A. Paula Monaghan

Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction

The Spemann organizer in amphibian embryos is a tissue with potent head-inducing activity, the molecular nature of which is unresolved. Here we describe dickkopf-1 (dkk-1), which encodes Dkk-1, a secreted inducer of Spemanns organizer in Xenopus and a member of a new protein family. Injections of mRNA and antibody indicate that dkk-1 is sufficient and necessary to cause head induction. dkk-1 is a potent antagonist of Wnt signalling, suggesting that dkk genes encode a family of secreted Wnt inhibitors.

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

Characterization of the mouse HNF-4 gene and its expression during mouse embryogenesis

Hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor gene superfamily with unknown ligand. It has been assumed to play an important role in the regulation of gene expression in the liver. Here, we report the cloning and characterization of the mouse HNF-4 gene, as well as its expression during embryogenesis. The HNF-4 protein is encoded by ten exons. The gene structure is unique in the steroid receptor superfamily in that the second zinc finger is encoded by two exons. HNF-4 mRNA is expressed in a limited number of mouse adult tissues: liver, kidney, intestine, stomach and skin. HNF-4 could play an important role in the formation and function of visceral yolk sac and in the development of the liver and kidney since its mRNA, as determined by in situ hybridization, appears upon primary differentiation of these organs. As a first step in the study of the regulatory elements of the HNF-4 gene, we mapped the transcription start site and carried out DNase I hypersensitive site (HS) analysis over a region of approximately 22kb upstream of the gene. The complexity of the HSs suggests that multiple elements might contribute to the transcriptional regulation of the HNF-4 gene.

Activating Transcription Factor 1 and CREB Are Important for Cell Survival during Early Mouse Development

ABSTRACT Activating transcription factor 1 (ATF1), CREB, and the cyclic AMP (cAMP) response element modulatory protein (CREM), which constitute a subfamily of the basic leucine zipper transcription factors, activate gene expression by binding as homo- or heterodimers to the cAMP response element in regulatory regions of target genes. To investigate the function of ATF1 in vivo, we inactivated the corresponding gene by homologous recombination. In contrast to CREB-deficient mice, which suffer from perinatal lethality, mice lacking ATF1 do not exhibit any discernible phenotypic abnormalities. Since ATF1 and CREB but not CREM are strongly coexpressed during early mouse development, we generated mice deficient for both CREB and ATF1. ATF1−/− CREB−/− embryos die before implantation due to developmental arrest. ATF1+/− CREB−/− embryos display a phenotype of embryonic lethality around embryonic day 9.5 due to massive apoptosis. These results indicate that CREB and ATF1 act in concert to mediate signals essential for maintaining cell viability during early embryonic development.

The mouse homolog of the region specific homeotic gene spalt of Drosophila is expressed in the developing nervous system and in mesoderm-derived structures

The region specific homeotic gene spalt (sal) of Drosophila determines the specification of terminal segments. Its mutation leads to an incomplete transformation of terminal segments into trunk-like segments. The gene product is a zinc finger protein with a novel structure. We have isolated the mouse homolog of the Drosophila spalt gene (msal). The msal cDNA sequence is similar to its Drosophila counterpart in that it contains seven C2H2-type zinc finger motifs grouped into three pairs plus a single zinc finger closely linked to the middle pair. The two genes exhibit high sequence similarity in the zinc finger regions and to a lower extent in the putative transactivation domains. We have analysed the expression pattern of msal and show that it is expressed in the developing neuroectoderm of the brain, the inner ear and the spinal cord and in urogenital ridge-derived structures such as testis, ovaries and kidneys. A weaker and transient expression is seen in early embryos in the branchial arches and in tissues like the notochord, the limb buds and the heart. Given its role in Drosophila melanogaster and its strong sequence conservation, this expression pattern suggests an important role for msal in the development of the nervous system.

Molecular genetic analysis of glucocorticoid signaling during mouse development

Glucocorticoids are important in a number of developmental processes in mammals around birth. The pathway of gluconeogenesis is activated in liver shortly after birth due to the combined effects of glucocorticoids and glucagon. We have defined the essential cis-regulatory elements directing hormone-dependent liver-specific expression of the gene for tyrosine aminotransferase, a key gluconeogenic enzyme. The hormone response elements synergize with cell-type specific elements. In the case of glucocorticoids, the glucocorticoid-dependent enhancer is composed of the glucocorticoid response element and binding sites for liver cell-enriched transcription factors, in particular hepatocyte nuclear factor-3. The dependence of the respective enhancer motifs on each other restricts the hormonal activation of the tyrosine aminotransferase gene in liver in response to a hormonal signal. To further understand the role of glucocorticoid signaling via the type II glucocorticoid receptor (GR) in the perinatal period and earlier during development, we have studied the expression of the mouse GR gene. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected as early as day 9.5 of mouse development. To specifically address the role of glucocorticoid signaling via the GR during development, we have disrupted the GR gene by homologous recombination in mouse embryonic stem cells. The majority of GR mutants die shortly after birth and analysis so far has revealed defects in lung, liver, and adrenal function.

Expression of the mouse Fkh1/Mf1 and Mfh1 genes in late gestation embryos is restricted to mesoderm derivatives

We have compared the expression patterns of the mouse Forkhead homologue 1/ mesoderm/mesenchyme forkhead 1 (Fkh1/Mf1) gene with that of the highly related winged helix gene Mfh1 in late gestation mouse embryos. Transcripts for both genes are restricted to derivatives of the mesoderm. Co-expression was found in cartilage primordia of the head, ribs, vertebra and bones. However, in several structures analyzed, Fkh1/Mf1 signals are lower in the inner layers of the developing cartilage than those of Mfh1.