BIOLOGICAL OVERVIEW

The adult phenotype in which the wings are held out at right angles to the body was named eagle when it was initially characterized in 1930 by Thomas Hunt Morgan. eagle was cloned in a search for genes related to Drosophila knirps, a member of the hormone receptor superfamily (Rothe, 1989). Interest in the gene was heightened when it became apparent that it is expressed in neuroblasts, and current fascination stems from Eagle's involvement in the determination of serotonergic neurons. Serotonin is an evolutionarily conserved neurotransmitter, found in both invertebrates and vertebrates, and involved in locomotor and behavioral roles. Serotonin is produced in descendents of neuroblast NB 7-3. NB7-3 expresses several genes including engrailed, huckebein, seven-up, pdm1 and eagle. The pair of neurons expressing serotonin send their projections contralaterally (to the opposite side) through the posterior commisure. Since the development of NB 7-3 is understood in some detail, it is of interest to examine gene expression in this lineage. It has been found that Eagle is required in one of two serotonin positive neuroblast products of the NB 7-3 lineage, but not in the other (Higashijima, 1996 and Lundell, 1998 and references).

Before discussing the role of Eagle in promoting serotonin neuroblast fate, a short digression will be made to describe to biogenesis of neurotransmitters serotonin and dopamine and the identity of the cells that make these neurotransmitters. Dopamine is a derivative of the amino acid tyrosine. The enzyme tyrosine hydroxylase (TH) is required for production of dopamine and catalyzes the first step in dopamine biosynthesis. TH is expressed only in the midline dopamine cell and the DL-cells, lateral dopamine cells. Serotonin is a derivative of the amino acid tryptophan. There are two serotonin positive cells (7-3 lateral and 7- 3 medial) that are derived from the neuroblast 7-3 lineage. Dopamine decarboxylase (Ddc) catalyzes the last step in the biosynthesis of both dopamine and serotonin and a Ddc antibody will detect both serotonergic and dopamine-synthesizing cells (Lundell, 1994).

When eagle is ectopically expressed throughout the central nervous system, approximately 70% of the hemineuromeres contain 1 to 2 ectopic serotonin-expressing cells (es-cells). This experiment shows that eagle is competent to promote ectopic serotonergic differentiation. These es-cells show neuronal cell morphology and occur at a reproducible, dorsolateral position. This suggests that ectopic serotonin expression does not occur randomly but in specific CNS neurons. It also suggests that ectopic expression of Eg protein forces specific additional CNS cells to enter the serotonergic differentiation pathway. The position and number of es-cells suggested that they might be identical to the dorsolateral dopamine cells (DL-cells). Serotonin, DDC and TH expression were examined in the first larval instar CNS of animals producing es cells. The es cells were found to produce TH, unlike normal serotonergic cells which fail to produce TH. Althought the es cells were initially thought to be DL cells, especially because of their production of TH, upon careful observation this presumption failed to be confirmed and the origin of the es cells is unknown. The ectopic TH production is likely to be due, however, to the presence of ectopic Eagle in the es cells (Dittrich, 1996).

At late embryonic stages, the serotonergic neurons are the only cells expressing en and hkb simultaneously, suggesting that these genes act in combination to determine the serotonergic cell fate. Loss-of-function mutations in these genes lead to loss of serotonin immunoreactivity (Lundell, 1996). Thus, it makes sense to investigate the functional relationship between en, hkb and eg in order to clarify the serotonergic differentiation pathway within the NB 7-3 lineage. To examine the functional relationship between eg and en loss-of-function en mutant flies were stained with the anti-Eg antiserum. In about 90% of mutant hemineuromeres, a complete loss of eg expression is observed at the position of the NB 7-3 derived neurons. This is in agreement with results obtained by Matsuzaki (1996), who found a loss of Eg expression in 81% of hemineuromeres. In the remaining hemineuromeres, eg expression is found in both lateral and medial NB 7-3 progeny. en function in NB 7-3 development must be upstream of eg and is therefore necessary for correct eg expression within this lineage (Dittrich, 1997).

Another transcription factor important for NB 7-3 development is huckebein. In hkb mutants, serotonin differentiation is severely disturbed (Lundell, 1996). In hkb mutants, eg expression is observed in all NB 7-3 progeny, both lateral and medial, in approximately 80% of mutant hemineuromeres. A complete loss of eg expression at the position of NB 7-3 neurons occurs in only 20% of mutant hemisegments. This loss of eg expression cannot exclusively account for the observed serotonin phenotype in hkb mutants, since earlier studies reveal a loss of 80% of the ventral ganglion serotonin cells in hypomorphic hkb mutants (Lundell, 1996). Thus, the majority of 7-3 Iateral cells in hkb mutant embryos express eg but fail to exhibit serotonin expression (Dittrich, 1997).

To investigate whether a combinatorial function of eg and hkb is generally necessary to promote serotonergic differentiation, ectopic eg expression was induced in a hkb-mutant background. This was designed to test whether the differentiation of es-neurons is also dependent on the function of hkb. Approximately 80% of the wild-type and ectopic serotonin cells are absent in the hkb mutant. This finding strongly suggests that the ectopic serotonergic neurons are generated by a hkb-positive NB and implies that the Eg and Hkb transcription factors are part of a combinatorial code made up of regulatory proteins necessary for promoting the serotonergic cell fate (Dittrich, 1997).

Although eg is expressed in both lateral and medial NB 7-3 derived serotonin cells, the eg loss-of-function mutants often affect the development of only one serotonin cell from each pair. The two cells can be distinguished from one another by differential expression of Zn finger homeodomain 2 (zfh-2). This dual domain transcription factor has been shown to bind to and activate the DDC gene (Lundell, 1992 and 1994). The differential expression of zfh-2 and of another gene, pdm-1, can be used to determine that the remaining serotonin positive single cell in eg mutants expresses markers characteristic of the more lateral serotonin cell. In a wild-type CNS, both zfh-2 and pdm1 are selectively expressed in the more lateral serotonin cell but not in the more medial cell. engrailed and eagle are expressed in both these serotonin cells. In eg mutants only the medial cell consistently fails to become a serotonin cell. Therefore, even though eg is normally expressed in both serotonin cells, the absence of Eg protein has a more dramatic effect on the fate of the more medial neuron. This important observation suggests that the lateral serotonin cell can maintain its fate in the absence of Eg (Lundell, 1998).

Analysis of gene expression in eg mutants shows that expression of zfh-2 and en is dependent on eg function but expression of pdm1 is independent of eg function. Loss of eg function appears to have no affect on the expression of pdm1. Clearly, the serotonin cell phenotype in eg mutants is not directly related to the expression of pdm1. Loss of eg function affects the expression of zfh-2 in the lateral serotonin neuron. Loss of eg function affects the expression of en in both serotonin neurons. Thus, eagle is necessary for the maintenance of both engrailed and zfh-2 expression in the serotonin neurons (Lundell, 1998).

The simplest explanation for the difference between the medial and lateral serotonin neurons is that the lateral cell contains a redundant mechanism that allows continued synthesis of serotonin in the absence of Eg protein. This redundant mechanism is not 100% efficient, since not all segments in an eg mutant CNS contain serotonin cells. Since zfh-2 but not pdm1, expression is affected in eg mutants, it is suggested that zfh-1 is a potential factor for this redundant pathway, which establishes eg-independent serotonin synthesis. In an eg-loss-of-function mutant, the loss of Ddc expression is always accompanied by the loss of en expression, but can occur independently in the two serotonin cells. In a hemisegment where both cells fail to express Ddc, neither cell shows en expression. In a hemisegment where only the lateral serotonin cells continues to express Ddc, this lateral cell shows en expression but the medial cell does not. It is concluded that the two serotonin cells have distinctive regulatory networks. In the medial cell, eagle is required for the serotonin fate, while in the lateral cell, engrailed and zhf-1 are required but eagle is not. It is shown that hypomorphic alleles of eagle can produce viable adults that have a dramatic reduction in the number of serotonin-producing neurons (Lundell, 1998).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 373

Structural Domains

Nucleotide sequence indicates an open reading frame encoding a polypeptide of 373 amino acids, with a 1421-nucleotide intron between codons 26 and 27. The first 90 amino acids, which contain the zinc-finger domain, show 80% and 86% similarity to the products of kni and knrl, respectively; the three proteins share a 19-amino-acid motif, the kni box, just downstream from the zinc-finger domain. These polypeptides share features with vertebrate steroid hormone receptors; their putative ligand-binding domains exhibit a low level of similarity (Rothe, 1989).

date revised: 5 April 98

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