Sergei I. Agulnik

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development

A novel family of genes, characterized by the presence of a region of homology to the DNA‐binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T‐box, and the new mouse genes that contain the T‐box domain have been named T‐box 1–6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1–Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3–5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2–5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T‐box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Expression of T-box genes Tbx2–Tbx5 during chick organogenesis

T-box genes encode putative transcription factors implicated in diverse developmental processes (Papaioannou, V.E. and Silver, L.M., 1998. BioEssays 20, 9-19). We have previously reported the embryonic expression patterns of T-box genes in mice (Chapman, D.L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S.I., Gibson-Brown, J.J., Cebra-Thomas, J., Bollag, R.J., Silver, L.M., Papaioannou, V.E., 1996. Dev. Dyn. 206, 379-390; Chapman, D.L., Agulnik, I., Hancock, S., Silver, L.M. and Papaioannou, V.E., 1996. Dev. Biol. 180, 534-542; Gibson-Brown, J.J., Agulnik, S.I., Chapman, D.L., Alexiou, M., Garvey, N., Silver, L.M., Papaioannou, V.E., 1996. Mech. Dev. 56, 93-101). Four of these genes (Tbx2-Tbx5) are represented in the mouse genome as two cognate, linked gene pairs (Agulnik, S.I., Garvey, N., Hancock, S., Ruvinsky, I., Chapman, D.L., Agulnik, I., Bollag, R., Papaioannou, V.E., Silver, L.M., 1996. Genetics 144, 249-254), and have all been implicated in playing important roles in limb development (Gibson-Brown, J.J., Agulnik, S.I., Chapman, D.L., Alexiou, M., Garvey, N., Silver, L.M., Papaioannou, V.E., 1996. Mech. Dev. 56, 93-101). To investigate the role of these genes in limb development, we cloned the chicken orthologs and report functional studies, as well as patterns of expression in the developing limbs, elsewhere (Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Niswander, L., Papaioannou, V.E., Development, in press). This report details the patterns of expression of Tbx2-Tbx5 in chick embryonic tissues other than the limbs.

Mapping and expression analysis of the mouse ortholog of Xenopus Eomesodermin

The T-box gene family has been conserved throughout metazoan evolution. The genes code for putative transcription factors which share a uniquely defining DNA binding domain, known as the T-box ([Bollag et al., 1994]). They are implicated in the control of diverse developmental processes by their highly specific expression patterns throughout gastrulation and organogenesis in mouse and other species ([Chapman et al., 1996]) ([Gibson-Brown et al., 1998]), and by mutations in T-box genes that have profound developmental effects ([Papaioannou, 1997]; [Chapman and Papaioannou, 1998]; [Papaioannou and Silver, 1998]). In this report, we describe the mapping and expression pattern of the mouse ortholog of a gene, Eomesodermin, first identified in Xenopus ([Ryan et al., 1996]). The mouse gene was previously reported ([Wattler et al., 1998]) under the name MmEomes. The gene maps to mouse chromosome 9 in a region syntenic with human chromosome 3p. Mouse eomesodermin is expressed in the trophoblast of the blastocyst and in its derivative, the chorionic ectoderm. At gastrulation, eomesodermin is expressed in the primitive streak and embryonic mesoderm as well, but this expression disappears prior to the end of gastrulation. Later, eomesodermin is expressed in the developing forebrain, in a pattern largely overlapping a closely related T-box gene, Tbr1 ([Bulfone et al., 1995]), and is also seen in a localized area of each limb.

Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans.

Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.

Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family.

The T-box motif is present in a family of gene whose structural features and expression patterns support their involvement in developmental gene regulation. Previously, sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes established a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, we focused our search for the human homolog, Hs-TBX2, within a region of synteny conservation on Chr 17q. We used Dm-Omb polymerase chain reaction (PCR) primers to amplify a 137-basepair (bp) product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product showed 89% DNA sequence identity and 100% petide sequence identity to the corresponding T-box segment of Mm-Tbx2. The putative Hs-TBX2 locus was isolated within a YAC contig that included three anonymous markers, D17S792, D17S794, and D17S948, located at Chr 17q21-22. Hybridization-and PCR-based screening of a 15-week fetal kidney cDNA library yielded several TBX2 clones. Sequence analysis of clone λcTBX2-1 confirmed homology to Mm-Tbx2-90% DNA sequence identity over 283 nt, and 96% peptide sequence identity over 94 amino acids. Similar analysis of Hs-TBX2 cosmid 15F11 confirmed the cDNA coding sequence and also identified a 1.7-kb intron located at the same relative position as in Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggested that the putative human TBX2 and mouse Tbx2 are true homologs. Northern blot analysis identified two major TBX2 transcripts of 3.5 and 2.8kb, with high levels of TBX2 expression in fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels were seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation.

Analysis of mutation rates in the SMCY/SMCX genes shows that mammalian evolution is male driven.

Mammalian evolution is believed to be male driven because the greater number of germ cell divisions per generation in males increases the opportunity for errors in DNA replication. Since the Y Chromosome (Chr) replicates exclusively in males, its genes should also evolve faster than X or autosomal genes. In addition, estimating the overall male-to-female mutation ratio (αm) is of great importance as a large αm implies that replication-independent mutagenic events play a relatively small role in evolution. A small αm suggests that the impact of these factors may, in fact, be significant. In order to address this problem, we have analyzed the rates of evolution in the homologous X-Y common SMCX/SMCY genes from three different species—mouse, human, and horse. The SMC genes were chosen because the X and Y copies are highly homologous, well conserved in evolution, and in all probability functionally interchangeable. Sequence comparisons and analysis of synonymous substitutions in approximately 1kb of the 5′ coding region of the SMC genes reveal that the Y-linked copies are evolving approximately 1.8 times faster than their X homologs. The male-to-female mutation ratio αm was estimated to be 3. These data support the hypothesis that mammalian evolution is male driven. However, the ratio value is far smaller than suggested in earlier works, implying significance of replication-independent mutagenic events in evolution.

Effect of sperm genotype on chromatid segregation in female mice heterozygous for aberrant chromosome 1

An aberrant chromosome 1 with two large homogeneously staining insertions was isolated from wild populations of Mus musculus musculus. The specific features of the aberrant chromosome have been described elsewhere (Agulnik et al. 1990). These include its preferential entry into the oocyte of heterozygous females, increased mortality of homozygotes and decreased fertility of homozygous females. Here we present data indicating that chromatid segregation in heterozygous females depends upon which sperm enters the oocyte before the second meiotic division: meiotic drive is powerful when it is sperm bearing normal chromosome 1, and segregation normalizes during MII when it is sperm bearing chromosome 1 with the extra segment. Experimental data are adduced and explanations offered for the observed phenomenon.

TBX10, a member of the Tbx1-subfamily of conserved developmental genes, is located at human Chromosome 11q13 and proximal mouse Chromosome 19

The T-box developmental gene family was originally described in the mouse (Bollag et al. 1994), and homologs were subsequently identified in a wide variety of metazoans (Agulnik et al. 1995). T-box genes encode transcription factors that are expressed differentially during embryogenesis and/or in tissue-specific fashion throughout adulthood (reviewed in Smith 1997). The importance of T-box genes recently was underscored by the identification of TBX gene mutations in two human developmental diseases— TBX5 in Holt-Oram syndrome (Li et al. 1997; Basson et al. 1997) and TBX3 in ulnar-mammary syndrome (Bamshad et al. 1997). Here we report the isolation and mapping of a new T-box family member, TBX10/Tbx10, in human and mouse. We isolated the human clone from a lambda Charon 4A lymph node genomic library (ATCC LI014), using reduced stringency hybridization with a mouse Tbx1 cDNA probe. This probe is comprised of sequence within the highly conserved T-box region of Tbx1 (nt258-nt439 of GenBank entry MMU57327), and, as expected, the isolated clone showed high sequence similarity to the T-box of both mouse Tbx1 and human TBX1 (Chieffo et al. 1997). Human TBX1 resides at Chromosome (Chr) 22q11, syntenic to the map location of mouse Tbx1 on Chr 16 (Chieffo et al. 1997). Our localization of human TBX10 to Chr 11q13 (Figs. 1, 2A) and gene sequence analysis (Fig. 3) confirm that the isolated gene is a related, but distinct, member of the Tbx1 subfamily, which we have designated TBX10. By FISH analysis, human TBX10 maps to Chr 11q13 (Fig. 1). To refine the map location, human TBX10 was isolated in a CEPH/Genethon YAC contig linked to anonymous markers spanning 11q13.1-q13.2 (Fig. 2A). YAC clones 894A10 and 809C9 were isolated using an STS based on TBX10 exon sequence (TBX10Exon-F:58-TTAGACAGCTCGGCCTGG-38 and TBX10Exon-R: 58-CATTGTCATCCAGCAGGTTG-38). The YAC contig in Fig. 2A shows that human TBX10 is within 100 kb of anonymous marker AFMa152yh1 at 11q13.2. This places TBX10 in close proximity to the locus for Bardet-Biedl syndrome 1 (BBS1), an autosomal recessive disorder characterized by retinitis pigmentosa, polydactyly, obesity, hypogenitalism, mental retardation, and renal malformations (Bruford et al. 1997; Leppert et al., 1994). Screening of the GenBank database with human TBX10 sequence identified mouse T-cell cDNA clones 550940 (GenBank Accession No. AA098449) as a highly significant match. A probe derived from this clone was mapped on the BSS backcross panel to proximal Chr 19 and co-segregated with marker D19Bir1 (Fig. 2B; MGD-JNUM-43729). This location is within a region of known synteny to human Chr 11q13 (DeBry and Seldin 1996). To further confirm that we were studying the mouse ortholog of the human TBX10 gene, we compared available gene sequence outside of the highly conserved T-box (Fig. 3). High homology in Correspondence to: D.J. Law

Sex-specific modifiers of tail development in mice heterozygous for the brachyury (T) mutation

The brachyury, or T, locus encodes a transcription factor that plays a crucial role in the early development of all animals. In the mouse, animals heterozygous for a null mutation at this locus are born with a characteristic short tail. Expressivity of the short tail phenotype is greatly affected by genetic background. As a genetic entry into the identification of genes that interact with the Brachyury locus, we have performed a QTL analysis for modifiers of this phenotype. Surprisingly, we discovered that the major modifiers uncovered all act in a sex-limited manner. We have identified two QTLs—Brm1 on Chr 9 and Brm2 on Chr 15—that act only in female offspring(N2) from female T/+ parents(F1) and are responsible together for most, or all, of the genetic variance in phenotypic expression observed between C57BL/10 and C3H/HeJ animals.

Genetics analysis of mouse mutations Abnormal feet and tail and rough coat, which cause developmental abnormalities and alopecia

Mutations in the mouse Brachyury (T) gene are characterized by a dominant reduction of tail length and recessive lethality. Two quantitative trait loci, Brachyury-modifier 1 and 2 (Brm1 and Brm2) are defined by alleles that enhance the short-tail Brachyury phenotype. Here we report on a genetic analysis of a visible dominant mutation Abnormal feet and tail (Aft) located in the vicinity of Brm1. Affected animals display kinky tails and syndactyly in the hindlimbs, both likely resulting from a defect in apoptosis. We observed an unusual genetic incompatibility between Aft and certain genetic backgrounds. We show that Aft and T are likely to interact genetically, since some double heterozygotes are tailless. In addition to the tail and hindlimb phenotypes, Aft-bearing mutants display characteristic late-onset skin lesions. We therefore tested for allelism between Aft and a closely linked recessive mutation rough coat (rc) and found that these two mutations are likely nonallelic. Our results provide a valuable resource for the study of mammalian skin development and contribute to the genetic analysis of Brachyury function.