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STRUCTURE: In promoter region of mouse Hox-1.4 gene six GC-rich sequences A to F are detected. Three of these contained a genuine Sp1 consensus binding site (GC-boxes C, E, F) and were shown to bind this transcription factor, whereas the others were related to it. The first 750 bp of the coding sequence or the additional upstream region may play a role in the gene response to Retinoic Acid.
EXPRESSION: The Hox-1.4 gene expressed from a rather anterior level in a subset of cellular types within different organs. In mouse embryo at early stages the expression of Hox-1.4 gene are restricted to Neural tube (CNS) and lateral plate mesoderm. Signal is also detected in the Limb buds. The expression is found in structures partially derived from lateral plate or para-axial mesoderm, including fetal lung, intestinae and kidney. In addition, signal detected in some cervical and thoracic regions of mesodermal origin: mesenchyme tissue located between the aortic arch and the mesenchyme surrounding the base of the larynx and the trachea. In the embryo lungs, Hox-1.4 expression is restricted to mesenchime. Expression in stomach and gut is also limited by mesenchyme cells.
See also Michael Tainsky' HoxA4 Page and the HoxA4 promoter
Targeted disruption of the mouse Hox-1.5 gene results in regionally restricted developmental defects. Hox-1.5(-)//Hox-1.5(-) homozygotes are athymic, aparathyroid, hae reduced thyroid and submaxillary tissue and exhibit a wide range of throat abnormalities. In addition, they often feature defects of the heart and arteries as well as craniofacial abnormalities. The genetic function of Hox-1.5 more spatially and temporally restricted than the gene expression pattern would predict. Hox-1.5 is expressed in the lung, stomach, spleen and kidney, however no mutant phenotype can be detected.
The Hox-1.6 gene disrupted in embryonic stem cells by homologous recombination was introduced into the mouse germline. Heterozygous mice were normal, but homozygous mice died at birth from anoxia and had numerous defects that were centered at the level of rhombomeres 4 to 7 and included delayed neural tube closure, absence of certain cranial nerves and ganglia, and malformed inner ears and bones of the skull. Thus, HOX-1.6 is involved in regional specification along the rostro-caudal axis, but only in its most rostral domain of expression. HOX-1.6 appears to specify neurogenic neural crest cells prior to specification of mesenchymal neural crest cells by Hox-1.5. Thus, within the same region of the presumptive hindbrain, two HOX-1 genes are involved in the patterning of two different populations of neural crest cells.
Hindbrain segments have a number of functional interconnections with Neural crest derivatives. A possible mechanism that might underlie these spatial relationships is that neural crest is patterned according to its rhombdomeric origin. Therefore, it may be significant that only Hox-2.8 and -2.9 are expressed in the VIIth/VIIIth ganglia, located adjacent to R4, as it is only these HOX-2 genes that are expressed in R4. Other HOX-2 genes also exibit restricted expression in the cranial ganglia, because Hox-2.7 expression occurs in the IXth and Xth ganglia, and Hox-2.1 expression occurs only in the Xth ganglion.
STRUCTURE: A promoter region of 3600 bp was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. Similar results have been obtained with Hox-1.1, -1.3, -2.3 and -3.1 promoters. These observations suggest that several or all of the homeo box genes in a cluster contain an independent functional promoter that is able to direct gene expression at least into subsets of the normal expression domains. In contrast, in all studies described to date, even large upstream regions of individual Hox genes do not contain all necessary regulatory elements, indicating that cis-acting regulatory regions are missing that may be located in introns or downstream regions, or can be found in more distal regions upstream or downstream of neighboring genes or at the ends of the HOX clusters. Hox-2.3 gene
STRUCTURE: In murine Hox-2.3 promoter region seven distinct protein binding sites were found, U1A, -1B, -1C, U2, U3, U4, U5, U6, D1. The core of U5, GGGTGGG, is present upstream from the human c-myc gene where it was shown to interact with the transcription factor P and F. The U4 contains two Sp1 binding sites. Both U3 and U6 resemble the consensus sequence established by comparing six similar G-rich sequences in Hox-1.4 promoter region. D1 is not similar to known transcription factors binding sites but seems plausible that it interacts with one of many DNA binding factors which are induced by Retinoic Acid in EmbryoCarcinoma cells. D1 was detected in a region downstream of the transcription start near the start of the open reading frame. In the case of U1 the DNA sequence fit the consensus sequence GNCCNCGTGACNNG of sites reported to be bound by USF/MLTF transcription factor. It is possible that Retinoic Acid Responsible Element (RARE) is present in the HOX-2 cluster outside the described regions. It is Known that a fragment extending 1316 bp upstream of the transcription start site, thus curresponding to the Hox-2.4/-2.3 inter- genic sequences is capable to mediating transgenic expression in mice. The transgene apparently lacks sequences needed for correct Hox-2.3 expression in somitic and lateral plate mesoderm and in neuroectoderm.
EXPRESSION: Expression of the murine Hox-2.3 gene suggests multiple time-dependent and tissue-specific roles during development. Using in situ hybridisation one first detect Hox-2.3 transcripts in the allantois primordium at 7.5 days embryo. One day later transcripts are found in embryonic ectoderm and mesoderm. In 9.5-10.5 day embryo Hox-2.3 expressi- on is observed in the CNS. Within this antero-posterior domain, Hox-2.3 expression is also found in the PNS, in the mesoderm and in the hindgut epithelium. During subsequent development, the initially broad expression pattern in the somitic, lateral plate and intermediate mesoderm becomes res- tricted to structures in the urogenital system. Expression of the Hox-2.3 gene in the forelimb bud mesenchyme was detected on 9.5 day, thus in the early phase of limb formation.
Sequence analysis revealed that the Hox-2.9 homeodomain is most similar to those of Drosophila lab and mouse Hox-1.6. In early mouse embryo prior to the start of neurulation Hox-2.9 is expressed within and posterior to the embryonic mesoderm that will participate in hindbrain formation. With the onset of neurulation, expression then becomes detectable in the neural plate as well, but only in the part that overlies the Hox-2.9-expressing mesoderm. A second phase of expression is detected in the Neural tube in which Hox-2.9 expression restricted by Rhombdomere 4 and also in developing branchial arch units of the hindbrain region.
It is known that a master promoter located into the HOX-3 complex controls the transcription of three entirely different homeo box genes lying over a region of approx. 35 Kbp of genomic DNA. These genes are Hox-3A, -3B, -3C.
The murine Hox-3.1 gene maps to a HOX-3 cluster of Chr.15. During embryogenesis Hox-3.1 transcript are detected first in the posterior neural tube of 9.5 day embryo. At later developmental stages, a ventral-dorsal gradient of Hox-3.1 transcript accumulation is established. Hox-3.1 also are detected in the thoracic sclerotomes from 6th to 10th thoracic pre- vertebrae. The establishment of the complex spatio-temporal expression pattern of Hox-3.1 gene is likely to require cis-regulatory regions containing multiple target sequences for numerous transcription factors. It is established that the Hox-3.1 promoter elements required to establish the correct pattern of Hox-3.1 expression in early embryo are separable from those required later. It is possible that elements that lie far from the coding region of Hox-3.1 are required to bring about an appropriate level of pattern of expression. A similar regulatory element within the HOX-3 locus could act alone or in concert with more proximal elements to influence expression of Hox-3.1 and possibly other genes within the cluster. In Hox-3.1 transcription unit and the Hox-3.2 - Hox-3.1 intragenic region there are several potential sequences for interactions with homeo domain and zinc finger proteins, which include a perfect match to the consensus sequences recognized by the Hox-1.3 protein. Several scattered G+C boxes representing potential binding sites for the Sp1 transcription factor have also been found. It is interesting that G+C-rich domain with several Sp1 bind sites has been found in the upstream region of the Hox-1.4 transcription start site and a similar region is located upstream of the Hox-3.1 translation start site. Sequence motifs resembling Hormone Response Elements, including a Glucocorticoid Response Element (GRE) located upstream of the transcription start region and a Thyroid Hormone Response Element (TRE) in the intron, have been identified. A TRE-like sequence contining 7-bp spacer is present about 220 bp upstream of the transcription start region. Such TRE-like sequences may mediate responses to the morphogen Retinoic Acid. In this respect it is important to note that expression of the Hox-1.3 gene is inducible by RA in F9 embryonic carcinoma cells. In addition, sequence motifs with homology to the Estrogen Response Element have been found in 5' region. Another response element pertinent to developmentally regulated gene expression is the Heat Shock Element (HSE). Besides, the sequence analyses showed the presence of several nucleotide repeat motifs, some of which may by involved in the formation of altered chromatin structure and/or binding of regulatory proteins (sites similar to Antp, Kr, ftz/en/eve response elements). One have identified two extended CT repeats, a proximal and distal, (GT)22 sequence motif, (T)26 sequence and GATA repeat.
The murine Hox-3.2 gene is the most 5' member of the HOX-3 complex in Chromosome 15 isolated to date. In early mouse embryo Hox-3.2 is detected in the Neural tube with a sharp anterior boundary at the level of the third thoracic prevertebra. In contrast to Hox-3.1, Hox-3.2 is not expressed in the dorsal horns containing the sensory neurons. Hox-3.2 are also detected in the posterior prevertebrae, the hind Limb buds and the cortex of the developing kidney. Unlike Hox-1.4 and Hox-1.3 and their paralogs, Hox-3.2, -2.5 and -4.4 show strikingly different anterior bound areas of expression in the CNS and prevertebrae.
The upstream region of the human homeo box gene Hox-3D is a target for regulation by Retinoic Acid and Hox homeoproteins.
COOPERATIVE EXPRESSION OF HOX-4 COMPLEX DURING LIMB DEVELOPMENT The HOX-4 complex are expressed in different but overlapping domains in limbs during murine development. The more 5' the position of these genes in the complex, the later and more distal is their expression. Comparison of these observations with the expression patterns of the genes of HOX complexes in the early embryo suggest that similar molecular mechanisms are involved in the positional signaling along the axes of both the embryonic trunk and the fetal limbs. The mechanism by which the genes are turned into an open state could be independent of cis-regulation in HOX-4 complex: it could for example rely on chromatin unwinding. Hox-4.3 gene Hox-4.3 transcripts are detected in the genital ridges, and, later, in the fetal gonads. Two other members of the HOX-4 complex (Hox-4.2 and -4.4) have been shown to be expressed in the fetal gonads in contrast to the genes located 5' to Hox-4.4. In this case the three genes expressed in the genital ridges are those that show specific expression in the epithelium of mesonephric tubules will give rise to the genital excretory ducts, while the major part of the mesonephros will degenerate before birth. Within the foreLimb, transcripts extend up to the very proximal and anterior part of the buds and the expression domain appears at least as wide as that of Hox-4.4. Thus, the Hox-4.3 expression pattern in the early limb buds perfectly fits on the expression of HOX-4 genes in these structures.
Two gene members of the murine HOX-4 complex (Hox-5.2 and -5.3) show regional and cell-type specific expression in developing Limbs (See HOX-4 cluster expression) and gonads. In 12.5 day old foetuses Hox-5.2 transcripts are found in the genital ridge region. This strong expression extends more anteriorly than that seen in the dorsally adjacent mesonephros. The Hox-5.2 pobe hybridizes to the presumptive gonadal region at 10.5 days embryo. Strong hybridization is restricted to the ventral edge of the mesonephros, close to coelomic epithelium. Comparison by hybridization with Hox-5.3 probe fails to reveal any labelling in the indifferent gonad except in its very posterior edge, in a level where hybridization is found in the adjacent mesonephros.
Hox-7.1 is first member of a new family of mouse homeo box containing genes. The putative homeodomain sequence shows 57% similarity to mouse Hox-1.6. It is of intertest that the mouse mutation luxate maps within the region of the Hox-7.1 locus. The phenotype of this mutation involves polydactyly and oligodactyly of the hind feet. Msh-like mice HOX-7 homeobox gene associate with areas of mesenchimal- epithelial interaction and cell migration especially in Neural crest ecto- mesenchymal cells. Aside from the expression patterns seen in the facial anlage of the 9.5 days embryo, HOX-7 transcripts were also detected in neuro-epithelium including cells of the dorsal midline of the Neural tube. High expression of HOX-7 was present in the mesenchyme cells invading the pouch formed by the involuting choroid plexus neuroepithelium. A second major site where HOX-7 was expressed was the anlage of the anterior pituitary; the Rathke's pouch. When these expression patterns are considered together with characterized human and mouse Retinoic Acid embryopathies and the congenital malformations seen in human children associated with deletion of chromosome 4p16.1 (Wolf-Hirschhorn syndrome), HOX-7 may be a good candidate as one of the genes involved in the initiation of the choroid plexus phenotype and its subsequent formation, the formation of the outer ear, formation of the dentition and the differentiation of the cell types of the anterior pituitary. The expression pattern of HOX-7 in the dorsal midline of the neural tube further suggests that it may also be involved in the specification of the dorsal-ventral axis of the developing nervous system.