BIOLOGICAL OVERVIEW

tailless and huckebein are known as terminal gap genes, due to their expression in both anterior and posterior ends (or terminals) of the egg. huckebein's efforts to regulate transcription are both positive and negative. As a repressor, HKB assures that the formation of mesoderm (by ventral invagination of the presumptive mesoderm) does not spread to the two poles of the egg. In hkb mutants, invagination is not normal, spreading both to the anterior and posterior beyond the usual limits.

The positive regulatory action of HKB results in the formation of proper endoderm, developed from invaginating gut primordia at either end of the egg. Gut invagination doesn't proceed normally in hkb mutants. Germ cells usually migrate through the gut to take up a position in the mesoderm. As a gap gene, hkb is also involved in regulating wingless and engrailed in the head, assuring proper subdivision of head somite compartments [Images].

Engrailed and Huckebein are essential for development of serotonin neurons in the Drosophila CNS. en and hkb coexpress uniquely in the serotonin neurons and in neuroblast 7-3 (NB7-3). In the grasshopper, the analogous serotonin neurons originate from the first ganglion mother cell produced from NB7-3. The corresponding NB7-3 in Drosophila can be identified by its time of birth, size, and relative position within each hemisegment. The serotonin neurons can be identified during late embryogenesis by the appearance of DOPA decarboxylase (DDC) immunoreactivity. The DDC enzyme catalyzes the last step in the biosynthesis of serotonin and dopamine and can be used as a marker for both cell types. In the ventral ganglion there are three anatomically distinguished types of DDC immunoreactive cells per segment, a pair of ventrolateral serotonin cells (VL), a single midline dopamine cell (M) and the dorsal lateral (DL) dopamine cells. en and hkb are coexpressed in the VL cells but not the DL or M cells. The high selectivity of coexpression of these two gene products suggests that their combined activities may be important for the development of NB7-3 progeny. Serotonin neuron differentiation is abnormal in en and hkb mutants. Although neither mutant shows a complete loss of DDC immunoreactive serotonin cells, the few escaper serotonin neurons may be due to low levels of functional hkb gene product in a hypomorphic allele. Since NB 7-3 appears normal in hkb mutants, the effect of hkb on development of the serotonin cell lineage must be at a later stage of development, either at division of the neuroblast or ganglion mother cells or on the identity of the GMC progeny (Lundell, 1996). For more information on serotonin and dopamine neurons see Islet and Zn finger homeodomain 1).

The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. Groucho has been implicated in Drosophila embryonic terminal patterning: it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. An additional requirement for Groucho in this developmental process has been revealed by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, a functional repressor domain in Huckebein that has been identified contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebeinís FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Thus Groucho-repression regulates sequential aspects of terminal patterning in Drosophila (Goldstein, 1999).

One proposed Hkb target is the snail (sna) gene, which is transcribed in the ventral-most portion of the embryo. sna expression is thought to be excluded from the posterior pole by hkb activity. Accordingly, sna and hkb expression domains abut in cellularizing wild-type embryos, whereas sna expression extends to, and includes, the posterior pole of hkb mutant embryos. In tor^D embryos, hkb expression expands towards the center of the embryo and the sna domain correspondingly retracts. By contrast, in gro^mat- embryos, the expression of sna does not respect the sna posterior border and spreads to the pole, overlapping extensively with the hkb expression domain. The expression of the T-related gene brachyenteron (byn; also called Trg) also seems to be repressed by Hkb. byn is not expressed at the most posterior region of wild-type (or tor^D) embryos, whereas it extends throughout the posterior cap of hkb mutant embryos, consistent with hkb setting the posterior limit of byn expression. However, it is found that byn is ectopically expressed at the posterior tip of gro^mat- embryos. Together, these results suggest that gro is, directly or indirectly, necessary for hkb repressor functions (Goldstein, 1999).

To establish whether Hkb can function as a repressor, a Hairy^Hkb chimera was constructed by replacing the C terminus of Hairy with Hkbís N-terminal 195 amino acids (lacking the Hkb Sp1-like zinc-finger DNA-binding domain). When expressed under the regulation of the hb promoter, the Hairy^Hkb chimera causes effective repression of Sxl (normally a target of Hairy) in the anterior region of female embryos. Furthermore, this repression also causes female-specific lethality, probably due to the role of Sxl in dosage compensation. These results indicate that Hkb contains a potent repression domain within its N-terminal 195 aminoacids (Goldstein, 1999).

Hkb also behaves genetically as a positive regulator of forkhead (fkh) and serpent (srp) expression. In hkb mutant embryos, the posterior fkh domain is smaller than in wild-type embryos and srp expression at the poles is not initiated. Perhaps Hkb functions as an activator of fkh and srp expression that, when associated with Gro, represses other target genes. Arguing against this possibility, there is no direct evidence that Hkb contains an activation domain. For example, it does not promote activation of reporter genes when introduced into yeast cells. Additionally, the Hairy Hkb chimera containing the N-terminal 195 residues of Hkb does not cause activation of Sxl in male embryos, whereas this does occur in a Hairy fusion containing the viral VP16 activation domain. These results suggest that Hkb regulates fkh and srp transcription indirectly, possibly by repressing a repressor of these genes (Goldstein, 1999 and references).

GENE STRUCTURE

Exons - two

Bases in 3' UTR - 518

PROTEIN STRUCTURE

Amino Acids - 296

Structural Domains

Huckebein encodes a triple zinc finger protein containing a glutamine rich activation domain and an alanine rich repressor domain (Bronner, 1994).

date revised: 5 August 97

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