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.     

Machine learning based tissue analysis reveals Brachyury has a diagnosis value in breast cancer

Background: The aim of the present study was to confirm the role of Brachyury in breast cancer and to verify whether four types of machine learning models can use Brachyury expression to predict the survival of patients.Methods: We conducted a retrospective review of the medical records to obtain patient information, and made the patient’s paraffin tissue into tissue chips for staining analysis. We selected 303 patients for research and implemented four machine learning algorithms, including multivariate logistic regression model, decision tree, artificial neural network and random forest, and compared the results of these models with each other. Area under the receiver operating characteristic (ROC) curve (AUC) was used to compare the results.Results: The chi-square test results of relevant data suggested that the expression of Brachyury protein in cancer tissues was significantly higher than that in paracancerous tissues (P=0.0335); patients with breast cancer with high Brachyury expression had a worse overall survival (OS) compared with patients with low Brachyury expression. We also found that Brachyury expression was associated with ER expression (P=0.0489). Subsequently, we used four machine learning models to verify the relationship between Brachyury expression and the survival of patients with breast cancer. The results showed that the decision tree model had the best performance (AUC = 0.781).Conclusions: Brachyury is highly expressed in breast cancer and indicates that patients had a poor prognosis. Compared with conventional statistical methods, decision tree model shows superior performance in predicting the survival status of patients with breast cancer.     

Tint maps to mouse chromosome 6 and may interact with a notochordal enhancer of Brachyury

At the proximal part of mouse chromosome 17 there are three well-defined genes affecting the axis of the embryo and consequently tail length: Brachyury, Brachyury the second, and the t-complex tail interaction (T1, T2, and tct). The existence of T1 and tct in fact defines the classical "t-complex" that occupies approximately 40 cM of mouse chromosome 17. Their relationship to each other and various unlinked interacting genes has been enigmatic. The tint gene was the first of the latter to be identified. We report here its genetic mapping using a microsatellite scan together with outcrosses to Mus spretus and M. castaneous followed by a subsequent testcross to T, T1, and T2 mutants. Surprisingly, tint interacts with T2 but not with T1. The implications of our data suggest that T2 may be part of the T1 regulatory region through direct or indirect participation of tint.     

Mouse Brachyury the Second (T2) is a gene next to classical T and a candidate gene for tct.

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.     

Involvement of EphA2 in the formation of the tail notochord via interaction with ephrinA1

Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. We generated EphA2 mutant mice using a gene trap method. Homozygous mutant mice developed short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 was expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, was at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells were spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface.     

Formation of the chordamesoderm in the amphioxus embryo: Analysis with Brachyury and fork head/HNF-3 genes

 The embryonic development of amphioxus (cephalochordates) has much in common with that of vertebrates, suggesting a close phylogenetic relationship between the two chordate groups. To gain insight into alterations in the genetic cascade that accompanied the evolution of vertebrate embryogenesis, we investigated the formation of the chordamesoderm in amphioxus embryos using the genes Brachyury and fork head/HNF-3 as probes. Am(Bb)Bra1 and Am(Bb)Bra2 are homologues of the mouse Brachyury gene isolated from Branchiostoma belcheri. Molecular phylogenetic analysis suggests that the genes are independently duplicated in the amphioxus lineage. Both genes are initially expressed in the involuting mesoderm of the gastrula, then in the differentiating somites of neurulae, followed by the differentiating notochord and finally in the tail bud of ten-somite stage embryos. On the other hand, Am(Bb)fkh/HNF3-1, an amphioxus (B. belcheri) homologue of the fork head/HNF-3 gene, is initially expressed in the invaginating endoderm and mesoderm, then later in the differentiating notochord and in the tail bud. With respect to these two types of genes, the formation of the notochord and tail bud in amphioxus embryos shows similarity and dissimilarity with that of the notochord and tail bud in vertebrate embryos. Received: 21 November 1996 / Accepted: 24 January 1997     

TCreERT2, a Transgenic Mouse Line for Temporal Control of Cre-Mediated Recombination in Lineages Emerging from the Primitive Streak or Tail Bud

The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the T (Brachyury) promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first in vivo tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ß-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ß-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.     

Characterization of NF-kappa B/I kappa B proteins in zebra fish and their involvement in notochord development

Although largely involved in innate and adaptive immunity, NF-kappa B plays an important role in vertebrate development. In chicks, the inactivation of the NF-kappa B pathway induces functional alterations of the apical ectodermal ridge, which mediates limb outgrowth. In mice, the complete absence of NF-kappa B activity leads to prenatal death and neural tube defects. Here, we report the cloning and characterization of NF-kappa B/I kappa B proteins in zebra fish. Despite being ubiquitously expressed among the embryonic tissues, NF-kappa B/I kappa B members present distinct patterns of gene expression during the early zebra fish development. Biochemical assays indicate that zebra fish NF-kappa B proteins are able to bind consensus DNA-binding (kappa B) sites and inhibitory I kappa B alpha proteins from mammals. We show that zebra fish I kappa B alphas are degraded in a time-dependent manner after induction of transduced murine embryo fibroblasts (MEFs) and that these proteins are able to rescue NF-kappa B activity in I kappa B alpha(-/-) MEFs. Expression of a dominant-negative form of the murine I kappa B alpha (mI kappa B alpha M), which is able to block NF-kappa B in zebra fish cells, interferes with the notochord differentiation, generating no tail (ntl)-like embryos. This phenotype can be rescued by coinjection of the T-box gene ntl (Brachyury homologue), which is typically required for the formation of posterior mesoderm and axial development, suggesting that ntl lies downstream of NF-kappa B . We further show that ntl and Brachyury promoter regions contain functional kappa B sites and NF-kappa B can directly modulate ntl expression. Our study illustrates the conservation and compatibility of NF-kappa B/I kappa B proteins among vertebrates and the importance of NF-kappa B pathway in mesoderm formation during early embryogenesis.     

Diplomyelia: caudal duplication of the neural tube in mice

Diplomyelia (duplication of the neural tube or spinal cord) was studied histologically in nine cases of T/+ mouse embryos at 10-15 days of gestation. Grüneberg investigated T/+ fetuses and interpreted the extra neural tube to be notochord, but a reexamination of this material demonstrated that the interpretation is incorrect. Diplomyelia is produced in the mutations Fused, Kinky, vestigial tail, homozygous Brachyury, and t-haplotype t9 and in the new mutation, NM 529, described here.     

Tail regeneration in the Xenopus tadpole

The tail of the Xenopus tadpole contains major axial structures, including a spinal cord, notochord and myotomes, and regenerates within 2 weeks following amputation. The tail regeneration in Xenopus can provide insights into the molecular basis of the regeneration mechanism. The regenerated tail has some differences from the normal tail, including an immature spinal cord and incomplete segmentation of the muscle masses. Lineage analyses have suggested that the tail tissues are reconstructed with lineage-restricted stem cells derived from their own tissues in clear contrast to urodele regeneration, in which multipotent blastema cells derived from differentiated cells play a major role. Comprehensive gene expression analyses resulted in the identification of a panel of genes involved in sequential steps of the regeneration. Manipulation of genes' activities suggested that the tail regeneration is regulated through several major signaling pathways.