Summary The developmental mechanisms underlying human congenital heart disease (CHD) are poorly understood. Atrial septal defects (ASDs) can result from haploinsufficiency of cardiogenic transcription factors including TBX5. We demonstrated that Tbx5 is required in the second heart field (SHF) for atrial septation in mice. Conditional Tbx5 haploinsufficiency in the SHF but not the myocardium or endocardium caused ASDs. Tbx5 SHF knockout embryos lacked atrial septum progenitors. We found that Tbx5 mutant SHF progenitors demonstrated cell-cycle progression defects and that Tbx5 regulated cell-cycle progression genes including Cdk6. Activated hedgehog (Hh) signaling rescued ASDs in Tbx5 mutant embryos, placing Tbx5 upstream or parallel to Hh in cardiac progenitors. Tbx5 regulated SHF Gas1 and Osr1 expression, supporting both pathways. These results describe a SHF Tbx5-Hh network required for atrial septation. A paradigm defining molecular requirements in SHF cardiac progenitors for cardiac septum morphogenesis has implications for the ontogeny of CHD.

Variants in SCN10A, which encodes a voltage-gated sodium channel, are associated with alterations of cardiac conduction parameters and the cardiac rhythm disorder Brugada syndrome; however, it is unclear how SCN10A variants promote dysfunctional cardiac conduction. Here we showed by high-resolution 4C-seq analysis of the Scn10a-Scn5a locus in murine heart tissue that a cardiac enhancer located in Scn10a, encompassing SCN10A functional variant rs6801957, interacts with the promoter of Scn5a, a sodium channel-encoding gene that is critical for cardiac conduction. We observed that SCN5A transcript levels were several orders of magnitude higher than SCN10A transcript levels in both adult human and mouse heart tissue. Analysis of BAC transgenic mouse strains harboring an engineered deletion of the enhancer within Scn10a revealed that the enhancer was essential for Scn5a expression in cardiac tissue. Furthermore, the common SCN10A variant rs6801957 modulated Scn5a expression in the heart. In humans, the SCN10A variant rs6801957, which correlated with slowed conduction, was associated with reduced SCN5A expression. These observations establish a genomic mechanism for how a common genetic variation at SCN10A influences cardiac physiology and predisposes to arrhythmia.

The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

The noncoding genome is pervasively transcribed. Noncoding RNAs (ncRNAs) generated from enhancers have been proposed as a general facet of enhancer function and some have been shown to be required for enhancer activity. Here we examine the transcription-factor-(TF)-dependence of ncRNA expression to define enhancers and enhancer-associated ncRNAs that are involved in a TF-dependent regulatory network. TBX5, a cardiac TF, regulates a network of cardiac channel genes to maintain cardiac rhythm. We deep sequenced wildtype and Tbx5-mutant mouse atria, identifying ~2600 novel Tbx5-dependent ncRNAs. Tbx5-dependent ncRNAs were enriched for tissue-specific marks of active enhancers genome-wide. Tbx5-dependent ncRNAs emanated from regions that are enriched for TBX5-binding and that demonstrated Tbx5-dependent enhancer activity. Tbx5-dependent ncRNA transcription provided a quantitative metric of Tbx5-dependent enhancer activity, correlating with target gene expression. We identified RACER, a novel Tbx5-dependent long noncoding RNA (lncRNA) required for the expression of the calcium-handling gene Ryr2. We illustrate that TF-dependent enhancer transcription can illuminate components of TF-dependent gene regulatory networks.

Atrioventricular septal defects (AVSDs) are a common severe form of congenital heart disease (CHD). In this study we identified deleterious non-synonymous mutations in two cilia genes, Dnah11 and Mks1, in independent N-ethyl-N-nitrosourea-induced mouse mutant lines with heritable recessive AVSDs by whole-exome sequencing. Cilia are required for left/right body axis determination and second heart field (SHF) Hedgehog (Hh) signaling, and we find that cilia mutations affect these requirements differentially. Dnah11 did not disrupt SHF Hh signaling and caused AVSDs only concurrently with heterotaxy, a left/right axis abnormality. In contrast, Mks1 disrupted SHF Hh signaling and caused AVSDs without heterotaxy. We performed unbiased whole-genome SHF transcriptional profiling and found that cilia motility genes were not expressed in the SHF whereas cilia structural and signaling genes were highly expressed. SHF cilia gene expression predicted the phenotypic concordance between AVSDs and heterotaxy in mice and humans with cilia gene mutations. A two-step model of cilia action accurately predicted the AVSD/heterotaxyu phenotypic expression pattern caused by cilia gene mutations. We speculate that cilia gene mutations contribute to both syndromic and non-syndromic AVSDs in humans and provide a model that predicts the phenotypic consequences of specific cilia gene mutations.

Insulin-like factor 5 (INSL5), a member of the insulin superfamily, is expressed in the colorectum and hypothalamus. To facilitate studies into the role of INSL5, we generated Insl5(-/-) mice by gene targeting. Insl5(-/-) mice were born in the expected Mendelian ratio, reached normal body weight, but displayed impaired male and female fertility that are due to marked reduction in sperm motility and irregular length of the estrous cycle. Furthermore, Insl5(-/-) mice showed impairment in glucose homeostasis with characteristic elevation of serum glucose levels at an advanced age. Glucose and insulin tolerance tests revealed that the increased blood glucose in Insl5(-/-) mice was due to glucose intolerance resulting from reduced insulin secretion. Morphometric and immunohistological analyses revealed that the Insl5(-/-) mice had markedly reduced average islets area and β-cell numbers. Furthermore, immunohistochemistry showed the expression of INSL5 in enteroendocrine cells in the colorectal epithelium and the presence of its putative receptor relaxin family peptide receptor 4 in pancreatic islet cells. These results suggest the potential role of INSL5 signaling in the regulation of insulin secretion and β-cell homeostasis.

The spermatogonial stem cells (SSCs) are responsible for the transmission of genetic information from an individual to the next generation. SSCs play critical roles in understanding the basic reproductive biology of gametes and treatments of human infertility. SSCs not only maintain normal spermatogenesis, but also sustain fertility by critically balancing both SSC self-renewal and differentiation. This self-renewal and differentiation in turn is tightly regulated by a combination of intrinsic gene expression within the SSC as well as the extrinsic gene signals from the niche. Increased SSCs self-renewal at the expense of differentiation result in germ cell tumours, on the other hand, higher differentiation at the expense of self-renewal can result in male sterility. Testicular germ cell cancers are the most frequent cancers among young men in industrialized countries. However, understanding the pathogenesis of testis cancer has been difficult because it is formed during foetal development. Recent studies suggest that SSCs can be reprogrammed to become embryonic stem (ES)-like cells to acquire pluripotency. In the present review, we summarize the recent developments in SSCs biology and role of SSC in testicular cancer. We believe that studying the biology of SSCs will not only provide better understanding of stem cell regulation in the testis, but eventually will also be a novel target for male infertility and testicular cancers.

Insulin-like factor 6 (INSL6), a member of the insulin-like superfamily, is predominantly expressed in male germ cells. Expression of the Insl6 is first detected in mouse testis at postnatal d 15 when the first wave of spermatogenesis progresses to pachytene spermatocytes. To elucidate the role of INSL6 in germ cell development, we generated Insl6-deficient mice. The majority of the Insl6-deficient males on a hybrid genetic background exhibited impaired fertility, whereas females were fertile. The number of mature sperm and sperm motility were drastically reduced in the epididymis. The reduced sperm count could be due to apoptotic death of a significant number of developing germ cells. Analysis of germ cell development during the juvenile life showed an arrest of the first wave of spermatogenesis in late meiotic prophase. RNA analysis revealed a significant decrease in expression of late meiotic- and postmeiotic-specific marker genes, whereas expression of early meiotic-specific genes remains unaffected in the Insl6(-/-) testes. These results demonstrate that INSL6 is required for the progression of spermatogenesis.