BIOLOGICAL OVERVIEW

The Drosophila Goosecoid homolog is expressed in two domains during embryogenesis. During early cellularization an expression domain anterior to the cephalic furrow gives rise to cells that ultimately will be located in the brain hemispheres, while a second domain invaginates inside the stomodeum along with ectodermal cells to give rise to the stomatogastric nervous system, ring gland and foregut.

Goosecoid expression in vertebrates takes place in the organizer, the compartment associated with the dorsal lip of the blastopore which functions in the recruitment of cells for involution through the blastopore and is responsible for self-determination of cells into dorsal mesoderm. Goosecoid expressing cells induce neighboring cells to migrate toward the anterior of the embryo and enhance involution. The migrating cells contribute mainly to the most anterior involuting tissues, namely anterior endoderm and head mesoderm. Thus Goosecoid appears to mimic properties of the "organizer" (Niehrs, 1993).

What is evolutionarily conserved with respect to GSC function when comparing between vertebrates and invertebrates? Any comparison is complicated by the fact that vertebrates are Deuterostomes (the mouth forms as a secondary consequence far away from the blastopore, which gives rise to the anus), while insects are Protostomes (the mouth forms at or near the blastopore). In all Protostomes, the stomodeum is the anterior end of the blastopore. In Drosophila the cells invaginating in the stomodeum (those that express Gsc) constitute the anterior-most structure of the embryo. The stomatogastric nervous system is believed to arise from the labrum, the most anterior embryonic segment (Schmidt-Ott, 1994).

In vertebrates, Gsc is expressed in the dorsal lip of the blastopore, the region leading the invagination of the mesoderm during gastrulation that will give rise to the head process and the anterior-most tissues of the vertebrate embryo (prechordal plate mesoderm). Thus Gsc is expressed in the foregut of both flies and vertebrates. Therefore Gsc expression in Drosophila and vertebrates could be evolutionarily related (Goriely, 1996).

Are the same genes involved in regulation of vertebrate Gsc as those involved in regulating Drosophila Gsc? In vertebrates, nodal, a member of the TGF-ß superfamily, has been implicated as a signal for induction of axial mesoderm during gastrulation. Activin too has been considered as a candidate for this role, but experiments with follistatin, an activin antagonist, lead to the conclusion that activin is not a requirement for mesoderm induction (Jones, 1995 and references). In the zebrafish, nodal expression leads to trunk duplication, but no obvious head duplications. Nodal induces ectopic Gsc expression and axis duplication in zebrafish. This observation is similar to results reported in Xenopus using other inducing factors in the TGF-ß superfamily. In contrast, complete axis duplications can be achieved by injection of certain WNT mRNAs in Xenopus (Toyama, 1995 and references). Interestingly, wingless (the Drosophila wnt prototype) expression is required not as an instructive signal, but as a permissive factor that coordinates the spatial activity of morphoregulatory signals within the stomatogastric nervous system anlage (González-Gaitán, 1995). Currently, the regulation of the stomodeal expression of Drosophila Gsc remains a mystery (Goriely, 1996).

The implication of this work is that the stomatogastric nervous system is of ancient origin, possibly older than the brain itself. The reason for this conclusion is its proximity to what is potentially the remnants of the organizer, a structure common to protostomes and deuterostomes. Expression in flies of Gsc in the brain anlage, albeit occurring earlier than expression in the stomatogastric anlage, represents an evolutionary expansion of a more primitive nervous system. A corollary to this argument is that both the spinal cord of vertebrates and the ventral cord of invertebrates are also derivative structures.

Study of goosecoid expression and the effects of gsc mutation have lead to a much better understanding of the development of the stomatogastric nervous system. goosecoid expression overlaps with one of the three SNS precursor groups invaginating from the foregut anlage. At stages 14 and 15, the SNS precursor cells that have invaginated migrate as three separate groups (or pouches) following apparently independent paths; their luminal sides are well separated. At the end of migration, the gsc expressing cells derived from the anterior-most pouch are aligned as a curved array of cells along the esophagus. These cells represent the first esophageal ganglion (EG1) of the SNS, a chain of 12-15 neurons associated with the dorsal surface of the esophagus. It is possible that the gsc expressing portion of the ring gland is derived from the anterior-most SNS invagination, since the ring gland in other insects in connected to the enteric nervous system via nerve connections (Hahn, 1996).

In embryos homozygous for a lethal allele of gsc, the anterior-most and middle pouches undergo a defective fusion event at stage 13, rather than remaining distinct. The two pouches stay fused while in migration, at least until stage 14. The two fused pouches in migration contain fewer than the normal number of gsc expressing cells. Subsequently, only one of these cells reaches the wall of the esophagus. It remains in this mutant condition and may represent a residual esophageal ganglion (EG1). The fusion of the pouches is most likely due to the exclusion of gsc expressing EG1 precursors from the anterior-most pouch. It is likely that gsc is required for the birth, survival or invagination (detachment from the foregut anlage) of EG1 precursor cells. The recurrent nerve, which connects the frontal ganglion to EG1 and the second esophageal ganglion (EG2) with two connectives in wild-type, has only one connective linked to EG2 in mutant embryos. In a second gsc mutant, representing a true null allele, a late forming (stage 17) hairpin loop region (normally found at the pharynx-esophagus junction) collapses and the esophagus is swayed anteriorly. Shortly thereafter, the midgut protrudes dorsally, compressing the dorsal-most tissues of the embryo. This defect could explain the embryonic lethality of the null allele. It is likely that cells under the control of gsc in the brain are not massively eliminated in gsc mutants, but there may be subtle defects in the brain region (Hahn, 1996).

GENE STRUCTURE

Bases in 5' UTR - 464

Exons - 3

Bases in 3' UTR - 635

PROTEIN STRUCTURE

Amino Acids - 419

Structural Domains

The homeodomain at residue position 284-345 contains a lysine at postion 50. This is a landmark of proteins encoded by genes like bicoid, orthodenticle and sine oculis that are involved in patterning the anterior region of the Drosophila embryo (Goriely, 1996).

The Engrailed homeoprotein is a dominantly acting, so-called 'active' transcriptional repressor, both in cultured cells and in vivo. When retargeted via a homeodomain swap to the endogenous fushi tarazu gene (ftz), Engrailed actively represses ftz, resulting in a ftz mutant phenocopy. Functional regions of Engrailed have been mapped using this in vivo repression assay. In addition to a region containing an active repression domain identified in cell culture assays, there are two evolutionarily conserved regions that contribute to activity. The one that does not flank the HD is particularly crucial to repression activity in vivo. This domain is present not only in all engrailed-class homeoproteins but also in all known members of several other classes, including goosecoid, Nk1, Nk2 (vnd) and muscle segment homeobox. The repressive domain is located in the eh1 region, known as 'region three', found several hundred amino acids N-terminal to the homeodomain. The consensus sequence, arrived at by comparing Engrailed, Msh, Gsc, Nk1 and NK2 proteins from a variety of species, consists of a 23 amino acid homologous motif found in all these proteins. Thus Engrailed's active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily. Using rescue transgenes it has been shown that the widely conserved in vivo repression domain is required for the normal function of Engrailed in the embryo (Smith, 1996).

date revised: 1 Dec 97

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