 throughout the palatal mesenchyme and pErk in the medial mesenchyme, but down-regulates pJak2 in the epithelium, suggesting that during normal palatogenesis, the medial mesenchymal cell proliferation is stimulated by FGF/Erk pathway, while the epithelial cell proliferation is maintained through FGF/Jak2 pathway.

FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro

The cranial neural crest (CNC) cells play a vital role in craniofacial development and regeneration. They are multi-potent progenitors, being able to differentiate into various types of tissues. Both pre-migratory and post-migratory CNC cells are plastic, taking on diverse fates by responding to different inductive signals. However, what sustains the multipotency of CNC cells and derivatives remains largely unknown. In this study, we present evidence that FGF8 signaling is able to sustain progenitor status and multipotency of CNC-derived mesenchymal cells both in vivo and in vitro. We show that augmented FGF8 signaling in pre-migratory CNC cells prevents cell differentiation and organogenesis in the craniofacial region by maintaining their progenitor status. CNC-derived mesenchymal cells with Fgf8 overexpression or control cells in the presence of exogenous FGF8 exhibit prolonged survival, proliferation, and multi-potent differentiation capability in cell cultures. Remarkably, exogenous FGF8 also sustains the capability of CNC-derived mesenchymal cells to participate in organogenesis such as odontogenesis. Furthermore, FGF8-mediated signaling strongly promotes adipogenesis but inhibits osteogenesis of CNC-derived mesenchymal cells in vitro. Our results reveal a specific role for FGF8 in the maintenance of progenitor status and in fate determination of CNC cells, implicating a potential application in expansion and fate manipulation of CNC-derived cells in stem cell-based craniofacial regeneration.

Genetic Regulation of Sinoatrial Node Development and Pacemaker Program in the Venous Pole

The definitive sinoatrial node (SAN), the primary pacemaker of the mammalian heart, develops from part of pro-pacemaking embryonic venous pole that expresses both Hcn4 and the transcriptional factor Shox2. It is noted that ectopic pacemaking activities originated from the myocardial sleeves of the pulmonary vein and systemic venous return, both derived from the Shox2+ pro-pacemaking cells in the venous pole, cause atrial fibrillation. However, the developmental link between the pacemaker properties in the embryonic venous pole cells and the SAN remains largely uncharacterized. Furthermore, the genetic program for the development of heterogeneous populations of the SAN is also under-appreciated. Here, we review the literature for a better understanding of the heterogeneous development of the SAN in relation to that of the sinus venosus myocardium and pulmonary vein myocardium. We also attempt to revisit genetic models pertinent to the development of pacemaker activities in the perspective of a Shox2-Nkx2-5 epistatic antagonism. Finally, we describe recent efforts in deciphering the regulatory networks for pacemaker development by genome-wide approaches.

Phosphorylation of Shox2 Is Required for Its Function to Control Sinoatrial Node Formation

Background Inactivation of Shox2, a member of the short‐stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. Methods and Results A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2‐hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p‐Shox2a) in cell culture as well as in the developing mouse and human SAN. Site‐directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal‐regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a‐S92AS110A, mutant in the developing heart resulted in down‐regulation of Nkx2.5 in wild‐type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. Conclusions Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis. Highlighted article: A Shox2-coordinated transcriptional program functions in the osteogenic lineage to regulate Hox-TALE factors and skeletogenesis, and to pattern the mouse limb.

Augmented Indian hedgehog signaling in cranial neural crest cells leads to craniofacial abnormalities and dysplastic temporomandibular joint in mice

Extensive studies have pinpointed the crucial role of Indian hedgehog (Ihh) signaling in the development of the appendicular skeleton and the essential function of Ihh in the formation of the temporomandibular joint (TMJ). In this study, we have investigated the effect of augmented Ihh signaling in TMJ development. We took a transgenic gain-of-function approach by overexpressing Ihh in the cranial neural crest (CNC) cells using a conditional Ihh transgenic allele and the Wnt1-Cre allele. We found that Wnt1-Cre-mediated tissue-specific overexpression of Ihh in the CNC lineage caused severe craniofacial abnormalities, including cleft lip/palate, encephalocele, anophthalmos, micrognathia, and defective TMJ development. In the mutant TMJ, the glenoid fossa was completely absent, whereas the condyle and the articular disc appeared relatively normal with slightly delayed chondrocyte differentiation. Our findings thus demonstrate that augmented Ihh signaling is detrimental to craniofacial development, and that finely tuned Ihh signaling is critical for TMJ formation. Our results also provide additional evidence that the development of the condyle and articular disc is independent of the glenoid fossa.