Deborah L. Chapman

Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue.

The expansion of GAG triplet repeats in the translated region of the human HD gene, encoding a protein (huntingtin) of unknown function, is a dominant mutation leading to manifestation of Huntingtons disease. Targeted disruption of the homologous mouse gene (Hdh), to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, we propose that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway.

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.

Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6

Somites, segmented mesodermal units of the vertebrate embryo, are the precursors of adult skeletal muscle, bone and cartilage. During embryogenesis, somite progenitor cells ingress through the primitive streak, move laterally to a paraxial position (alongside the body axis) and segment into epithelial somites. Little is known about how this paraxial mesoderm tissue is specified,. We have previously described a mouse T-box gene, Tbx6 (ref. 3), which codes for a putative DNA-binding protein,. The embryonic pattern of expression of Tbx6 in somite precursor cells suggests that this gene may be involved in the specification of paraxial mesoderm. We now report the creation of a mutation in Tbx6 that profoundly affects the differentiation of paraxial mesoderm. Irregular somites form in the neck region of mutant embryos, whereas more posterior paraxial tissue does not form somites but instead differentiates along a neural pathway, forming neural-tube-like structures that flank the axial neural tube. These paraxial tubes show dorsal/ventral patterning that is characteristic of the neural tube, and have differentiated motor neurons. These results indicate that Tbx6 is needed for cells to choose between a mesodermal and a neuronal differentiation pathway during gastrulation; Tbx6 is essential for the specification of posterior paraxial mesoderm, and in its absence cells destined to form posterior somites differentiate along a neuronal pathway.

Critical role for Tbx6 in mesoderm specification in the mouse embryo.

Tbx6 is a member of the T-box family of transcription factor genes. Two mutant alleles of this gene establish that Tbx6 is involved in both the specification and patterning of the somites along the entire length of the embryo. The null allele, Tbx6(tm1Pa), causes abnormal patterning of the cervical somites and improper specification of more posterior paraxial mesoderm, such that it forms ectopic neural tubes. In this study, we use this allele to further investigate the mechanism of action of the Tbx6 gene and investigate possible genetic interactions. We have tested the developmental and differentiation potential of Tbx6(tm1Pa)/Tbx6(tm1Pa) cells in ectopic sites, in vitro, and in chimeras in vivo. We have also documented cell proliferation and cell death in mutant tail buds in an attempt to explain the mechanism of tail bud enlargement in the Tbx6 mutant embryos. Our results indicate specific developmental restrictions on the differentiation of posterior cells lacking Tbx6, once they have traversed the primitive streak, but no restrictions in differentiation of anterior somites, or of Tbx6 null embryonic stem (ES) cells. We further demonstrate that Tbx6 null ES cells fail to populate posterior somites in chimeric embryos. To discover whether different T-box proteins interact on the same down stream targets in areas of expression overlap, we have explored potential interactions between Tbx6 and T (Brachyury) in genetic crosses. Our results reveal that the T(Wis) mutation is epistatic to the Tbx6(tm1Pa) mutation and that there is no apparent genetic interaction. However, homozygosity for Tbx6(tm1Pa) and heterozygosity for T(Wis) mutation shows a combinatorial interaction at the phenotypic level.

TET-mediated DNA demethylation controls gastrulation by regulating Lefty–Nodal signalling

Mammalian genomes undergo epigenetic modifications, including cytosine methylation by DNA methyltransferases (DNMTs). Oxidation of 5-methylcytosine by the Ten-eleven translocation (TET) family of dioxygenases can lead to demethylation. Although cytosine methylation has key roles in several processes such as genomic imprinting and X-chromosome inactivation, the functional significance of cytosine methylation and demethylation in mouse embryogenesis remains to be fully determined. Here we show that inactivation of all three Tet genes in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signalling. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signalling contributes to the gastrulation failure of Tet mutants. Increased Nodal signalling is probably due to diminished expression of the Lefty1 and Lefty2 genes, which encode inhibitors of Nodal signalling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty–Nodal signalling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphological and molecular abnormalities as the null mutation. Taken together, our results show that TET-mediated oxidation of 5-methylcytosine modulates Lefty–Nodal signalling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signalling pathways in early body plan formation.

Expression of Proto-Oncogenes and Protein Kinases in the Testis

Specificity of expression is regarded as a strong indication that genes play a role in determining the phenotype or function of the cells in which they are expressed. A molecular genetic approach to understanding the control of male germ cell differentiation involves, in part, identifying genes that are expressed in a spermatogenic cell type-specific manner. Previous observations from our laboratory and others have shown that some testis-specific genes are expressed in spermatogenic cells in a stage-specific manner. Similarly, more ubiquitously expressed genes can exhibit particularly abundant expression in germ cells, often of uniquely sized transcripts. Both patterns of expression suggest a role for these genes in spermatogenesis or subsequent function of male gametes (rev. in Willison and Ashworth 1987; Hecht 1990; Erickson 1990; Wolgemuth and Watrin 1991).

Expression and Function of Protein Kinases During Mammalian Gametogenesis

Publisher Summary This chapter reviews critical stages of mammalian germ cell differentiation during which protein kinases (and their corresponding phosphatases) may be functioning. The chapter emphasizes on those kinases involved with regulating the eukaryotic cell cycle with the bias that gametogenesis represents a specialized and highly regulated series of cell cycle events. The chapter reviews the main classes of protein kinases that are the tyrosine kinases and the serine/threonine kinases. It emphasizes the results from the mouse system and includes results from other organisms, especially in particular stages of gametogenesis in which the mammalian system is poorly understood. It has been established that growth factors and their respective receptor tyrosine kinases function in the mitogenic pathway. Receptor tyrosine kinases consist of three distinct domains: (1) the extracellular ligand binding domain, (2) a transmembrane region, and (3) the intracellular cytoplasmic tyrosine kinase catalytic domain. In the cytoplasmic domain, serine/threonine and tyrosine residues serve as phosphorylation sites that regulate the activity of the receptor tyrosine kinase. Many non-receptor tyrosine kinases have been identified as products of retrovirally encoded oncogenes. Non-receptor tyrosine kinases are divided into two groups: (1) transmembrane and (2) cytosolic families. Cytoplasmic serine/threonine protein kinases catalyze the transfer of phosphate groups to serine and threonine residues of target proteins.