Stephen Gaunt
Tel. (01223) 496347

• Contact via email


• Recent, selected Publications

Caudal (cdx) and Hox proteins as regulators of the embryonic body plan

During the early development of a mouse or human embryo, the correct location of body parts along the head-to-tail axis is specified by the expression boundaries of 39 special genes known as Hox genes. Each Hox protein is expressed in a unique spatial domain along the body where, functioning as a transcription factor, it instructs embryonic cells on a particular route of morphogenesis. Hox expression domains thus provide a pre-pattern to the body plan. How are these domains established? The three caudal (cdx) proteins are upstream activators of Hox genes, and they are expressed in posterior-to-anterior gradients along the embryonic axis. Could these gradients function as instructional (morphogen) gradients for the setting of Hox domains?

Expression of the Hoxa-7 gene can be detected experimentally by looking at Hoxa-7/lacZ transgenes expressed in transgenic mice. Wherever the Hoxa-7/lacZ protein is produced it can be detected as a blue stain. When the transgene, like the normal gene, contains one copy of the enhancer element (red box in Figure 1A) the anterior boundary of the expression domain lies at the levels of spinal ganglion 5 in neural tissue and vertebra 13 in mesoderm (transgene1; Figure 1B). The addition of three extra copies of the enhancer causes a forward shift in the anterior boundary of the expression domain to the levels of spinal ganglion 3 in neural tissue and vertebra 8 in mesoderm (transgene 2; Figure 1B).

The Hoxa-7 enhancer element contains the motif TTTATG, which is known to be the binding site for cdx transcription factors. To test the significance of this in the difference between the expression of transgenes 1 and 2 (Figure 1), we produced transgene 3 which is identical to transgene 2 except that the three additional copies of the enhancer are each mutated in the binding motif (green boxes; Figure 1A). The expression boundaries of transgene 3 are located at the levels of spinal ganglion 4 and vertebra 11 (Figure 1B). Thus, much, although not all, of the anteriorizing effect of additional enhancers (transgene 2 relative to transgene 1) is nullified by destruction of the additional cdx protein binding sites (transgene 3).

These results can be explained if firstly: there is normally an instructional (morphogen) gradient of cdx proteins along the length of the embryo to regulate each Hox gene's expression boundaries within specific threshold limits, and secondly: multiple copies of the cdx binding motif make a Hox gene more sensitive to cdx concentration, thereby enabling activation at lower (more anterior) positions along the cdx gradient.

Gradients of caudal gene activity formed by time-dependent decay

The three vertebrate caudal (cdx) proteins are upstream activators of the Hox genes. The Hox genes become expressed in a series of partially overlapping domains along the embryo. Hox proteins, functioning as transcription factors, then coordinate development of the embryo along its anteroposterior axis.

In an attempt to unravel the mechanism of cdx gene function, particularly with regard to Hox gene activation, we have studied the mouse cdx-1 gene and its chick orthologue, cdx-A, by use of lacZ reporter constructs expressed in transgenic mouse embryos. Both chick and mouse constructs give similar results: at 8.7 days, cdx/lacZ reporter activity is seen as a clear gradient over somites 5 to 11, and over the anterior neural tube (Figure 2A, C-F).

We have suggested that such gradients of cdx gene activity may function as morphogen gradients to specify the expression domains of the Hox genes. In this model, different Hox genes would become activated at different threshold concentrations of cdx gene activity, and each Hox gene would therefore acquire its own anterior boundary of expression along the embryo. The range of somites over which we observe the gradient (somites 5 to 11, contributing to neck vertebrae 1 to 7) is the same as those over which there is derangement of Hox expression, and accompanying homeotic mutation, in cdx-1 knockout mice.

The mouse embryo forms by the progressive emergence of somites from posterior tissues (the primitive streak). We found that the gradient forms by a time-dependent decay of cdx/lacZ activity within somites once they have emerged from the streak. Newly formed (more posterior) somites therefore have a higher activity than older (more anterior) somites. Evidence for this is seen by comparing Figure 2A and B. Thus, it is seen that at 8 days newly formed somite 5 is labelled as strongly as more posterior parts, yet by 8.7 days somite 5 is only weakly labelled.

By deletion of sequence within the cdx/lacZ reporter constructs we found that an essential cdx enhancer element lies within the first intron. Comparison of mouse and chick sequences over this region shows conserved motifs, potentially responsive to both retinoic acid and Wnt/β-catenin signalling. We found that specific mutation of either of these two binding motifs results in a posterior shift in the position of the cdx/lacZ activity along the embryo, indicating roles for both retinoic acid and Wnt proteins in the upstream activation of cdx-1.

Evolutionary shifts of vertebrate Hox gene expression

During evolution, animals may change their body shape by shift (or 'transposition') of anatomical structures up or down their body axes. We earlier showed how this shift in mesodermal structures (e.g. the vertebral column) could be attributed to a prior shift in the embryonic expression patterns of Hox genes. More recently, we presented evidence that shifts in embryonic Hox expression can similarly account for evolutionary transposition in neural components. Mouse and chick display a relative transposition in the position of their forelimb and accompanying brachial plexus.

Figure 3 shows how this is presaged by similar transposition in the neural expression of the Hoxa-7 gene. The brackets depict the embryonic expression domains of various Hoxa genes. Spinal ganglia, vertebrae and somites are shown as circles, squares and lines respectively. Cervical, thoracic, lumbar and sacral structures are coloured pale blue and yellow alternately along the body. Arrows show anterior boundaries of Hox gene expression in vertebrae.

Recent, selected publications

Gaunt SJ, Drage D, Trubshaw RC (2005) cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse.
International Journal of Developmental Biology 49 901-908
http://dx.doi.org/10.1387/ijdb.052021sg

Gaunt SJ, Cockley A, Drage D (2004) Additional enhancer copies, with intact cdx protein binding sites, anteriorize Hoxa-7/lacZ expression in mouse embryos: evidence in keeping with an instructional cdx gradient.
International Journal of Developmental Biology 48 613-622
http://dx.doi.org/10.1387/ijdb.041829sg

Gaunt SJ, Drage D, Cockley A (2003) 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.
Mechanisms of Development 120 573-586
http://dx.doi.org/10.1016/S0925-4773(03)00023-6

Reid A, Gaunt SJ (2002) Colinearity and non-colinearity in the expression of Hox genes in developing chick skin.
International Journal of Developmental Biology 46 209-215
http://www.ijdb.ehu.es/web/paper.php?doi=11934149

Gaunt SJ (2001) Gradients and forward spreading of vertebrate Hox gene expression detected by using a Hox/lacZ transgene.
Developmental Dynamics 221 26-36
http://dx.doi.org/10.1002/dvdy.1122

Gaunt SJ (2000) Evolutionary shifts of vertebrate structures and Hox expression up and down the axial series of segments: a consideration of possible mechanisms.
International Journal of Developmental Biology 44 109-117
http://www.ijdb.ehu.es/web/paper.php?doi=10761855