BIOLOGICAL OVERVIEW

There are three GATA homologs in Drosophila: Serpent (dGATAb), found in the midgut and ovary (Lossky, 1995), Pannier (GATA-2), found in the dorsal epidermis, and dGATAc, found in the procephalic [Image] region, posterior spiracles, gut, and central nervous system.

Pannier acts as a local repressor of achaete and scute and is required for the normal pattern of sensory bristles in those parts of the epithelium where it is expressed. pannier mutation may take one of two forms: either an over- or under-expression of achaete-scute. Consequently the phenotype shows either an overabundance or a lack of bristles.

Studies suggest that Pannier's action on achaete-scute is negative. In this view, mutants with too few bristles would be due to a hyperactive enzyme, resulting in accentuated repressive activity. These mutations are dominant, affecting the primary structure of Pannier and suggest that mutant Pannier protein acts as a non-functional heterodimer with another transcription factor (Ramain, 1993).

Two other proteins act as repressors of achaete-scute: hairy and extramachrochaete. Loss of these two functions also results in overexpression of achaete-scute and ectopic bristles.

To identify genes involved in the patterning of adult structures, Gal4-UAS (upstream activating site) technology was used to visualize patterns of gene expression directly in living flies. The gene yellow (y) was made sensitive to Gal4 control and a Gal4-containing P element was inserted randomly into the fly genome. A large number of Gal4 insertion lines were generated and their expression patterns studied. In addition to identifying several characterized developmental genes, the approach revealed previously unsuspected genetic subdivisions of the thorax, which may control the disposition of pattern elements. For example, the pannier expression domain marks a dorsal band along the length of the body, from the occipital head region to the end of the abdomen but excluding the terminalia. In the notum (derived from the wing imaginal disc), the gene labels a territory extending from the dorsal midline laterally to a longitudinal (anterior to posterior) straight line defined by the dorsocentral bristles. Another insertion em462 shows y+ rescue in a territory adjacent to the pannier domain; it is also demarcated medially by the position of the dorsocentral bristles. The em462 domain extends laterally but does not reach the more lateral region of the notum. Possibly, the gene iroquois defines a distinct, more lateral domain.

The subdivision of the notum demarcated by the dorsocentral bristles may be significant because this same line also appears to demarcate the most medial expression border of the wingless gene. In the notum, wingless is expressed in a narrow stripe and the wingless domain is included within the em462 domain, extending laterally from the dorsocentral bristles. Apparently there is no overlap between pnr and wg in the scutum (the anterior part of the notum), but they do overlap in the more posterior scutellum. Genetic interaction experiments show that pannier acts as a negative regulator of wingless in the notum and suggest that some of the effects of pannier mutants are produced through an alteration of wingless function. Interestingly, the dorsocentral line is known not to function as a cell lineage border. In addition to bristles, another frequent pattern element in insects is pigmentation, often disposed in longitudial bands and used as a diagnostic criterion for the taxonomy of dipteran species. The medial boundary of a diagnostic pigment band is exactly delimited by the same longitudinal line straddling the dorsocentral bristles that in the Drosophila demarcates pannier, wingless and em462 (Calleja, 1996).

The pannier gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. Novel aspects of the regulation and function of pnr during embryogenesis are reported in this study. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but it has been found that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila (Herranz, 2001).

In early development, pnr is activated in response to dpp activity in a broad dorsal domain, which extends from parasegments 2/3 to the border between 13/14, although the borders are not strictly parasegmental. The control by dpp is consistent with the effect of brk mutations on early pnr expression. The original expression domain is substantially modified during embryogenesis. By germ band extension (stage 10) pnr activity is limited dorsally by the border between the epidermis and the amnioserosa, and laterally by the dorsal border of iro. It is not known which factor(s) is responsible for the loss of expression in the amnioserosa, although likely candidates are several genes specifically active in this region, such as Race, zen, hindsight or serpent. In addition, it is not known how the late expression is regulated at the lateral border. It is not achieved by iro, since the loss of the entire Iroquois complex does not affect pnr expression (Herranz, 2001).

Another modification occurs between stages 10 and 11, and is the loss of expression in the A8 segment. Expectedly, it is under the control of Abd-B; in Abd-B mutants the gap in A8 does not appear. However, none of the known Abd-B target genes (sal, ems and grn) is involved in the regulation, since their mutations do not affect pnr expression. The finding that lin, which is considered as a co-factor of Abd-B, is involved, suggests that downregulation of pnr in the A8 segment is mediated either by an unknown Abd-B target or directly by interaction between the Abd-B and Lin products. It is not clear why pnr activity has to be eliminated precisely in the A8 segment. This segment gives rise to the spiracles, protruding structures that are very different from those differentiated by the other abdominal segments where pnr remains active. In fact, there are several Abd-B target genes specifically activated in the spiracles. It is possible that the formation of these structures demands that the pnr activity, which specifies larval epidermis of very different morphology, be turned off (Herranz, 2001).

Interestingly, whereas early pnr expression is under dpp control, the late expression is not. Late inactivation of the Dpp pathway, using a dominant negative form of thick veins, does not modify pnr expression. In addition, mutations at brk, which allow higher response levels to Dpp signaling fail to affect pnr expression in late development, although they affect early expression. This indicates that pnr expression is controlled independently in early and late development, and by different factors (Herranz, 2001).

There is already evidence that pnr has distinct functions during embryogenesis. Its activity in the dorsal epidermis is required for dorsal closure and it is also expressed in the dorsal mesoderm where it is involved in the specification of cardiac cells. Evidence is provided for another and more general function of pnr; it specifies the identity of a dorsomedial body region that spans from the labial segment to the end of the abdomen. This is clearly demonstrated by the effects seen in mutant embryos and after ectopic expression experiments. In pnr^VX6 embryos, the dorsomedial cuticle does not form, and there is an expansion of the dorsolateral epidermis, suggesting that the cells of the dorsomedial domain acquire a dorsolateral fate. The ectopic expression experiments also point to the same conclusion. In larvae ectopically expressing pnr (arm-Gal4/UAS-pnr) the entire larval epidermis acquires dorsomedial features, whereas using more restricted drivers (Ubx-Gal4, wg-Gal4) the transformation is limited to the region where the Pnr protein is present, suggesting that the effect of pnr is cell autonomous. Thus, the Pnr protein is able by itself to trigger a developmental pathway, a typical property of selector gene products. In addition, it induces a ventral to dorsal transformation, corresponding to each segment, indicating that it acts in combination with Hox genes. These observations indicate that selector genes in the AP and DV axes have to co-operate to determine the different spatial patterns (Herranz, 2001).

The transformation of ventral and dorsolateral epidermis towards dorsomedial observed after ectopic pnr expression is also reflected in the activity of marker genes of the distinct regions. Characteristic genes of the ventral neuroectoderm such as BP102 for the CNS or buttonhead are suppressed. In addition, pnr is able to suppress iro activity, a property that, as in the adult cells, is important to keep the dorsomedial and dorsolateral domains separate during embryogenesis (Herranz, 2001).

The developmental effects observed after either the loss or gain of pnr function in the larval epidermis resemble those reported for the adult cuticle. In the latter, it has been shown that the activity of pnr maintains the segregation of the dorsal cuticle into medial and lateral domains, and also specifies the identity of a medial domain. This indicates that pnr has a general function involved in the subdivision of the body along the DV axis. The longitudinal stripe of pnr expression established during embryogenesis is probably a major constituent of the body and represents a zone of common identity (Herranz, 2001).

In addition, Pnr has other more concrete functions connected with the specification of cardiac cells and embryonic dorsal closure. The involvement of pnr in dorsal closure is exerted through its activation of dpp in late embryogenesis, which is responsible for the formation of the Dpp stripe at the junction of the epidermis with the amnioserosa. Normal functioning of the Dpp pathway in this region is required for dorsal closure, suggesting that defects in dorsal closure observed in pnr mutant embryos is the result of the lack of the dorsal dpp stripe (Heranz, 2001).

There is evidence that this dpp expression requires function of the JNK kinase pathway, and it also requires pnr activity. The observation that in the absence of pnr activity the expression of puc, the end element of the JNK pathway is normal, indicates that in pnr mutants the JNK pathway is normally active. In turn, it shows that the activation of dpp in the dorsal stripe requires independent inputs from both the JNK pathway and pnr (Herranz, 2001).

One intriguing aspect of pnr function is that it is able to induce a developmental modification in all ectodermal structures along the DV body axis except in the amnioserosa, the most dorsal tissue. Even under conditions in which pnr is transcribed and translated in all the amnioserosa cells, it does not appear to elicit any developmental effect; none of the amnioserosa marker genes is affected by forcing pnr activity and the retraction of the germ band [a morphological indicator of the function of specific amnioserosa genes is also normal. Similarly, pnr is able to induce dpp activity all over the body except in the amnioserosa, where the presence of the Pnr protein appears to be inconsequential. This situation resembles the phenotypic suppression/posterior prevalence phenomenon discovered in the Hox genes specifying the AP body axis. It consists of a functional inactivation of a Hox protein by the presence of another normally expressed in a more posterior region of the body. It is conceivable that there might be a ‘dorsal prevalence’ in the DV axis, by which dorsal expressing genes are functionally dominant over the ventral expressing ones. It would be expected that genes specifying amnioserosa would be able to transform all structures since they would be ranking highest in the functional hierarchy (Herranz, 2001).

GENE STRUCTURE

Bases in 5' UTR - 689

Exons - four

Bases in 3'UTR - 688

PROTEIN STRUCTURE

Structural Domains

Pannier has two zinc fingers, a polyglutamine stretch and two helicies rich in hydrophobic amino acids (Ramain, 1993 and Winick, 1993).

date revised: 20 December 99

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