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During human placental development, specialized cells allocated to the extraembryonic lineage (cytotrophoblasts) invade the uterus, anchoring the conceptus to the decidua and tapping a supply of maternal blood. This unusual behavior requires cytotrophoblasts to assume highly specialized characteristics; some are commonly associated with tumor cells, while others are typical of endothelia. Here we investigated the transcriptional mechanisms that control cytotrophoblast differentiation/invasion. Specifically, we examined the cells' expression of a number of transcription factors, at the RNA level, as they differentiated along the invasive pathway in vitro. Since basic helix-loop-helix (bHLH) proteins play important roles in murine trophoblast differentiation, we first examined their expression by cytotrophoblasts. As in murine placental development, expression of the human homologue of Mash-2 was confined to progenitor cells. But expression of Hand-1, which promotes differentiation of murine trophoblast giant cells, was not detected. We also found that cytotrophoblasts upregulated the expression of bHLH/PAS factors that function in adaptive responses to hypoxia, including hEPAS-1, which is expressed primarily in endothelial cells. Quite unexpectedly, we discovered that cytotrophoblasts express high levels of mRNA encoding the human homologue of the Drosophila neuronal fate gene, glial cells missing-1 (gcm-1). We also found evidence of crosstalk between the bHLH and GCM-1 regulatory networks. Together, these results offer insights into the transcriptional mechanisms that govern cytotrophoblast differentiation/invasion. Interestingly, these mechanisms suggest analogies with those that govern differentiation of murine stem cells allocated to both the intra- and extraembryonic lineages.

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Following the discovery of the homeobox as a conserved sequence in developmentally important genes of Drosophila, a plethora of such sequences have been identified in evolutionarily distant organisms. Among mammals, the mouse homeobox genes have been studied most intensively with a hope of deciphering basic mechanisms of embryonic development. The genomic arrangement of many mouse homeobox genes is similar to the organization of the Drosophila genes, suggesting that they arose as a consequence of gene duplication and divergence from a primordial cluster during evolution. Homeobox genes encode proteins that may form a part of the autoregulatory and transregulatory network specifying positional value in the embryo. Supporting this view, the more diverged members of this growing family function as transcription factors, some of which regulate the expression of tissue-specific genes. Mouse homeobox genes are expressed during embryonic development in a spatially restricted manner and alterations in their expression pattern can disrupt embryonic development. The implications of these findings will be discussed in the context of the role of homeobox genes in the embryonic development of Drosophila and other organisms.

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The region-specific patterns of expression of mouse homeobox genes are considered important for establishing the embryonic body plan. A 5-kilobase (kb) DNA fragment from the Hox-3.1 locus that is sufficient to confer region-specific expression to a beta-galactosidase reporter gene in transgenic mouse embryos has been defined. The observed reporter gene expression pattern closely parallels endogenous Hox-3.1 expression in 8- to 9.5-day postcoitum (p.c.) embryos. At 10.5 days p.c. and later, the pattern of beta-galactosidase activity diverges from the Hox-3.1 pattern, and an inappropriately high level of reporter gene expression is observed in posterior spinal ganglia. Inclusion of an additional 2 kb of upstream sequences is sufficient to suppress this aberrant expression in the developing spinal ganglia. Together, these results show that the control of early Hox-3.1 expression is complex, involving at least one positively acting and one negatively acting element.

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To examine the possible role of homeo box genes in murine development we have studied the structure and expression pattern of Hox-2.5, a newly isolated homeo box gene that maps to the left end of the mouse Hox-2 locus on chromosome 11. The sequence of the Hox-2.5 homeo box has been determined. It is highly homologous to Hox-1.7 and Hox-3.2, demonstrating extended conservation among three homeo box complexes in the mouse. Northern and in situ hybridization analyses of Hox-2.5 demonstrate a novel, regionally restricted pattern of expression in developing mesoderm and neurectoderm. We detect localized Hox-2.5 transcripts as early as 8.5 days postcoitum. The expression pattern of Hox-2.5 was analyzed over the next 3 days of ontogeny, as well as in later embryonic, newborn, and adult stages. Three-dimensional reconstruction of Hox-2.5 transcript localization within the central nervous system of early embryos clearly illustrates the neural expression domain. Although the Hox-2.5 expression pattern is regionally restricted during all of these stages of development, the pattern changes along the anteroposterior and dorsoventral axes of the CNS as the embryo undergoes complex morphogenetic movements and cytodifferentiation.

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The Hox-2.2 gene is one of a cluster of homeobox-containing genes on mouse chromosome 11. A cDNA clone containing the Hox-2.2 homeobox has been isolated from an adult spinal cord library. Our analysis of the Hox-2.2 cDNA and genomic clones indicates that there are at least two oxons and one intron. The largest open reading frame includes the homeobox and codes for a 224 amino acid protein of molecular weight 25,312. Comparisons of the predicted Hox-2.2 protein with other homeodomain-containing proteins revealed four regions of sequence similiarity: an N-terminal octapeptide, a hexapeptide upstream of the homeodomain, the homeodomain, and a glutamic acid-rich region at the C terminus. Possible functions of these regions are discussed. The Hox-2.2 gene is expressed in 13.5-day embryos in the developing hindbrain and spinal cord. The expression patterns of Hox-2.2 and Hox-2.1 in 13.5-day embryos are compared.

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The isolation of multiple Na+,K+-ATPase cDNAs from rat brain has led to the discovery of a family of alpha-isoform genes. Using A1 (alpha), A2 (alpha+), and A3 (alpha III) Na+,K+-ATPase gene probes, we have analyzed the distribution of Na+,K+-ATPase mRNAs in adult and fetal rat tissues by RNA blot and hybridization histochemistry. A1 Na+,K+-ATPase mRNA was found ubiquitously among various tissues, with highest levels in transport epithelial and neural tissues. A2 mRNA was found in adult neural and muscle tissues, and A3 mRNA was found only in neural tissues and fetal heart muscle. Both A1 and A2 mRNAs were less abundant in fetal brain than in adult brain; in contrast, A3 mRNA was abundant at both stages. In situ mapping of brain areas that contain A3 mRNA suggests that this Na+,K+-ATPase isoenzyme is expressed predominantly by neural cells. Analysis of Na+,K+-ATPase proteins generated by cell-free translation of synthetic mRNAs suggests that the A3 protein has properties similar to A2 (alpha+).

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Mammalian homeo box genes have been identified on the basis of sequence homology to Drosophila homeotic and segmentation genes. These studies examine the distribution of transcripts from two mouse homeo box genes, Hox-2.1 and Hox-3.1, throughout the latter third of prenatal development. Transcripts from these genes are regionally localized along the rostro-caudal axis of the developing central nervous system, yielding expression patterns very similar to patterns of Drosophila homeotic gene expression.

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Considerable information has accumulated on mouse homeo box gene organization and expression. Homeo box genes are expressed in a wide variety of tissues, developmental stages, and cell lines. How can this be interpreted in view of the relationship of these genes to Drosophila morphogenetic loci? One view is that homeo box genes control determinative decisions by modulating transcription of as yet unidentified target genes. Proponents of this view are faced with two tasks: to identify developmental processes that are controlled by homeo box genes, and to identify the target genes that mediate this control. Such target genes might be identified on the basis of in vitro homeo domain-DNA interactions. Candidate morphogenetic processes might be identified on the basis of the observed patterns of homeo box gene expression. It must be stressed that finding expression in a given tissue in no way demonstrates that the expression is necessary for the determination of that tissue. The role of Drosophila homeo box genes in determinative decisions is based upon analysis of mutants to demonstrate that the pattern of homeo box gene expression determines the morphogenetic outcome. To test whether the expression of a mouse homeo box gene is involved in a determinative decision, one must disrupt the normal pattern of expression of that gene and observe the resulting morphogenetic effect. In mouse this can be approached by looking for allelism with known morphogenetic loci, by isolating mutants in homeo box genes through large-scale mutagenesis screens, or by introducing altered homeo box genes into transgenic mice. One of the most intriguing possibilities is that homeo box genes are involved in regional specification along the anteroposterior axis. In situ hybridization and Northern blot analysis have demonstrated that at least four different homeo box genes display distinct regional patterns of expression along the anteroposterior axis of the developing CNS. The expression of each of these genes has a unique anterior boundary from which expression extends posteriorly within the CNS. Hox 1.5 expression has an anterior boundary within the hindbrain just posterior to the pontine flexure. The anterior boundary of Hox 2.1 expression lies more posteriorly within the medulla of the hindbrain. Weak expression of Hox 2.5 is detected in the spinal cord just posterior to the first cervical vertebra, and maximal expression is found posterior to the second cervical vertebra.(ABSTRACT TRUNCATED AT 400 WORDS)

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A common feature of Drosophila homoeo box genes appears to be their spatially restricted expression patterns during morphogenesis. Using Northern blot analysis and in situ hybridization to mouse tissue sections, the spatially restricted expression of a newly identified mouse homoeo box locus, Hox-3, within the central nervous system of newborn and adult mice has been demonstrated.

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