Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease.

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

Tbx2 and Tbx3 regulate cell fate progression of the otic vesicle for inner ear development.

The morphogenesis of the otic vesicle (OV) to form inner ear organs serves as an excellent model system to understand cell fate acquisition on a single cell level. Tbx2 and Tbx3 (Tbx2/3) encode closely related T-box transcription factors that are expressed widely in the mammalian OV. Inactivation of both genes in the OV (Tbx2/3cKO) results in failed morphogenesis into inner ear organs. To understand the basis of these defects, single cell RNA-sequencing (scRNA-seq) was performed on the OV lineage, in controls versus Tbx2/3cKO embryos. We identified a multipotent population termed otic progenitors in controls that are marked by expression of the known otic placode markers Eya1, Sox2, and Sox3 as well as new markers Fgf18, Cxcl12, and Pou3f3. The otic progenitor population was increased three-fold in Tbx2/3cKO embryos, concomitant with dysregulation of genes in these cells as well as reduced progression to more differentiated states of prosensory and nonsensory cells. An ectopic neural population of cells was detected in the posterior OV of Tbx2/3cKO embryos but had reduced maturation to delaminated neural cells. As all three cell fates were affected in Tbx2/3cKO embryos, we suggest that Tbx2/3 promotes progression of multipotent otic progenitors to more differentiated cell types in the OV.

Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

GATA6 is a regulator of sinus node development and heart rhythm.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration.

BACKGROUND: The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. RESULTS: We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. CONCLUSIONS: TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

Identification of key variants correlated with susceptibility of primary osteoporosis in the Chinese Han group.

BACKGROUND: Primary osteoporosis is a systemic skeletal disease characterized by reduced bone mass and vulnerability to fractures. The genetics of osteoporosis in the Chinese population remain unclear, which hinders the prevention and treatment of osteoporosis in China. This study aimed to explore the susceptibility genes and the roles played by their variants in osteoporosis. METHODS: Blood samples were collected from 45 osteoporosis patients and 30 healthy individuals, and genome-wide association study was performed on array data. The expression levels of the candidate gene in different genotypes were further determined by using quantitative real-time PCR. Moreover, the differentiation capacity of bone marrow mesenchymal stem cells under different genotypes from osteoporosis patients was investigated. RESULTS: The most significant variant rs1891632 located in the upstream (918 bp) region of CRB2, which could down-regulate the expression levels of CRB2 in genotype-tissue expression database and played an essential role in the regulation of osteoblastic and osteoclastic differentiation during skeletal development. Another significant variant rs1061657 located within the 3'UTR region of TBX3 gene. We found that the mRNA levels of TBX3 decreased in the bMSCs of old osteoporosis patients. Interestingly, osteoblast differentiation capacity and TBX3 mRNA levels were similar between the young healthy individuals carrying derived and ancestral allele of rs1061657, whereas the differentiation capacity and TBX3 mRNA levels dramatically declined in elderly patients with osteoporosis. CONCLUSIONS: The variant rs1061657 might affect the osteogenesis of bMSCs in an age-dependent manner and that TBX3 may be a key susceptibility gene for primary osteoporosis. In conclusion, CRB2 and TBX3 may influence the development of osteoporosis; additionally, rs1891632 and rs1061657, as the key variants first reported to be associated with primary osteoporosis, may potentially contribute to predicting the risk of osteoporosis (especially for older individuals) and may serve as therapeutic targets.

Efficient generation of TBX3+ atrioventricular conduction-like cardiomyocytes from human pluripotent stem cells.

Atrioventricular conduction cardiomyocytes (AVCCs) regulate the rate and rhythm of heart contractions. Dysfunction due to aging or disease can cause atrioventricular (AV) block, interrupting electrical impulses from the atria to the ventricles. Generation of functional atrioventricular conduction like cardiomyocytes (AVCLCs) from human pluripotent stem cells (hPSCs) provides a promising approach to repair damaged atrioventricular conduction tissue by cell transplantation. In this study, we put forward the generation of AVCLCs from hPSCs by stage-specific manipulation of the retinoic acid (RA), WNT, and bone morphogenetic protein (BMP) signaling pathways. These cells express AVCC-specific markers, including the transcription factors TBX3, MSX2 and NKX2.5, display functional electrophysiological characteristics and present low conduction velocity (0.07 ± 0.02 m/s). Our findings provide new insights into the understanding of the development of the atrioventricular conduction system and propose a strategy for the treatment of severe atrioventricular conduction block by cell transplantation in future.

Characteristic of Przewalski horses population from Askania-Nova reserve based on genetic markers.

Przewalski horses are considered the last living population of wild horses, however, they are secondarily feral offspring of herds domesticated ~ 5000 years ago by the Botai culture. After Przewalski horses were almost extinct at the beginning of the twentieth century, their population is about 2500 individuals worldwide, with one of the largest breeding centers in Askania-Nova Biosphere Reserve (Ukraine). The research aimed to establish the maternal variation of Przewalski horses population maintained in Askania-Nova Reserve based on mitochondrial DNA hypervariable 1 and hypervariable 2 regions profiling, as well as, analysis of Y chromosome single nucleotide polymorphism unique for Przewalski horses, and coat color markers: MC1R and TBX3. The mtDNA hypervariable regions analysis in 23 Przewalski horses allowed assigning them to three distinctly different haplotypes, showing the greatest similarity to the Equus caballus reference, the Equus przewalskii reference, and to extinct species-Haringtonhippus. The Y chromosome analysis using fluorescently labelled assays differentiated horses in terms of polymorphism (g731821T>C) characteristic of Equus przewalskii. All male individuals presented genotype C characteristics for Przewalski horses. The polymorphisms within the coat color genes indicated only native, wild genotypes. The Y chromosome and coat color analysis denied admixtures of the tested horses with other Equidae.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system.

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3+/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3+/- mutants. Notably, Tbx3+/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

Complex functional redundancy of Tbx2 and Tbx3 in mouse limb development.

The limb phenotypes of Tbx2 and Tbx3 mutants are distinct: loss of Tbx2 results in isolated duplication of digit 4 in the hindlimb while loss of Tbx3 results in anterior polydactyly and posterior oligodactly in the forelimb. In the face of such disparate phenotypes, we sought to determine whether Tbx2 and Tbx3 have functional redundancy during development of the mouse limb. We found that sequential loss of alleles generates defects that are not simply additive of those observed in single mutants and that multiple structures in both the forelimb and hindlimb display compound sensitivity to decreased gene dosage.

Transcriptional Patterning of the Ventricular Cardiac Conduction System.

RATIONALE: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. OBJECTIVE: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. METHODS AND RESULTS: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. CONCLUSIONS: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

Genetic architectures and selection signatures of body height in Chinese indigenous donkeys revealed by next-generation sequencing.

Donkeys are widely distributed labour animals in the world. During the process of the domestication and artificial selection of domestic donkeys, body sizes show significant differences among different breeds of donkeys. Based on the genome resequencing data of 103 Chinese indigenous donkeys from 11 breeds (Biyang, Dezhou, Guangling, Hetian, Jiami, Kulun, Qingyang, Turfan, Tibetan, Xinjiang, and Yunnan), seven Spanish donkeys from two breeds (Zamorano~Leonés and Andalusian), and three wild donkeys, we investigated the population structures of Chinese domestic donkeys with different body sizes. We used FST and XP-EHH analyses to explore the selected regions related to body sizes. The results showed that Chinese indigenous donkeys have a closer relationship with African wild donkeys than with Asian wild donkeys. LCORL/NCAPG, FAM184B, TBX3, and IHH were identified as genes with strong signals in analysis of selection signature (FST and XP-EHH) in large and small donkeys. The seven identified variants can be served as candidate loci affecting the body size of Chinese donkeys. Five of seven loci were located in intron 9 of FAM184B and were in a haplotype block, and one of the identified variants (Chr03:112664848) located in the CDS region of the LCORL gene was found to cause stop-loss. These candidate genes and variants shed new light on the molecular basis of donkey body size and will facilitate the breeding activities of donkeys.

Ulnar-Mammary syndrome with TBX3 gene mutation in a Chinese family: A case report and literature review. / TBX3åŸºå› çªå˜è‡´Ulnar-Mammary综合征1ä¾‹å®¶ç³»æŠ¥é“å¹¶æ–‡çŒ®å¤ä¹ .

Ulnar-Mammary syndrome (UMS) is a rare monogenic disorder caused by mutations of the TBX3 gene. This paper reported a family of UMS. The proband, a 15-year old man, was presented with mammary gland dysplasia, ulnar limb defect, short stature, and delayed growth. Whole exome sequencing revealed a 1294_1301dup mutation in exon 6 of the TBX3 gene. Sanger sequencing was used to verify other members of the family, which suggested his mother also carried the same mutation, but merely resulting in the dysplasia of her left little finger. Notably, unilateral finger involvement without any systemic organ involvement was unusual in UMS patients. The proband then was treated with recombinant human growth hormone (rhGH) and human chorionic gonadotropin (hCG). After a year and a half, his height and secondary sexual characteristics were significantly improved. The clinical manifestations of the disease are highly heterogeneous, which is easy to be misdiagnosed and missed. When the diagnosis is unclear, genetic testing is helpful for auxiliary diagnosis.

Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction.

A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

HOTAIR maintains the stemness of ovarian cancer stem cells via the miR-206/TBX3 axis.

The poor prognosis of ovarian cancer is partly attributed to the frequent chemo-resistance and recurrence, which may be mediated by ovarian cancer stem cells (OCSCs). In the present study, we investigated the mechanisms contributing to the stemness of OCSCs, focusing on the long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR). Ovarian cancer cells were tested for high aldehyde dehydrogenase (ALDH) activity or high in vitro sphere-formation ability to identify OCSCs. HOTAIR was highly expressed in the OCSCs and its depletion caused a decrease in sphere-formation ability, along with reduced resistance to cisplatin and in vivo tumorigenicity. T-box transcription factor 3 (TBX3) was highly expressed in the OCSCs and was confirmed to be positively regulated by HOTAIR. Moreover, TBX3 maintained cell stemness, whereas elevating TBX3 could relieve the weakened sphere-formation ability caused by HOTAIR depletion. Subsequently, miR-206 was found to mediate the expression regulation of TBX3 by HOTAIR, and functionally involved in the regulation of stemness in OCSCs. In line with these findings, circulating HOTAIR expression was up-regulated in ovarian cancer patients. Collectively, our findings suggest that HOTAIR relieves the inhibition of TBX3 expression mediated by miR-206 in OCSCs and provide novel therapeutic targets for the treatment of ovarian cancer.

The special stemness functions of Tbx3 in stem cells and cancer development.

The T-box factors belong to an ancient protein family, which comprises a cluster of evolutionarily-conserved transcription factors that regulate gene expression and that are crucial to embryonic development. T-box transcription factor 3 (Tbx3) is a member of this family, is expressed in some tissues, and is a key regulator in many critical organs, including the heart, mammary gland, and limbs. Overexpression of Tbx3 is associated with a number of cancers, including head and neck squamous cell carcinoma, gastric, breast, ovary, cervical, pancreatic, bladder and liver cancers, as well as melanoma. Tbx3 promotes tumor development by modulating cell proliferation, tumor formation, metastasis, cell survival and drug resistance. Moreover, there is strong evidence that Tbx3 regulates stem cell maintenance by controlling stem cell self-renewal and differentiation. Verification of the upstream regulatory factors and potential molecular mechanism of Tbx3, being able to explain the function of Tbx3 in carcinogenic effects and stem cell maintenance, will make a valuable contribution to stem cell and cancer research. This review provides an insight into the current research on Tbx3 and explores the significance of Tbx3 in stem cells and tumorigenesis.

TOPK promotes epithelial-mesenchymal transition and invasion of breast cancer cells through upregulation of TBX3 in TGF-ß1/Smad signaling.

TOPK has been suggested to contribute to invasion of lung, prostate, gastric, pancreatic or breast cancer cells. However, how TOPK mediates TGF-ß1/Smad signaling leading to epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells remains unknown. Here we report that TOPK upregulates T-box transcription factor TBX3 to enhance TGF-ß1-induced EMT and invasion of MDA-MB-231 breast cancer cells. Expression of endogenous TOPK was promoted by TGF-ß1 treatment of MDA-MB-231 cells time-dependently. In addition, knockdown of TOPK attenuated TGF-ß1-induced phosphorylation or transcriptional activity of Smad3. Meanwhile, levels of both mRNA and protein of TBX3 induced by TGF-ß1 were abolished by TOPK depletion. Also, knockdown of TBX3 inhibited TGF-ß1 induction of EMT-related genes Snail, Slug or Fibronectin. Furthermore, ablation of TOPK or TBX3 suppressed TGF-ß1-induced MDA-MB-231 cell invasion. Collectively, we conclude that TOPK positively regulates TBX3 in TGF-ß1/Smad signaling pathway, thereby enhancing EMT and invasion of breast cancer cells, implying a mechanistic role of TOPK in TGF-ß1/Smad signaling.

Bcl-2 Overexpression Induces Neurite Outgrowth via the Bmp4/Tbx3/NeuroD1 Cascade in H19-7 Cells.

Bcl-2 is overexpressed in the nervous system during neural development and plays an important role in modulating cell survival. In addition to its anti-apoptotic function, it has been suggested previously that Bcl-2 might act as a mediator of neuronal differentiation. However, the mechanism by which Bcl-2 might influence neurogenesis is not sufficiently understood. In this study, we aimed to determine the non-apoptotic functions of Bcl-2 during neuronal differentiation. First, we used microarrays to analyze the whole-genome expression patterns of rat neural stem cells overexpressing Bcl-2 and found that Bcl-2 overexpression induced the expression of various neurogenic genes. Moreover, Bcl-2 overexpression increased the neurite length as well as expression of Bmp4, Tbx3, and proneural basic helix-loop-helix genes, such as NeuroD1, NeuroD2, and Mash1, in H19-7 rat hippocampal precursor cells. To determine the hierarchy of these molecules, we selectively depleted Bmp4, Tbx3, and NeuroD1 in Bcl-2-overexpressing cells. Bmp4 depletion suppressed the upregulation of Tbx3 and NeuroD1 as well as neurite outgrowth, which was induced by Bcl-2 overexpression. Although Tbx3 knockdown repressed Bcl-2-mediated neurite elaboration and downregulated NeuroD1 expression, it did not affect Bcl-2-induced Bmp4 expression. While the depletion of NeuroD1 had no effect on the expression of Bcl-2, Bmp4, or Tbx3, Bcl-2-mediated neurite outgrowth was suppressed. Taken together, these results demonstrate that Bcl-2 regulates neurite outgrowth through the Bmp4/Tbx3/NeuroD1 cascade in H19-7 cells, indicating that Bcl-2 may have a direct role in neuronal development in addition to its well-known anti-apoptotic function in response to environmental insults.