The function of T and its role in embryogenesis

A m ajor source of inform ation about the in vivo function o f T protein comes from studies o f the phenotypes o f Tm utant and chim aeric mice.

The m ost com m only studied Tm utant is T Brachyury

w hich is due to a large deletion (1 8 0 -2 0 0 kb) encom passing the entire gene

and extensive flanking sequences. Hom ozygous m ice die at m id

gestation due to a defective allantois. The prim itive streak is thickened and the node is not properly formed. As a result, somites posterior to the seventh pair are absent or abnorm al and the notochord does not form. T acts in a dose

dependent fashion such that heterozygous m ice have short tails and the notochord in the caudal region is branched or fused with the neural tube or gut (Grüneberg, 1958). Thus, with the exception o f the allantois, the expression pattern of T is consistent with the m utant phenotype (see section 1.1.2).

Sim ilarly, zebrafish hom ozygous no tail mutants (functional T null alleles) are lethal and lack a tail, a differentiated notochord and more than one third o f the m ost posterior somites (H alpem et al., 1993; Schulte-M erker et al., 1994). These observations suggest that T plays a role in norm al posterior m esoderm form ation and notochord differentiation.

The short-tail phenotype of the heterozygous m ice can be rescued by the addition of a single copy o f a T transgene, verifying the dose dependent action of T. Furtherm ore, transgenic studies suggest that a dosage o f T higher than normal can increase embryonic lethality (Stott et al., 1993). It was proposed that over-expression of T could lead to aberrant cell type specification in the mesoderm, eventually leading to death. Interestingly, the prom oter used to drive the expression of the transgene in these experim ents has subsequently been shown to drive expression only in the prim itive streak (Clem ents et al., 1996).

Studies o f other T mutants, which are not due to large deletions, also highlight the im portance of T dosage and led to hypotheses that T interacts with other proteins. is a m utant due to an insertion o f a retroviral-like elem ent in the seventh exon and 7^ involves a 19 bp deletion in exon 8 (review ed in Herrm ann and Kispert, 1994). Both these m utations alter the open reading fram e resulting in proteins that retain their D N A binding property but are truncated at the carboxy terminus of the regulatory domain. T^*® is lacking the TA 2 and R2 domains and T^ is lacking the R2 dom ain (see previous section). Both and T m utant m ice exhibit a m ore severe

phenotype than mice; homozygotes have no somites, the rostral boundary o f em bryonic defects extends into the cervical region and heterozygotes are tailless and have additional skeletal abnorm alities

(M acM urray and Shin, 1988; Herrmann, 1991; K ispert and Herrm ann, 1994). In addition, the T / + phenotype, but not the 7^/7^ phenotype, is dependent on the num ber of wild type T copies and two are required to rescue the tailless phenotype (M acM urray and Shin, 1988; Stott et a l , 1993). It is possible that

and proteins antagonise the wild type protein, effectively reducing the am ount o f functional protein. There is an alternative explanation for the m ore severe phenotype o f and ' f . If T interacts with other proteins to form a transcriptional complex, then a truncated T protein, which nevertheless binds DNA, could interfere with the assembly or function o f this transcriptional com plex. This interference in not a feature of mutants w hose T protein is com pletely absent.

Further clues about the way that T protein exerts its influence on m esoderm cell differentiation and m igration come from studies o f chim aeric

m ice generated by injecting embryonic stem (ES) cells into

blastocysts. chimaeras with high levels (>70% ) o f null alleles are alm ost indistinguishable from intact hom ozygous mutants, w hereas low level chim aeras show a range of defects that can vary from a severe

phenotype, sim ilar to that of mice, to localised defects in the tail (truncation, branching) or allantois. In all chimaeras there is a rostrocaudal increase in T null cell contribution and the level of chim aerism in the

neuroectoderm is higher than that in paraxial mesoderm. W here heterozygous

jSnuii/_^ cells w ere used to generate chimaeras, 7^”“^V+x+/+ mice are norm al with m ild tail defects (kinking, abnormal tail tip) and cells can colonise rostral regions efficiently. The phenotypes of these chim aeric m ice support the idea that T acts in a dose dependent, cell autonom ous w ay (Rashbass et al.,

1991; Beddington et a l , 1992; W ilson et al., 1993; W ilson et al., 1995). Careful analysis o f chimaeras and com parison with wild type and

jBnuiiijBnuii j^ytants showed that the cells fail to m igrate aw ay from the prim itive streak and subsequently accum ulate in the node, prim itive streak and later in the tail bud, leading to abnorm alities in the notochord and

posterior m esoderm (Rashbass et a l , 1991; Beddington et al., 1992; W ilson et a l , 1993; W ilson et a l , 1995). It was suggested that T affects cell m igration by regulating genes involved in cell adhesion, an idea supported by other observations. Brachyury m ouse mutants have a thickened prim itive streak and a reduced m esoderm /ectoderm ratio, which appears to be due to abnorm al cell m ovem ent during gastrulation (Y anagisawa et a l , 1981). In a separate series o f cell culture experim ents (reviewed in W ilson et a l , 1993), it was shown that w hen cells were com pared with + /+ cells, they show reduced m otility on extracellular m atrix and form smaller aggregates. However,

jSnuiiijBnuii tiave the same rate o f DNA synthesis and m itotic index as wild type cells, and in culture, they have the same differentiative capacity and

their life-span extends beyond the tim e of death in the embryo.

Thus, it seems unlikely that the abnormal behaviour o f cells in vivo can be explained by differential cell growth, aberrant cell differentiation, or low survival time.

Interestingly, a 5 integrin deficient mice resem ble hom ozygous

Brachyury m ice (Yang et a l , 1993). a5(3l integrin is a cell surface receptor that binds fibronectin and is thought to be involved in m any cellular processes including cell migration. In hom ozygous a 5 integrin null m ice the anterior part o f the embryo from somites 1 -1 0 develops normally, but the posterior regions are highly defective. Posterior somites are not form ed, paraxial m esoderm is reduced and the neural tube is kinked. The notochord is formed, but it is not know n if it is functional.

Analysis of chimaeras of wild-type cells and cells carrying a w ild type T transgene, showed that the expression o f T in cells restores their normal behaviour in the prim itive streak and tail bud. Furtherm ore, these experiments suggested that the level o f T expression determ ines the speed with which cells move away from the prim itive streak. High levels of T favour a quick passage o f cells through and away from the

streak, w hereas cells with low levels of T tend to stay in the prim itive streak for a longer time and populate the tail bud (W ilson and Beddington, 1997).

M uch o f w hat has been proposed regarding T and m esoderm

differentiation and migration refers to the situation in the prim itive streak and node. However, it is not clear how this applies to the notochord or tail bud. A lthough, the supply of cells to the axial mesoderm, and thus the notochord, is directly dependent on the prim itive streak and node, it is likely that T protein m ay have a distinct function in the differentiation and survival of notochordal cells. D isruption o f these functions would lead to the notochord abnorm alities seen in zebrafish and mouse T mutants and chim aeric m ice (Beddington et al.,

1992; W ilson et al., 1995). Similarly, although the prim itive streak is directly related to the tail bud, it has been proposed that T has a distinct role in tail bud function and affects tail differentiation and elongation. It follows from this proposal that the abnormal tail m esoderm form ation seen in m ice is a prim ary outcom e o f T deficiency and not a secondary response to a

degenerate notochord (W ilson et al., 1993; W ilson et al., 1995).

The im portance o f T in m esoderm form ation is also highlighted by expression studies in Xenopus. O ver-expression of Xbra in animal caps cause the ectopic form ation of posterior mesoderm. M esoderm al markers that are indirectly activated by X bra are Xsna, a zinc-finger transcription factor,

m uscle-specific actin and Xhox3, a hom eobox transcription factor expressed in posterior m esoderm (Cunliffe and Smith, 1992). The type o f m esoderm

form ed depends on the dose o f Xbra RNA, with low am ounts inducing m ésothélial smooth m uscle and m esenchym e and high am ounts inducing somitic m uscle (O'Reilly et a l , 1995).

Interestingly, the outcom e of Xbra over-expression is influenced by co­ expression with a variety o f other transcription factors and inducers, im plying that in vivo several genes co-operate with Xbra to specify dorsal m esoderm . These collaborating factors probably include Pintallavis, a m em ber o f the forkhead/H N F3p transcription factor fam ily (O'Reilly et al., 1995), noggin, a bone m orphogenetic protein (BMP) antagonist and Xwnt-8, a m em ber o f the

w nt fam ily o f secreted glycoproteins (Cunliffe and Smith, 1994). Changing the level of co-expression and the order o f expression of these factors

influences whether muscle, notochord and/or neural tissue are form ed and w hether dorsalisation of m esoderm occurs.

Recently, expression studies in the ascidian Halocynthia roretzi have shown that the role o f T in notochord form ation is probably conserved in all chordates (Yasuo and Satoh, 1998). O ver-expression of A s -T is sufficient for notochord differentiation of notochord precursors in a cell-autonom ous way. However, in a sim ilar way to Xbra, the response to over-expression in non­ notochord lineages depends on the presence of other factors.