Seeking Sense in the Hox Gene Cluster.

The Hox gene cluster, responsible for patterning of the head-tail axis, is an ancestral feature of all bilaterally symmetrical animals (the Bilateria) that remains intact in a wide range of species. We can say that the Hox cluster evolved successfully only once since it is commonly the same in all groups, with -like genes at one end of the cluster expressed in the anterior embryo, and -like genes at the other end of the cluster expressed posteriorly. This review attempts to make sense of the Hox gene cluster and to address the following questions.

Mouse embryo Hox gene enhancers assayed in cell culture: Hoxb4, b8 and a7 are activated by Cdx1 protein.

Mouse Hox gene enhancer elements have typically been identified and characterized using Hox/lacZ transgenic mouse embryos. Such studies have, for example, identified Cdx responsive binding motifs in the enhancers of Hoxb8 and Hoxa7. Production of transgenic mouse embryos involves issues of cost, welfare, and considerable technical skill.

Hox cluster genes and collinearities throughout the tree of animal life.

The discovery of Hox gene clusters, first in Drosophila (a protostome) and then as homologues in vertebrates (deuterostomes), was a major step in our understanding of both developmental and evolutionary biology. Hox genes in both species perform the same overall function: that is, organization of the body along its head-tail axis. The conclusion is that the protostome-deuterostome ancestor, founder of 99% of all described animal species, must already have had this same basic Hox cluster, and that it probably used it in the same way to establish its body plan.

Gdf11/Smad signalling and Cdx proteins cooperate to activate the Hoxc8 early enhancer in HepG2 cells.

Developing anatomy along the head-tail axis of bilaterian embryos is specified, to a large extent, by the overlapping patterns of expression of the Hox genes. Hox gene enhancers respond to a variety of signals in order to regulate these discreet domains of expression. For mouse Hoxc8, the 399bp "early enhancer" plays a major role.

Possible rules for the ancestral origin of Hox gene collinearity.

The Hox gene cluster is believed to have formed from a single ProtoHox gene by repeated cycles of the following events: tandem gene duplication, mutation to generate a new expression boundary along the embryonic axis, and acquisition of a new Hox patterning function. The Hox cluster in Bilateria evolved in compliance with the so-called collinearity rule. That is, the order of the genes along the chromosome corresponds with the order of their embryonic expression domains along the head-tail axis.

The significance of Hox gene collinearity.

Arthropods and vertebrates inherited their Hox clusters from an ancestral cluster of at least six genes already present in their last common ancestor, Urbilateria. Clustering and a common transcriptional direction are both likely features of the way that the gene complex first arose in a process of tandem gene duplication. Spatial collinearity (correspondence between ordering of Hox genes along the chromosome and their expression patterns along the head-tail axis) has been conserved in many animal groups and is likely to have been already present in Urbilateria.

Synergistic action in P19 pluripotential cells of retinoic acid and Wnt3a on Cdx1 enhancer elements.

Cdx1 encodes a homeodomain protein that regulates expression of some Hox genes. Cdx1 itself is known to be regulated in the primitive streak/tailbud by both retinoic acid (RA) and Wnt3a. Cdx1 in eutherian mammals has two retinoic acid response elements (RAREs), located upstream and in the first intron, and each is adjacent to structural Lef/Tcf motifs.

Ossification sequence and genetic patterning in the mouse axial skeleton.

We provide novel data on vertebral ontogeny in the mouse, the mammalian model-of-choice for developmental studies. Most previous studies on ossification sequences in mice have focused on pooled elements of the spine (cervicals, thoracics, lumbars, sacrals, and caudals). Here, we contribute data on ossification sequences in the neural arches and centra to provide a comparative basis upon which to evaluate mammalian diversity of the axial skeleton.

Direct activation of a mouse Hoxd11 axial expression enhancer by Gdf11/Smad signalling.

A Hoxd11/lacZ reporter, expressed with a Hoxd11-like axial expression pattern in transgenic mouse embryos, is stimulated in tailbud fragments when cultured in presence of Gdf11, a TGF-β growth/differentiation factor. The same construct is also stimulated by Gdf11 when transiently transfected into cultures of HepG2 cells. Stimulation of the reporter in HepG2 cells is enhanced where it contains only the 332 bp Hoxd11 enhancer region VIII upstream or downstream of a luciferase or lacZ reporter.

Changes in Cis-regulatory Elements during Morphological Evolution.

How have animals evolved new body designs (morphological evolution)? This requires explanations both for simple morphological changes, such as differences in pigmentation and hair patterns between different Drosophila populations and species, and also for more complex changes, such as differences in the forelimbs of mice and bats, and the necks of amphibians and reptiles. The genetic changes and pathways involved in these evolutionary steps require identification. Many, though not all, of these events occur by changes in cis-regulatory (enhancer) elements within developmental genes.

Origins of Cdx1 regulatory elements suggest roles in vertebrate evolution.

Cdx1, an upstream regulator of Hox genes, is best characterized for its homeotic effects upon the developing axial skeleton, particularly in the neck. It responds to retinoic acid (RA) in both mouse embryos and embryonal carcinoma (EC) cells. By use of beta-galactosidase chemiluminescence, we show that a mouse Cdx1/lacZ reporter expressed in P19 EC cells responds to RA by the combined activities of an intron retinoic acid response element (RARE) and an upstream RARE.

Down-regulation of Cdx2 in colorectal carcinoma cells by the Raf-MEK-ERK 1/2 pathway.

Cdx2 is a homeodomain transcription factor that regulates normal intestinal cell differentiation. Cdx2 is frequently lost during progression of colorectal cancer (CRC) and is widely viewed as a colorectal tumour suppressor. A previous study suggested that activation of protein kinase C (PKC) may be responsible for Cdx2 down-regulation in CRC cells.

Multiple regulatory regions control the complex expression pattern of the mouse Cdx2 homeobox gene.

Background & Aims: The Cdx2 homeobox gene exerts multiple functions including trophectoderm specification, antero-posterior patterning, and determination of intestinal identity. The aim of this study was to map genomic regions that regulate the transcription of Cdx2, with a particular interest in the gut.

Methods: Genomic fragments covering 13 kilobase (kb) of the mouse Cdx2 locus were analyzed in transgenic mice and in cell assays.

Increased Cdx protein dose effects upon axial patterning in transgenic lines of mice.

To investigate the link between Cdx protein concentration and axial patterning in embryos, we made lines of mice OE1, OE2 and OE4 that overexpress each of the Cdx genes Cdx1, Cdx2 and Cdx4, respectively. The lines carry Cdx transgenes under the transcriptional control of their own promoter/enhancer elements. Transgenic embryos show Cdx transcription at 8.

cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse.

Expression of the mouse caudal genes cdx4 and cdx2 is examined by use of lacZ reporter constructs expressed in transgenic mouse embryos. During early gastrulation, up to at least 8.5 days of development, reporter mRNA distributions are apparently similar to those of endogenous cdx mRNAs.

Additional enhancer copies, with intact cdx binding sites, anteriorize Hoxa-7/lacZ expression in mouse embryos: evidence in keeping with an instructional cdx gradient.

Expression of a Hoxa-7/lacZ reporter construct in transgenic mouse embryos is shifted anteriorly when the upstream enhancer is multimerized. The shift occurs in spinal ganglia, neurectoderm and in both paraxial and lateral plate mesoderms. Much of the multimer effect is inhibited by destruction of a single caudal (cdx) binding motif in the additional copies of the enhancer.

Vertebrate caudal gene expression gradients investigated by use of chick cdx-A/lacZ and mouse cdx-1/lacZ reporters in transgenic mouse embryos: evidence for an intron enhancer.

The vertebrate caudal proteins, being upstream regulators of the Hox genes, play a role in establishment of the body plan. We describe analysis of two orthologous caudal genes (chick cdx-A and mouse cdx-1) by use of lacZ reporters expressed in transgenic mouse embryos. The expression patterns show many similarities to the expression of endogenous mouse cdx-1.

Colinearity and non-colinearity in the expression of Hox genes in developing chick skin.

Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. We found that these characteristics apply to several Hox genes expressed in developing chick skin (Hoxb-4, Hoxa-7 and Hoxc-8), and we classed this group of genes as regionally restricted. To our surprise, we found that most of the Hox genes we examined are regionally unrestricted in their expression in the embryonic chick skin.