Control of Hox gene regulation and function during anteroposterior patterning in Xenopus laevis

Abstract

The Hox genes form a subset of the homeobox containing genes. The homeobox encodes a DNA binding motif, called the homeodomain. It has been firmly established that Hox expression boundaries along the AP and other embryonic axes are correlated with structural identities. Generating correct Hox expression patterns is thus clearly essential for correct AP axis patterning. The aim of investigations presented in this thesis has been to gain more insight into the processes controlling expression and function of Hox genes during anteroposterior patterning. Recent work in our research group has shown that a temporally colinear expression sequence of Hox genes is already present in the mesoderm of Xenopus gastrulae. Most, if not all, previous work concerning Hox colinearity has been focused on colinear Hox gene expression in the neurectoderm. In chapter two, we present data to show that Xwnt8 is directly upstream of Hoxd1 in mesoderm. This is the first example of an initiator of expression of a 3 Hox gene in a vertebrate embryo. An upstream regulator of Hox gene expression in the neurectoderm of vertebrates is retinoic acid (RA) (and or its derivatives). Retinoids can act via the nuclear receptors of the RAR and RXR family. Retinoic Acid Response Elements (RAREs) have been found in the regulatory sequences of a number of labial- and deformed group Hox genes. We searched for conserved RAREs in the Hox clusters of mouse and human, and the results are reported in chapter three. Most Hox proteins contain a second conserved domain, in addition to the homeodomain, the hexapeptide. This domain is mediates Hox/PBC interaction; this interaction leads to increased binding specificity and -affinity. Sequence analysis of Hox proteins demonstrates interspecies sequence conservation among paralog group members in the hexapeptide-flanking sequences, which is reported in chapter four. Meis/PBC interaction leads to shielding of the nuclear export signal of PBC proteins, resulting in a net influx into the nucleus, modifying the activity of Hox proteins present. In zebrafish hindbrain development, a synergistic relation between Hoxb1, Pbx4, and Meis3 has been shown, and was argued to directly induce the expression of Hoxb1. Since recent discoveries have shown that Hox genes are expressed in a colinear sequence in marginal zone mesoderm we investigated whether a Xenopus Meis homolog, XMeis3, cooperates with Hox function during gastrula stages. In chapter five, we report that XMeis3 is necessary for mesodermal and ectodermal Hox expression, and the progression of gastrulation. In chapter six, conservation of hexapeptide-flanking sequences of Pdx1 and Cdx proteins, present in a wide range of species, resembling the conservation found in Hox group 1 through 8 proteins (Chapter four) is reported. More generally the presence of a hexapeptide sequences and conservation of flanking sequences in all of the members of the Antp-class of homeodomain proteins was investigated, and found to be widely distributed, in addition, hexapeptide-flanking sequence conservation was found. Identifying the downstream targets of Hox genes is necessary if their function in development is to be understood in molecular terms. XRap1 joins a very short list as to date very few Hox targets have been identified. In chapter seven, the small GTPase XRap1 is reported as a direct target of Hoxb4 regulation.