Ulnar-mammary Syndrome Research Articles

TBX3, the gene mutated in ulnar-mammary syndrome, promotes growth of mammary epithelial cells via repression of p19ARF, independently of p53

TBX3, the gene mutated in ulnar-mammary syndrome (UMS), is involved in the production of a transcription factor of the T-box family, known to inhibit transcription from the p14ARF (p19ARF in mouse) promoter in fibroblasts and to contribute to cell immortalization. One of the main features of the UMS phenotype is the severe hypoplasia of the breast, associated with haploinsufficiency of the TBX3 gene product. In mice homozygous for the targeted disruption of Tbx3, the mammary glands (MGs) are nearly absent from early stages of embryogenesis, whereas in heterozygous adults, the MGs show reduced ductal branching. All these data strongly suggest a specific role of TBX3 in promoting the growth of mammary epithelial cells (MECs), although direct evidence of this is lacking. Here, we provide data showing the growth-promoting function of Tbx3 in several models of MECs, in association with its ability to repress the ARF promoter. However, no effect of Tbx3 on cell differentiation or apoptosis has been observed. The growth promoting function also entails the down-regulation of p21 ( CIP1/WAF ) and an increase in cyclin D1 but is independent of p53 and Mdm2 cell-cycle regulatory proteins, as p53-null MECs show similar growth responses associated with the up- or down-regulation of Tbx3. This is the first direct evidence that the level of Tbx3 expression positively controls the proliferation of MECs via pathways alternative to Mdm2-p53.

Molecular interactions between Tbx3 and Bmp4 and a model for dorsoventral positioning of mammary gland development.

The formation of the dorsoventral (DV) boundary is central to establishing the body plan in embryonic development. Although there is some information about how limbs are positioned along the DV axis and how DV skin color pattern is determined, the way in which mammary glands are positioned is unknown. Here we focus on Bmp4 and Tbx3, a gene associated with ulnar-mammary syndrome, and compare their expression along the DV axis in relation to mammary gland initiation in mouse embryos. Tbx3 is expressed in the mammary gland-forming region with Tbx15, a gene involved in a DV coat color being expressed more dorsally and Bmp4 being expressed more ventrally. When Tbx3 was overexpressed, formation of mammary gland epithelium was extended along the DV axis. In contrast, overexpression of Bmp4 inhibited both Tbx3 and Tbx15 expression. In addition, when BMP signaling was inhibited by NOGGIN, Lef1 expression was lost. Thus, we propose that mutual interactions between Bmp4 and Tbx3 determine the presumptive DV boundary and formation of mammary glands in early mouse embryogenesis. 1,19-Dioctadecyl-3,3,39,39-tetramethyl indocarbocyanine perchloride labeling experiments showed that cells associated with mammary glands originate more dorsally and then move ventrally. This finding, together with previous findings, suggests that the same DV boundary may not only position limbs and determine coat color but also position mammary glands. Furthermore, Bmp signaling appears to be a fundamental feature of DV patterning.

Tbx3, a transcriptional factor, involves in proliferation and osteogenic differentiation of human adipose stromal cells

Tbx3 is a transcription factor, the mutation of which causes ulnar mammary syndrome (UMS) characterized by abnormality and hypoplasia of the mammary gland, teeth, limbs, hair and genitalia. Tbx3 has been reported to be related to apoptosis and proliferation of rat bladder carcinoma cell and to regulate proliferation and differentiation of mouse osteoblast cells. Human adipose tissue stromal cells (hADSC) have been defined as multipotential adult stem cells, capable of differentiating into a variety of cell types such as osteoblasts, chondrocytes, adipocytes, muscle cells, and neural cells. To determine the functional roles of Tbx3 expression in hADSC, we used lentivirus siRNA vector. Expression of Tbx3 was downregulated during culture expansion. Downregulation of Tbx3 in hADSC by transduction of siTbx3 lentivirus decreased proliferation and osteogenic differentiation of hADSC. Expression of Tbx3 and the ratio of Tbx3 + 2a to Tbx3 increased during osteogenic differentiation. This report shows that Tbx3 plays an important role on osteogenic differentiation and proliferation of human mesenchymal stem cell derived from adipose tissue.

Ulnar–mammary syndrome with dysmorphic facies and mental retardation caused by a novel 1.28 Mb deletion encompassing the TBX3 gene

Ulnar-mammary syndrome (UMS) is a rare autosomal-dominant disorder caused by mutations in TBX3. The condition is characterized by hypoplasia or aplasia of upper limbs on the ulnar side, mammary glands and nipples, and of apocrine glands in both sexes (MIM #181450). We report on a girl presenting with an UMS like phenotype, a dysmorphic facies, and mental retardation. Mutation analysis of TBX3 and G-banded chromosome analysis from lymphocytes were performed. We used microarray-based comparative genomic hybridization (array CGH) to investigate the patient's genomic DNA for submicroscopic aberrations. No mutation of the TBX3 gene was detected in our patient and chromosome analysis revealed a normal female karyotype (46,XX). Hybridization of a whole-genome tiling path array consisting of more than 36 000 BAC clones revealed an interstitial 1.28 Mb deletion within chromosomal band 12q24.21. The deleted region encompasses one known gene, TBX3. The deletion and haploinsufficiency of TBX3 was confirmed by fluorescence in situ hybridization using BAC clones representing the deletion on the BAC array. To our knowledge, this is the first description of TBX3 haploinsufficiency caused by a genomic deletion in a patient with UMS. We suggest that the UMS phenotype in conjunction with the characteristic facial changes and mental retardation observed in our patient is owing to the deletion of TBX3 and the involvement of neighbouring genes.

Isl1Cre reveals a common Bmp pathway in heart and limb development

A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 (Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor, Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo.

Tbx3, the ulnar‐mammary syndrome gene, and Tbx2 interact in mammary gland development through a p19Arf/p53‐independent pathway

The closely related T-box genes Tbx2 and Tbx3 are both expressed in the developing mammary glands of mouse embryos and both have been implicated in mammary carcinogenesis. Tbx3 is essential for induction of the mammary placodes in mice. In humans, mutations in TBX3 are responsible for ulnar-mammary syndrome. Here, we show a haploinsufficiency effect of Tbx3 on maintenance of the mammary placodes and on the extent of branching of the ductal tree in mice. Loss or heterozygosity for Tbx2, on the other hand, has no effect on either induction or maintenance of the placodes, although a small effect was seen on branching morphogenesis in adult heterozygotes. However, the deficiency in maintenance of the mammary placodes in Tbx2, Tbx3 double heterozygous mice is more marked than in Tbx3 single heterozygotes, indicating a genetic interaction between the two genes. In spite of a large body of evidence implicating these genes in cell cycle control through the p19(Arf)/p53 pathways, we find no evidence for involvement of these pathways either in embryonic lethality of homozygous mutants or in the mammary gland phenotype of Tbx3 heterozygous mice.

P4: Ulnar-mammary syndrome with mental retardation caused by a novel 0.8 Mb deletion encompassing the TBX3 gene

P4: Ulnar-mammary syndrome with mental retardation caused by a novel 0.8 Mb deletion encompassing the TBX3 gene

Congenital palmar polyonychia with postaxial limb defects may be the same as the ulnar–mammary syndrome

Congenital palmar polyonychia with postaxial limb defects may be the same as the ulnar–mammary syndrome

Novel TBX3 mutation data in families with Ulnar–Mammary syndrome indicate a genotype–phenotype relationship: mutations that do not disrupt the T-domain are associated with less severe limb defects

We describe a family affected by Ulnar–Mammary syndrome (UMS) in which typical UMS traits (hypoplasia of the breast and axillary hair, upper limbs and genital defects) are present together with cardiac malformations and pulmonary stenosis. Sequence analysis of TBX3 shows a new heterozygous mutation that causes a frame-shift (Nt.1586-1587-insC) in exon 6, resulting in a truncated ORF. Recently the expression of Tbx3 has been described also in the septal region of the embryonic murine heart. This observation may establish a link between the congenital heart defects and the TBX3 mutation in this family. Combining the TBX3 mutation data in the literature with this novel mutation we find an association between mutations that disrupt the DNA-binding domain and a higher frequency of severe upper limb malformations and teeth defects. A possible explanation is that mutant TBX3 proteins that retain the T-domain, if translated, might be minimally active in promoting/repressing transcription of target genes in the limbs and in other embryonic tissues.

Familial progressive sinoatrial and atrioventricular conduction disease of adult onset with sudden death, dilated cardiomyopathy, and brachydactyly. A new type of heart‐hand syndrome?

We identified a family with 10 affected members in four generations suffering from adult-onset progressive sinoatrial and atrioventricular conduction disease, sudden death due to ventricular tachyarrhythmia, dilated cardiomyopathy, and a unique type of brachydactyly with mild hand involvement (short distal, middle, proximal phalanges and clinodactyly) and more severe foot involvement (short distal, proximal phalanges and metatarsal bones, short or absent middle phalanges, terminal symphalangism, duplication of the bases of the second metatarsals, extra ossicles, and syndactyly). The phenotype differences from other reported genetic abnormalities and linkage exclusion of Holt-Oram syndrome, ulnar-mammary syndrome, brachydactyly type B or Robinow syndrome, and cardiac conduction disease or Brugada syndrome loci suggest that we report on a new hereditary heart-hand syndrome.

Evaluating the Domain-Specific Functions of Tbx3 in Limb Development in a Mouse Model of Ulnar-Mammary Syndrome

Evaluating the Domain-Specific Functions of <i>Tbx3</i> in Limb Development in a Mouse Model of Ulnar-Mammary Syndrome

ATCUN‐like metal‐binding motifs in proteins: Identification and characterization by crystal structure and sequence analysis

The amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif is a small metal-binding site found in the N-terminus of many naturally occurring proteins. The ATCUN motif has been implicated in DNA cleavage and has been shown to have antitumor activity. In proteins, the ATCUN motif is formed from a histidine in the third position, its preceding residue and the free N-terminus. Four nitrogen atoms from these three residues act as metal ligands. Knowledge of metal-binding geometry helps in the design of metal-binding peptides and in understanding of the mechanisms of metal-mediated functions. Since the N-terminus region of ATCUN-containing proteins is highly disordered, no geometrical features can be derived from the protein structures. However, the crystal structure of a small metal-bound ATCUN peptide shows that the nitrogen ligands form a distorted square planar geometry. Distance constraints derived from this designed peptide were used to search 1949 polypeptide chains to find ATCUN-like motifs in any position along the polypeptide chain. Only approximately 1.9% and approximately 0.3% of histidines are involved in partial and full ATCUN-like geometric features, respectively. These two datasets were compared with the dataset of all histidines. None of the ATCUN-like motifs occur in the middle of an alpha-helix or a beta-strand. Further sequence analysis revealed total conservation of ATCUN histidines in four proteins including the transcription factor TBX3, implicated in Ulnar-Mammary Syndrome. Our analysis suggests that the ATCUN-like motif in TBX3 is a potential metal-binding site, although a structural role was not completely ruled out. Metal-binding activity in TBX3, if confirmed, will help us to understand the role of metals in transcriptional regulation and is likely to cast light on the causes of some serious genetic disorders. A conformational role is suggested for ATCUN-like motifs in other proteins.

TBX3 and its isoform TBX3+2a are functionally distinctive in inhibition of senescence and are overexpressed in a subset of breast cancer cell lines.

TBX3 is a transcription factor of the T-box gene family. Mutations of TBX3 cause ulnar-mammary syndrome (MIM 181450) in humans, an autosomal dominant disorder characterized by the absence or underdevelopment of the mammary glands and other congenital anomalies. It recently was found that TBX3 was able to immortalize mouse embryo fibroblast (MEF) cells. In addition, TBX2, a homologue of TBX3, is active in preventing senescence in rodent cells and was found to be amplified in some human breast cancers, suggesting TBX3 plays a role in breast cancer. This study examined the function of TBX3 and its isoform, TBX3 + 2a. TBX3 + 2a differs from TBX3 in the DNA binding domain with an extra 20 amino acids produced by alternative splicing. We first examined the tissue expression and alternative splicing patterns of these two isoforms. We found that TBX3 and TBX3 + 2a are widely expressed in humans and mice, and alternative splicing could be tissue specific and species specific. Overexpression of TBX3 is able to immortalize MEF cells, whereas TBX3 + 2a shows an acceleration of senescence, a functional difference that may be explained by the fact that these two isoforms may have different downstream targets. TBX3, but not TBX3 + 2a, is able to bind to the previously identified T-box binding site in a gel shift assay. A subset of human breast cancer cell lines overexpresses TBX3. Our results indicate that TBX3 and TBX3 + 2a are functionally distinctive in inhibition of senescence of MEF cells and may play a role in breast cancer.

Interactions between FGF and Wnt signals and Tbx3 gene expression in mammary gland initiation in mouse embryos.

Interactions between Wnts, Fgfs and Tbx genes are involved in limb initiation and the same gene families have been implicated in mammary gland development. Here we explore how these genes act together in mammary gland initiation. We compared expression of Tbx3, the gene associated with the human condition ulnar-mammary syndrome, expression of the gene encoding the dual-specificity MAPK phosphatase Pyst1/MKP3, which is an early response to FGFR1 signalling (as judged by sensitivity to the SU5402 inhibitor), and expression of Lef1, encoding a transcription factor mediating Wnt signalling and the earliest gene so far known to be expressed in mammary gland development. We found that Tbx3 is expressed earlier than Lef1 and that Pyst1 is also expressed early but only transiently. Patterns of expression of Tbx3, Pyst1 and Lef1 in different glands suggest that the order of mammary gland initiation is 3, 4, 1, 2 and 5. Consistent with expression of Pyst1 in the mammary gland, we detected expression of Fgfr1b, Fgf8 and Fgf9 in both surface ectoderm and mammary bud epithelium, and Fgf4 and Fgf17 in mammary bud epithelium. Beads soaked in FGF-8 applied to the flank of mouse embryos, at a stage just prior to mammary bud initiation, induce expression of Pyst1 and Lef1 and maintain Tbx3 expression in flank tissue surrounding the bead. Grafting beads soaked in the FGFR1 inhibitor, SU5402, abolishes Tbx3, Pyst1 and Lef1 expression, supporting the idea that FGFR1 signalling is required for early mammary gland initiation. We also showed that blocking Wnt signalling abolishes Tbx3 expression but not Pyst1 expression. These data, taken together with previous findings, suggest a model in which Tbx3 expression is induced and maintained in early gland initiation by both Wnt and Fgf signalling through FGFR1.

The role of Tbx2 and Tbx3 in mammary development and tumorigenesis.

TBX2 and TBX3 are members of a family of genes encoding developmental transcription factors, characterized by a 200 amino acid DNA binding domain (T-box). Tbx2 and Tbx3 are closely related T-box proteins that have been implicated in development of a number of different tissues including the mammary gland. TBX3 is required for normal mammary development in mouse models and in patients with ulnar-mammary syndrome (UMS). In addition to a role in development, TBX2 and TBX3 have been implicated in tumor development through downregulation of the alternative reading frame (ARF) tumor suppressor and an associated bypass of senescence. Here we review the current information on the roles of Tbx2 and Tbx3 in mammary gland development and tumorigenesis.

Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome.

Spontanteous mutations in the T-box gene TBX3, result in the human ulnar-mammary syndrome, a dominant developmental disorder characterized by abnormal forelimb and apocrine gland development. In order to develop a mouse model to study the role of this gene during development and disease, we produced a mutation in the mouse ortholog, Tbx3. The phenotype of the mutant mice verifies the role of this gene in limb and mammary gland development, and, in addition, reveals a previously unknown role for the gene in the yolk sac, a fetal membrane that is the site of hematopoiesis and is essential for survival during gestation. In homozygous mutant embryos, the yolk sac undergoes cell death and degeneration at midgestation and the fetuses die over a range of several days; none survive to birth. Tbx3 is the first T-box gene implicated in yolk sac development. Homozygous embryos show a deficiency of mammary gland induction, and exhibit both forelimb and hindlimb abnormalities. Although heterozygous mice, unlike their heterozygous human counterparts, have no apparent phenotype in limb or mammary gland, the homozygous defects in the development of these organs represent more severe manifestations of the defects characteristic of the ulnar-mammary syndrome.

Regulation of Tbx3 Expression by Anteroposterior Signalling in Vertebrate Limb Development

Tbx3 , a T-box gene family member related to the Drosophila gene optomotor blind ( omb ) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar–mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarising region which controls anteroposterior pattern. Here we carried out tissue transplantations, grafted beads soaked in Shh, Bmps, and Noggin in chick limb buds, and analysed Tbx3 expression. We also investigated Tbx3 expression in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. We show that Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centred on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. Our results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number.

Haploinsufficiency of TBX3 causes ulnar-mammary syndrome in a large Turkish family

We present a large Turkish family with autosomal dominant inherited ulnar-mammary syndrome in which 10 affected family members, spanning three generations, were diagnosed. The phenotypic expression of the disease was found to be highly variable among the affected family members showing posterior-limb deficiencies and/or duplications, mammary-gland hypoplasia, apocrine dysfunction, dental and genital abnormalities. Mutation analysis of the TBX3 gene showed a novel one base-pair insertion at position 89 (designated 88_89insA) in the coding region. The mutation leads to a shift of the open reading frame and causes a premature truncation of the protein (M30fsX110). The truncated protein lacks almost all functional important parts of TBX3, most likely leading to a complete loss of functional protein. Our findings indicate that ulnar-mammary syndrome shows a wide range of phenotypes even within the same family and provide further evidence that haploinsufficiency of TBX3 is the disease-causing mechanism.

Regulation of Tbx3 Expression by Anteroposterior Signalling in Vertebrate Limb Development

Tbx3, a T-box gene family member related to the Drosophila gene optomotor blind ( omb) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar–mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarising region which controls anteroposterior pattern. Here we carried out tissue transplantations, grafted beads soaked in Shh, Bmps, and Noggin in chick limb buds, and analysed Tbx3 expression. We also investigated Tbx3 expression in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. We show that Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centred on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. Our results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number.

The T-box Repressors TBX2 and TBX3Specifically Regulate the Tumor Suppressor Genep14ARF via a Variant T-site in the Initiator

The murine tumor suppressor p19(ARF) (p14(ARF) in humans) is thought to fulfill an important protective role in preventing primary cells from oncogenic transformation via its action in the p53 pathway. Several disease-implicated regulators of p19(ARF) are known to date, among which are the T-box genes TBX2, which resides on an amplicon in primary breast tumors, and TBX3, which is mutated in the human developmental disorder Ulnar-Mammary syndrome. Here we identify a variant T-site, matching 13 of 20 nucleotides of a consensus T-site, as the essential TBX2/TBX3-binding element in the human p14(ARF) promoter. Mutant analysis indicates that both the consensus T-box and a C-terminal conserved repression domain are essential for p14(ARF) repression. Whereas the core nucleotides required for interaction of the archetypal T-box protein Brachyury with a consensus T-site are conserved in the variant site, additional flanking nucleotides contribute to the specificity of TBX2 binding. This is illustrated by the inability of TBX1A or Xbra to activate via the variant p14(ARF) T-site. Importantly, this suggests a hitherto unsuspected level of specificity associated with T-box factors and corresponding recognition sites in regulating their target genes in vivo.