BIOLOGICAL OVERVIEW

Arrowhead (Awh) function is related to special developmental structures termed histoblast nests, precursors of certain abdominal structures in the adult fly. Unlike the process in vertebrate development, the adult fly is not formed as a result of the continuous development of embryonic tissues, rather imaginal cells from which various adult structures eventually arise are set apart from embryonic tissues early in embryonic development. Imaginal precursor cells are established as discrete groups of cells localized to specific regions of the embryo. The precursors of the adult head structures, appendages and genitalia form from invaginations of the embryonic epithelium and make up the imaginal discs; these are groups of cells not directly associated with the larval integument. The precursors of the abdomen and the internal organs of the adult, such as the gut, salivary glands and brain, arise from nests or rings of cells intimately associated with larval structures. For example, the salivary gland imaginal rings are embedded in the larval salivary glands; the midgut imaginal histoblast nests arise in the larval midgut and the abdominal histoblast nests form among the cells of the larval abdomen (Curtiss, 1995).

Each adult abdominal segment forms from four pairs of histoblast nests: the anterior and posterior dorsal pairs (which produce the tergites); the ventral pair (which produce the sternites and pleurites), and the spiracular pair (which form the spiracle and the surrounding pleurite tissues). Each anterior dorsal and ventral histoblast nest is composed of approximately 16 cells; each posterior dorsal histoblast nest consists of approximately five cells, and each spiracle histoblast nest has approximately three cells. The abdominal histoblasts do not divide during the larval stages, but begin to divide within the first 3 hours after pupariation. They continue to divide until approximately 15 hours of pupal development without displacing the larval cells. At about 15 hours of pupal life, the abdominal histoblast cells begin to migrate and displace the larval cells, which are then histolyzed. Following proliferation and migration, cells of adjacent segments fuse at the dorsal/ventral and segmental borders. During the terminal stages of abdominal development the cells differentiate to produce epidermal tissues, including the microchaetae and macrochaetae, and tosecrete the adult cuticle (Curtiss, 1995 and references).

In the Awh mutant pharate adult, a single row of bristles develop in the anterior-most segment. No other development of the abdominal epithelium occurs, as evidence by the absence of bristles and cuticle. Nevertheless, when partial development of abdominal epithelium occurs in mutant pupae, the cuticle and bristles appear normal. It has been concluded that Awh does not affect differentiation of the cells, but does affect the establishment or proliferation of the precursors. Examination of escargot (a gene required for cell cycle regulation of imaginal tissue) expression in abdominal histoblasts and other imaginal precursors shows that Awh mutants have significantly fewer cells in each histoblast nest. This suggests that Awh is necessary to generate the proper number of abdominal histoblasts in the embryo (Curtiss, 1995).

Awh appears to have a role in salivary gland formation as well. The adult salivary glands develop from imaginal rings located at the anterior end of each larval salivary gland. The imaginal ring cells resume mitosis at the molt between second and third instar. The larval salivary gland degenerates during the early pupal stages until, by approximately 24 hours of pupal development, it is completely dissolved. At approximately 10 hours of pupal development the imaginal ring cells begin to grow out anteriorly and posteriorly to form the adult salivary gland. Approximately 10 cells in each ring of wild-type late second instar constitute the embryonic founder cells of the salivary gland imaginal ring. Each imaginal ring contains approximately 150 cells in mature third instar larvae. In Awh mutant larvae, imaginal ring cells are often completely absent or reduced in number, and their arrangement is disorganized. Thus Awh is required for development of both abdominal histoblasts and salivary gland imaginal ring cells (Curtiss, 1995).

Expression of Awh in histoblast and imaginal ring tissue, and the requirement for Awh for the proliferation of these tissues, points to a clear distinction between two types of imaginal tissues: (1) imaginal discs that give rise to adult structures such as wings, legs and gonads do not require Awh; this is in contrast to (2) histoblasts and imaginal ring tissue that do require Awh function for establishment or proliferation. Curtiss and Helwig (1995) define as incorporate those imaginal precursor cells, including the abdominal histoblasts and salivary gland imaginal rings, that are embedded in larval tissue. During metamorphosis, incorporate imaginal cells replace the cognate larval organ in which the precursor cells are located. excorporate imaginal precursor cells are defined as imaginal discs, which develop separately from larval larval tissue. During metamorphosis, excorporate imaginal cells elaborate structures unique to the adult.

Several experimental results point to a clear distinction between these two types of imaginal tissue. Whereas expression of Awh is required for the development of incorporate imaginal precursors, ectopic expression of Awh promotes programmed cell death in excorporate imaginal cells. Expression of Awh in the wing disc in regions that give rise to the adult wing blade and part of the wing hinge give rise to adult flies that are missing all structures which derive from the portions of the imaginal discs that ectopically express Awh. However, ectopic expression of Awh in developing salivary glands results in normal functional tissues. A mutant allele, Awh1, a spontaneous gain-of-function allele that initially defined the Awh locus, exhibits no defect other than a reduction in size for compound eyes. In Awh1 mutant eyes, genes properly expressed in the developing eye (such as hairy, decapentaplegic, scabrous and glass), fail to be expressed in patterns corresponding to compoud eye development. Since the compound eye develops normally in Awh mutant pharate adults, it is not believed that Awhplays a requisite role in retinal development. The phenotype of the neomorphic Awh1 mutation is likely the result of altered expression of Awh that interferes with retinal development. This phenotype resembles that derived from ectopic expression of Awh in imaginal discs (Curtiss, 1995 and Curtiss, 1997).

Other evidence points to a significant difference between excorporate and incorporate imaginal tissue. The two types of tissues exhibit different modes of cell proliferation control. string, a mitotic inducer that encodes a fly homolog of cdc25 phosphatases, is essential for the G2 phase to mitosis transition in the 14th cell cycle during embryogenesis. string is also essential for the generation of imaginal tissue. Clones of a strong mutant allele were generated in growing imaginal tissue. Normal bristle formation is prevented by induction of string clones, indicating that string is essential for the generation of the adult cuticle, the tissue generated by division of histoblast nests. String's role in cell cycle regulation is illustrated by the observation that ectopic expression of string in the imaginal discs of the thorax induces mitoses in G2-arrested cells. Ectopic String also induces premature and extra mitoses in the developing eye-antennal imaginal disc. In contrast to the ability of String to induce mitosis in imaginal discs, imaginal histoblasts prove to be refractory to ectopic String. These experiments suggest that in wandering third istar larvae, a factor or factors other than String are limiting for entry of abdominal histoblasts into mitosis This is the first example of G2 arrest during fly development that is not mediated by string transcriptional regulation (Kylsten, 1997).

It is concluded that Arrowhead expression is required for development of one set of imaginal cells, the incorporate cells that are embedded in larval tissue, and is incompatible with development of another, the excorporate cells that develop as imaginal discs. Future work could elucidate a mechanism whereby Arrowhead functions as an upstream regulator of cell cycle.

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 214

Structural Domains and Evolutionary Homologs

Awh has two LIM domains and a homeodomain. The LIM-2 domain of Awh differs from that of other members of the family in that the first and second cysteines are separated by three residues, rather than the usual two. The atypical spacing of these cysteines in Awh LIM-2 is not likely to affect the overall structure of the domain. The protein can be thought of as being divided into three parts. The LIM-1 domain stretches from amino acids 8 through 60; the LIM-2 domain from amino acids 69 through 122, and the homeodomain from amino acids 152 through 207. Awh has no identifiable close affinities to Islet or Apterous type LIM homeodomain proteins; outside the LIM- and homeo-domains, the protein contains no significant similarity to other proteins currently in the database (Curtiss, 1997).

The C. elegans AWA, AWB, and AWC olfactory neurons are each required for the recognition of a specific subset of volatile odorants. lim-4 mutants express an AWC reporter gene inappropriately in the AWB olfactory neurons and fail to appropriately express an AWB reporter gene. The AWB cells are morphologically transformed toward an AWC fate in lim-4 mutants, adopting cilia and axon morphologies characteristic of AWC. AWB function is also transformed in these mutants: Rather than mediating the repulsive behavioral responses appropriate for AWB, the AWB neurons mediate attractive responses, like AWC. LIM-4 is a predicted LIM homeobox gene that is expressed in AWB and a few other head neurons. Ectopic expression of LIM-4 in the AWC neuron pair is sufficient to force those cells to adopt an AWB fate. The AWA nuclear hormone receptor ODR-7 also represses AWC genes, as well as inducing AWA genes. It is proposed that the LIM-4 and ODR-7 transcription factors function to diversify C. elegans olfactory neuron identities, driving them from an AWC-like state into alternative fates (Sagasti, 1999).

date revised: 23 January 98

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