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.

Cloning, mapping, and expression analysis of TBX15, a new member of the T-Box gene family.

The T-box gene family has been conserved throughout metazoan evolution and codes for putative transcription factors that share a uniquely defining DNA-binding domain. We have previously uncovered six mouse T-box genes with discrete spatial and temporal patterns of expression during embryogenesis. Here, we report a novel mouse T-box gene, Tbx15. The Tbx15 gene produces a 3.7-kb transcript with an open reading frame coding for a polypeptide with 602 amino acid residues. Phylogenetic analysis places the Tbx15 gene into a T-box subfamily that also includes mouse Tbx1, Drosophila H15, and nematode Ce-tbx-12 genes. We have mapped mouse Tbx15 to chromosome 3, at a position 49 cM from the centromere. During development, Tbx15 transcripts are first detected at embryonic day 9.5. The gene is expressed primarily in the cranio-facial region and in the developing limbs. An isolated human homolog, TBX15, has been mapped by in situ hybridization to chromosomal band 1p13. TBX15 appears to be an excellent candidate for the dominantly expressed acromegaloid facial appearance syndrome, which also maps to the short arm of human chromosome 1 and, like TBX15, is expressed prominently in the eyebrow regions.

Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development.

We have recently shown in mice that four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and that expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. In this report, we investigate the role of these genes in limb specification and development, using the chick as a model system. We induced the formation of ectopic limbs in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. We found that, whereas bud regions expressing Tbx4 developed characteristic leg structures, regions expressing Tbx5 developed characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retained autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryological manipulations. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, we found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH)-expressing cell pellet under the anterior AER, we found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target of SHH. Our findings suggest that these genes are intimately involved in limb development and the specification of limb identity, and a new model for the evolution of vertebrate appendages is proposed.

Sequence divergence within the sperm-specific polypeptide TCTE1 is correlated with species-specific differences in sperm binding to zona-intact eggs.

The T-complex-associated testes-expressed (TCTE1) gene encodes a novel sperm cell-specific polypeptide (TCTE1) that is conserved across vertebrate species. TCTE1 is absolutely required for fertilization and is expressed in earlier stages of spermatogenesis. When the amino acid sequence of the TCTE1 gene product is compared among various mammalian species, a large, highly conserved domain is observed, along with a divergent domain encoding the 56-58 residues at the N terminus. In this study, the N-terminal regions of the TCTE1 polypeptide from three rodent species--mouse, gerbil, and rat--were compared. The results show that while the gerbil and mouse species are most distant in evolutionary terms, their TCTE1 homologs have not undergone significant divergence. In contrast, the N-terminal region of the rat TCTE1 homolog has evolved rapidly, a finding that indicates positive Darwinian selection. We have tested the correlation between TCTE1 divergence and heterospecific sperm-egg binding ability in the three species under study. Gerbil sperm bind to mouse eggs, while no significant binding is observed between rat sperm and mouse eggs. The results obtained support the hypothesis that the sperm-specific polypeptide TCTE1 may facilitate species-specific divergence of sperm function.

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.

Three novel T-box genes in Caenorhabditis elegans.

The T-box gene family consists of members that share a unique DNA binding domain. The best characterized T-box gene, Brachyury or T, encodes a transcription factor that plays an important role in early vertebrate development. Seven other recently described mouse T-box genes are also expressed during development. In the nematode Caenorhabditis elegans, four T-box genes have been characterized to date. In this study, we describe three new C. elegans T-box genes, named Ce-tbx-11, Ce-tbx-12, and Ce-tbx-17. Ce-tbx-11 and Ce-tbx-17 were uncovered through the sequencing efforts of the C. elegans Genome Project. Ce-tbx-12 was uncovered through degenerate PCR analysis of C. elegans genomic DNA. Ce-tbx-11 and Ce-tbx-17 are located in close proximity to the four other previously described T-box genes in the central region of chromosome III. In contrast, Ce-tbx-12 maps alone to chromosome II. Phylogenetic analysis of all known T-box domain sequences provides evidence of an ancient origin for this gene family.

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 (alpha m) is of great importance as a large alpha m implies that replication-independent mutagenic events play a relatively small role in evolution. A small alpha m 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 alpha m 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.

Evolution of mouse T-box genes by tandem duplication and cluster dispersion.

The T-box genes comprise an ancient family of putative transcription factors conserved across species as divergent as Mus musculus and Caenorhabditis elegans. All T-box gene products are characterized by a novel 174-186-amino acid DNA binding domain called the T-box that was first discovered in the polypeptide products of the mouse T locus and the Drosophila melanogaster optomotor-blind gene. Earlier studies allowed the identification of five mouse T-box genes, T, Tbx1-3, and Tbr1, that all map to different chromosomal locations and are expressed in unique temporal and spatial patterns during embryogenesis. Here, we report the discovery of three new members of the mouse T-box gene family, named Tbx4, Tbx5, and Tbx6. Two of these newly discovered genes, Tbx4 and Tbx5, were found to be tightly linked to previously identified T-box genes. Combined results from phylogenetic, linkage, and physical mapping studies provide a picture for the evolution of a T-box subfamily by unequal crossing over to form a two-gene cluster that was duplicated and dispersed to two chromosomal locations. This analysis suggests that Tbx4 and Tbx5 are cognate genes that diverged apart from a common ancestral gene during early vertebrate evolution.

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.

Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity.

Tetrapod fore-and hindlimbs have evolved from the pectoral and pelvic fins of an ancient vertebrate ancestor. In this ancestor, the pectoral fin appears to have arisen following the rostral homeotic recapitulation of an existing pelvic appendage (Tabin and Laufer (1993), Nature 361, 692-693). Thus the basic appendage outgrowth program is reiterated in both tetrapod fore- and hindlimbs and the pectoral and pelvic fins of extant teleost fishes (Sordino et al. (1995) Nature 375, 678-681). Recently a novel family of putative transcription factors, which includes the T (Brachyury) locus, has been identified and dubbed the "T-box' family. In mice, all of these genes have expression patterns indicative of involvement in embryonic induction (Chapman et al. (1996) Dev. Dyn., in press), and four (Tbx2-Tbx5) are represented as two cognate, linked gene pairs (Agulnik et al., (1996), Genetics, in press). We now report that, whereas Tbx2 and Tbx3 are expressed in similar spatiotemporal patterns in both limbs, Tbx5 and Tbx4 expression is primarily restricted to the developing fore- and hindlimb buds, respectively. These observations suggest that T-box genes have played a role in the evolution of fin and limb morphogenesis, and that Tbx5 and Tbx4 may have been divergently selected to play a role in the differential specification of fore- (pectoral) versus hind- (pelvic) limb (fin) identity.

The Cairo spiny mouse Acomys cahirinus shows a strong affinity to the Mongolian gerbil Meriones unguiculatus.

The classification of the African spiny mice (genus Acomys) within the Muridae family of rodents has been fraught with controversy. Morphological data suggest a close affinity between this group and true old world mice of the genus Mus. However, the combined results of immunological, biochemical, and DNA melting studies suggest that spiny mice should not even be considered as members of the Murinae subfamily. To further elucidate the position of the spiny mice within the rodent phylogenetic tree, we performed a direct sequence comparison of a 583-nucleotide homologous region from the spiny mouse type species Acomys cahirinus and five other representative rodent species. Our results provide incontrovertible evidence to support the contention that the spiny mouse is more closely related to the Mongolian gerbil Meriones unguiculatus than it is to Mus musculus.

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 genes 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-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% peptide 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, 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 lambda omicron TBX2-1 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 154F11 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 expression 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.

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.

Evolution of a long-range repeat family in chromosome 1 of the genus Mus.

Copy numbers and variation of a clustered long-range repeat family on chromosome (Chr) 1 have been studied in different species of the genus Mus. The repeat sequence was present in all, as inferred from cross-hybridization with probes derived from the Mus musculus repeat family. Copy numbers determined by dot blot hybridization were very low, from three to six per haploid genome in M. caroli, M. cervicolor, and M. cookii. These species form one branch of the phylogenetic tree in the genus Mus. In the other group of phylogenetically related species--M. spicilegus, M. spretus, M. musculus and M. macedonicus--copy numbers ranged from 6 to 1810 per haploid genome. The repeat cluster is cytogenetically visible as a fine C-band in M. macedonicus and as a C-band positive homogeneously staining region (HSR) in several populations of M. m. domesticus and M. m. musculus. When cytogenetically visible, the clusters contained from 179 to 1810 repeats. Intragenomic restriction fragment length polymorphisms (RFLPs), which reflect sequence variation among different copies of the long-range repeat family, increased with higher copy numbers. The high similarity of the RFLP pattern among genomes with C-band positive regions in Chr 1 of M. m. musculus, M. m. domesticus, and M. macedonicus points to a close evolutionary relationship of their Chr 1 repeat families.

Population dynamics of aberrant chromosome 1 in mice.

Natural populations of two semispecies of house mouse, Mus musculus domesticus and M.m. musculus, were found to be polymorphic for an aberrant chromosome 1 bearing a large inserted block of homogeneously staining heterochromatin. Strong meiotic drive for the aberrant chromosome from M.m. musculus was previously observed in heterozygous female mice. There are at least three meiotic drive levels determined by different allelic variants of distorter. Homozygotes had low viability and females showed low fertility. Both homo- and heterozygous males had normal fertility and their segregation patterns did not deviate from normal. Computer simulations were performed of the dynamics of aberrant chromosome 1 in demes and populations. The data demonstrate that a spontaneous mutation (inversion) of an aberrant chromosome 1, once arisen, has a high probability of spreading in a population at high coefficients of meiotic drive and migration. In the long-term, the population attains a stationary state which is determined by the drive level and migration intensity. The state of stable genotypic equilibrium is independent of deme and population size, as well as of the initial concentration of the aberrant chromosome. As populations initially polymorphic for the distorters approach the stationary state, the stronger distorter is eliminated. The frequencies of the aberrant chromosome determined by computer analysis agree well with those obtained for the studied Asian M.m. musculus populations. The evolutionary pathways for the origin and fixation of the aberrant chromosome in natural populations are considered.

Meiotic drive on aberrant chromosome 1 in the mouse is determined by a linked distorter.

An aberrant chromosome 1 carrying an inverted fragment with two amplified DNA regions was isolated from wild populations of Mus musculus. Meiotic drive favouring the aberrant chromosome was demonstrated for heterozygous females. Its cause was preferential passage of aberrant chromosome 1 to the oocyte. Genetic analysis allowed us to identify a two-component system conditioning deviation from equal segregation of the homologues. The system consists of a postulated distorter and responder. The distorter is located on chromosome 1 distally to the responder, between the ln and Pep-3 genes, and it acts on the responder when in trans position. Polymorphism of the distorters was manifested as variation in their effect on meiotic drive level in the laboratory strain and mice from wild populations.

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.

Zoogeography of the chromosome 1 HSR in natural populations of the house mouse (Mus musculus).

A polymorphism of the central part of chromosome 1 has been described from natural populations of the house mouse (Mus musculus). The region shows up as a C band-positive homogeneously staining region (HSR) under the light microscope. M. m. domesticus mice carry single band HSRs, whereas M. m. musculus animals have double band HSRs. HSR size variations have been described in both subspecies. The frequency of the HSR chromosome 1 in populations varies from 4% to 81%, but none of the large samples examined consisted only of homozygotes. In the subspecies M. m. domesticus, HSRs were found in North Africa and Western Europe, mainly in the hilly regions of Southern Germany and Switzerland. Localities with double HSRs are distributed all over the area of M. m. musculus. Based on the population data presented and DNA similarity of different HSRs, the origin and distribution of HSR chromosomes in the house mouse are discussed.

Chromosome pairing and recombination in mice heterozygous for different translocations in chromosomes 16 and 17.

In order to clarify the relationship between meiotic pairing and recombination, an electron microscopic (EM) study of synaptonemal complexes (SC) and an analysis of chiasma frequency and distribution were made in male mice singly and doubly heterozygous for Robertsonian [Rb(16.17)7Bnr] and reciprocal [T(16:17)43H] translocations and also in tertiary trisomics for the proximal region of chromosome 17. In all these genotypes an extensive zone of asynapsis/desynapsis around the breakpoints was revealed. At the same time a high frequency of non-homologous pairing was observed in precentromeric regions of acrocentric chromosomes. The presence in the proximal region of chromosome 17 of the t haplotype did not affect the synaptic behaviour of this region. Chiasma frequency in the proximal region of chromosome 17 in the T(16:17)43H heterozygotes and trisomics was increased when compared with that in Robertsonian heterozygotes.