Fiona Stennard

T-box transcription factors and their roles in regulatory hierarchies in the developing heart

T-box transcription factors are important players in the molecular circuitry that generates lineage diversity and form in the developing embryo. At least seven family members are expressed in the developing mammalian heart, and the human T-box genes TBX1 and TBX5 are mutated in cardiac congenital anomaly syndromes. Here, we review T-box gene function during mammalian heart development in the light of new insights into heart morphogenesis. We see for the first time how hierarchies of transcriptional activation and repression involving multiple T-box factors play out in three-dimensional space to establish the cardiac progenitors fields, to define their subservient lineages, and to generate heart form and function.

Characterisation of six additional human metallothionein genes.

Human metallothionein (MT) genes are clustered in a locus on chromosome 16, and this report presents the characterisation of the remaining six univestigated members of the family. Nucleotide sequencing in whole or part suggested that four of these genes, MT1I, MT1J, MT1K and MT1L do not encode expressed MT proteins, based on the presence of structural faults or atypical amino acid assignments. On the other hand, the structures of MT1H and MT1X are consistent with these genes being functional and encoding unique type 1 isoforms. The promoters of both genes conferred activity to CAT expression constructs when transfected into HeLa cells, and showed differential responses to inducers MT synthesis. Endogenous MT1H and MT1X genes were expressed at the mRNA level in HeLa cells following cadmium treatment. This work brings the number of functional class 1 and 2 MT genes in the human to eight, and confirms that each encodes structurally unique proteins.

DIFFERENTIAL EXPRESSION OF VEGT AND ANTIPODEAN PROTEIN ISOFORMS IN XENOPUS

The VegT/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, we have generated affinity purified polyclonal antibodies to Apod, and for comparison, to the other early T-box proteins Xbrachyury and Eomesodermin. An anti-VegT/Apod antibody reveals that there are two protein isoforms in Xenopus, one that we refer to as VegT and a smaller molecular weight isoform that we refer to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT/Apod gene. VegT is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg until gastrulation in the presumptive endoderm. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm and is inducible by activin. We conclude that the developmental role of VegT/Apod is mediated by two different proteins, with entirely different patterns of expression and response to growth factors.

The Xenopus eomesodermin promoter and its concentration-dependent response to activin.

Eomesodermin is an essential early gene in Xenopus mesoderm formation and shows a morphogen-like response to activin. Here we define the regions of the Eomesodermin promoter required for mesodermal expression and for concentration-dependent response to activin. We find an activin response element (ARE) located between -5.6 and -5.0 kb which contains two critical FAST2 binding sites. The ARE alone is necessary and sufficient for concentration-dependent response to activin. A 5.6 kb promoter recapitulates Eomes expression in normal mesoderm cells. A repressor element extinguishes Eomes expression in the endoderm. We relate our results to mesoderm patterning in early Xenopus development and to a mechanism of morphogen gradient response.

Markers of vertebrate mesoderm induction

Mesoderm formation is the first major differentiative event in vertebrate development. Many new mesoderm-specific genes have recently been described in the mouse, chick, frog and fish and belong to classes comprising T-domain genes, homeobox genes and those encoding secreted proteins. The T-domain genes have different but overlapping expression patterns and, in Xenopus, can ectopically activate nearly all other mesodermal genes. Several new homebox genes seem to mediate the ventralising activity of bone morphogenetic protein. New genes encoding secreted proteins induce dorsal mesoderm, in some cases by antagonizing ventralising factors.

Localisation and expression of metallothionein immunoreactivity in the developing sheep brain

Metallothioneins are small cysteine‐rich proteins that bind heavy metals. In higher mammals there are complex families of metallothionein isoforms, which are well characterised at the DNA level but less so in terms of their cellular expression and function. In particular, little is known about the localisation of metallothionein in the developing mammalian brain. In this study, using sheep fetuses, we have shown that metallothionein 1 and 2 isoform expression undergoes shifts in regional and cellular localisation during development of the brain. Metallothionein l and 2 expression is first detected by embryonic days E72–E73 (gestation is 150 days) at the mRNA level and the metallothionein protein is observed in cells of the proliferating ventricular zones. Subsequent expression is detected in radial glial cells, oligodendrocytes and astrocytes in several regions of the brain, most notably the cerebral cortex. In the adult brain, metallothionein is expressed in astrocytes but not in oligodendrocytes. Double‐labelling immunohistochemistry using the glial fibrillary acidic protein (GFAP), an astrocyte marker, and metallothionein revealed that although there is an overlap in the profiles of the two proteins, there is no simple correlation in their expression. These observations are consistent with metallothionein, under physiological conditions, being regulated mainly by intracellular factors.

Xenopus differentiation: VegT gets specific

Depletion of the maternal store of the localised mRNA encoding the T-box transcription factor VegT in Xenopus embryos has recently been shown to dramatically block endoderm formation and change the normal position of the mesodermal and ectodermal germ layers.

Localization of c-myc protooncogene expression in the rat heart in vivo and in the isolated, perfused heart following treatment with norepinephrine.

We have investigated the expression of the protooncogene c-myc in rat hearts following exposure to norepinephrine, both in vivo and in isolated perfused preparations. Both chronic and acute norepinephrine treatment produced a rapid, transient elevation of c-myc mRNA in adult rat hearts, but chronic infusion produced a second, larger increase. This expression profile was characteristic for c-myc since it was not found for four other protooncogenes. In the isolated, perfused heart, addition of norepinephrine to the perfusion buffer and elevation of perfusion pressure separately increase c-myc mRNA suggesting both direct hormonal and hemodynamic factors might be important in vivo. Immunocytochemistry showed that Myc protein accumulated predominantly in the nuclei of non-myocyte cells following norepinephrine treatment indicating that expression at the mRNA level culminated in protein synthesis. These findings suggest that the c-myc expression observed in the hypertrophying adult heart following exposure to norepinephrine may be associated with proliferating cells like fibroblasts rather than cardiomyocytes.

Human metallothionein gene MT1L mRNA is present in several human tissues but is unlikely to produce a metallothionein protein

© 1997 Federation of European Biochemical Societies.

Metallothionein expression in the mammalian brain

The expression of metallothionein (MT) in the mammalian brain is of great current interest, and several reports localising MT immunoreactivity to specific regions of the adult brain have appeared [1-5]. These have found that MT-I and MT-II expression is widespread in the CNS and is mainly in astrocytes. It appears that both astrocytes [5] and neurons [6] are able to express MT-III, and these findings have been confirmed independently [7].