Androgen-independent expression of hoxb-13 in the mouse prostate.

BACKGROUND: Hox genes encode transcriptional regulatory proteins that are largely responsible for establishing the body plan of all metazoan organisms. A subset of Hox genes is expressed during the period of organogenesis and into adulthood. hoxb-13 is a recently-described member of the Hox gene family that is expressed in the spinal cord, hindgut, and urogenital sinus during embryogenesis. METHODS: Northern blot and in situ hybridization analyses of hoxb-13 expression in adult mouse tissues were performed. RESULTS: hoxb-13 mRNA is restricted to the prostate gland and distal colon in adult animals. In situ hybridization of mouse prostate tissue demonstrated that hoxb-13 is expressed in the epithelial cells of the ventral, dorsal, lateral, and anterior prostate lobes. Accumulation of hoxb-13 mRNA is not diminished following castration. CONCLUSIONS: These data demonstrate that hoxb-13 expression is androgen-independent in mouse prostate glands. The identification of hoxb-13 as an androgen-independent gene expressed in adult mouse prostate epithelial cells provides a new potential target for developing therapeutics to treat advanced prostate cancer.

Hematopoietic progenitor cell abnormalities in Hoxc-8 null mutant mice.

The mammalian Hox genes encode a family of conserved transcription factors that control the establishment of the body plan during embryogenesis. Many Hox genes are also known to be expressed in hematopoietic cells. We found that Hoxc-8, a member of the Hox C cluster, is expressed in the mouse hematopoietic organs, fetal liver and adult bone marrow. To determine the role of Hoxc-8 gene in hematopoiesis, we compared progenitor cell numbers in the fetal liver and adult bone marrow cells. We observed a significant reduction in the number of erythroid burst-forming unit (BFU-E) and in granulocyte/macrophage colony-forming unit (CFU-GM) in the Hoxc-8 null mice, although the peripheral blood cell counts were normal. The hematopoietic cells from the homozygote animals exhibited normal expansion capability in a liquid culture system, suggesting that the decreased number of progenitor cells may be due to a defect extrinsic to the hematopoietic cells, such as in the interaction with the microenvironment.

Prostate-specific and androgen-dependent expression of a novel homeobox gene.

A new member of the mouse NK family of homeobox genes that is related to Drosophila NK-3 has been identified. Expression of this gene, termed Nkx-3.1, is largely restricted to the prostate gland in adult animals. The level of Nkx-3.1 mRNA decreases markedly in response to castration, suggesting that its expression is androgen-dependent. In situ hybridization analyses demonstrated that expression of Nkx-3.1 in the prostate is confined to epithelial cells. In newborns, Nkx-3.1 mRNA is detected in the urethral epithelium that is being induced by the surrounding mesenchyme to invaginate to form prostatic buds. Together, these observations suggest that the Nkx-3.1 protein, which likely functions as a transcription factor, plays a prominent role both in the initiation of prostate development and in the maintenance of the differentiated state of prostatic epithelial cells.

Functional specificity of Hoxa-4 in vertebral patterning lies outside of the homeodomain.

The Hox family of proteins plays a central role in establishing the body plan of a wide range of metazoan organisms. Each member of this family of transcriptional regulators has a distinct functional specificity, yet they bind to similar DNA target sequences through their conserved homeodomain. The mechanisms whereby Hox proteins achieve their diverse specificities in vivo remain undefined. Using the opposing effects of Hoxa-4 and Hoxc-8 in vertebral patterning, we demonstrate by replacing the homeodomain of Hoxa-4 with that of Hoxc-8 that the functional specificity of Hoxa-4 does not track with the homeodomain. These observations provide evidence that other regions of Hox proteins play an important role in mediating functional specificity during mammalian embryogenesis.

Regulation of Hoxc-8 during mouse embryonic development: identification and characterization of critical elements involved in early neural tube expression.

We have characterized cis-acting elements that direct the early phase of Hoxc-8 expression using reporter gene analysis in transgenic mice. By deletion we show that a 135 bp DNA fragment, located approximately 3 kb upstream of the coding region of Hoxc-8, is capable of directing posterior neural tube expression. This early neural tube (ENT) enhancer consists of four separate elements, designated A, B, C and D, whose nucleotide sequences are similar to binding sites of known transcription factors. Nucleotide substitutions suggest that element A is an essential component of the ENT enhancer. However element A by itself is incapable of directing neural tube expression. This element requires interactions at any two of the other three elements, B, C or D. Thus, the components of the ENT enhancer direct neural tube expression in an interdependent manner. We propose that Hoxc-8 is activated in the neural tube by combinatorial interactions among several proteins acting within a small region. Our transgenic analyses provide a means to identify transcription factors that regulate Hoxc-8 expression during embryogenesis.

Gain of function mutations for paralogous Hox genes: implications for the evolution of Hox gene function.

To investigate the functions of paralogous Hox genes, we compared the phenotypic consequences of altering the embryonic patterns of expression of Hoxb-8 and Hoxc-8 in transgenic mice. A comparison of the phenotypic consequences of altered expression of the two paralogs in the axial skeletons of newborns revealed an array of common transformations as well as morphological changes unique to each gene. Divergence of function of the two paralogs was clearly evident in costal derivatives, where increased expression of the two genes affected opposite ends of the ribs. Many of the morphological consequences of expanding the mesodermal domain and magnitude of expression of either gene were atavistic, inducing the transformation of axial skeletal structures from a modern to an earlier evolutionary form. We propose that regional specialization of the vertebral column has been driven by regionalization of Hox gene function and that a major aspect of this evolutionary progression may have been restriction of Hox gene expression.

The Alzheimer's A beta peptide induces neurodegeneration and apoptotic cell death in transgenic mice.

To test whether the hypothesis that the Alzheimer's A beta peptide is neurotoxic, we introduced a transgene into mice to direct expression of this peptide to neurons. We show that the transgene is expressed in brain regions which are severely affected in Alzheimer's disease resulting in extensive neuronal degeneration. Morphological and biochemical evidence indicates that the eventual death of these cells occurs by apoptosis. Coincident with the cell degeneration and cell death is the presence of a striking reactive gliosis. Over 50% of the transgenic mice die by 12 months of age, half the normal life span of control mice. These data show that A beta is neurotoxic in vivo and suggest that apoptosis may be responsible for the accompanying neuronal loss, the principal underlying cellular feature of Alzheimer's disease.

Evolving concepts in molecular pathology.

During the last decade, an understanding of the causes of many human diseases has progressed rapidly, in large measure because of the development of technologies that allow us to identify the genes that are involved. Identification of a gene that is suspected to play an important role in a particular disease opens up a whole new dimension of research to understand the molecular events that underlie the cause of that disorder. A crucial step in this process is often the development of an animal model of the disease. Again, the last decade has seen rapid advances in our ability to create such models, particularly in mice. Technologies that allow for the addition, alteration, or elimination of individual genes from the genome to create a transgenic mouse are now routine. The advantages of having a transgenic mouse model of a human disease are many. These animals often provide the first unequivocal proof that a particular gene is responsible for causing the pathological changes that occur with disease. They also can provide a system to carefully dissect the successive events that lead to the disease state, and can provide a custom-designed whole animal system to test potential therapies to treat and eventually cure the disease. Most important, new concepts relating to gene expression and gene function in disease often emerge from such transgenic studies. This review will illustrate several examples in which transgenic animals have contributed significantly to the evolution of concepts of the underlying mechanisms of human disease.

A transgenic model of transactivation by the Tax protein of HTLV-I.

The human T-lymphotropic virus type I (HTLV-I) Tax protein is a transcriptional regulatory protein that has been suggested to play a causal role in the development of several HTLV-I-associated diseases. Tax regulates expression of its own LTR and of certain cellular promoters perhaps by usurping the function of the host transcriptional machinery. We have established a transgenic mouse model system to define the spectrum of tissues in vivo that are capable of supporting Tax-mediated transcriptional transactivation. Transgenic mice carrying the HTLV-I LTR driving expression of the Escherichia coli beta-galactosidase (beta gal) gene were generated, and this LTR-beta gal gene was transcriptionally inactive in all tissues. When LTR-beta gal mice were mated to transgenic mice carrying the same LTR driving expression of the HTLV-I tax gene, mice that carried both transgenes showed restricted expression of the beta gal reporter gene in several tissues including muscle, bone, salivary glands, skin, and nerve. In addition, a dramatic increase in the number of beta gal-expressing cells was seen in response to wounding. These observations provide direct evidence for viral transactivation in vivo, delimit the tissues capable of supporting that transactivation, and provide a model system to study the mechanism of gene regulation by Tax.

Altering the boundaries of Hox3.1 expression: evidence for antipodal gene regulation.

To investigate the function of region-specific patterns of mouse homeobox gene expression during embryogenesis, we programmed a minimal change in the distribution of Hox3.1 transcripts along the anteroposterior body axis in transgenic mice. Regulatory sequences from Hox1.4, a gene normally expressed more anteriorly than Hox3.1, were chosen to direct expression of a Hox3.1 transgene. Offspring of independent transgenic lines expressed the transgene more anteriorly than the Hox3.1 gene. Rather than predicted posterior transformations, we observed anterior transformations of vertebrae in newborn mice. Transgenic mice also developed profound gastrointestinal tissue malformations, which may provide a molecular explanation for human developmental disorders often involving these same two regions. Paradoxically, vertebral transformations in the transgenic mice were strikingly similar to those reported in mice homozygous for a null mutation of the Hox3.1 gene. This observation suggests that Hox genes may be regulated antipodally, with over- or underexpression resulting in similar phenotypes.

Regulating gene expression in transgenic animals.

Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes.

A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs.

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.

Evidence for positive and negative regulation of the Hox-3.1 gene.

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.