The Su(var)2-5 locus, an essential gene in Drosophila, encodes the heterochromatin-associated protein HP1. The Su(var)2-5 lethal period is late third instar. Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein. Heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in heterochromatin-mediated transcriptional regulation in these larvae. However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations. These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes (Lu, 2000).

Both the dominant and recessive phenotypes of mutations in HP1 were examined to look for an essential requirement for HP1 in development. It is proposed that reduced expression of one or more essential heterochromatic genes results in the recessive late larval lethality of Su(var)2-5. In support of this hypothesis, the essential heterochromatic genes rolled and light are misregulated in Su(var)2-5 mutants. rolled transcription at its normal chromosomal location is reduced in Su(var)2-5 mutant flies. Since no maternal Rolled protein is detectable in third instar larvae homozygous for rolled deficiencies, the RNA levels that are detected in mutant larvae and adults reflect zygotic gene expression. In the case of the heteroallelic mutant larvae, it should be emphasized that at the time the larvae were collected for Northern analysis, the Su(var)2-5 mutant larvae appeared healthy and would have lived on for several more days as third instar larvae before dying; indeed, a further decline in rolled RNA preceding larval death cannot be ruled out. Thus, reduced expression of rolled could contribute to the defects associated with loss of HP1. Of course, reduced expression of other heterochromatic genes probably also contributes to lethality due to HP1 deficiency (Lu, 2000 and references therein).

light also experiences variegated inactivation in Su(var)2-5 larval Malpighian tubules, and light transcripts are dramatically reduced overall in Su(var)2-5 mutant larvae. It is important to stress that the repressed light locus in these experiments is also in its normal chromosomal location. It is concluded that silencing of light in these experiments is a direct consequence of HP1 depletion, depriving the light locus of the heterochromatin context required for its normal expression. Several other genes reside in heterochromatin, and it will be interesting to see whether dependence on HP1 is a general attribute of gene expression in heterochromatin (Lu, 2000).

Mutations in rolled, like Su(var)2-5 mutations, lead to late larval or early pupal lethality with defective or missing imaginal discs. At the cytological level, rolled mutations cause defects in mitosis, including overcondensed and/or lagging anaphase chromosomes. Intriguingly, neuroblasts of larvae doubly mutant for hypomorphic alleles of rl and abnormal spindles (encodes a microtubule-associated protein) show telomeric stickiness and increased frequency of aneuploid mitotic figures. These phenotypes are also seen in neuroblasts of larvae heteroallelic for Su(var)2-5 mutations; indeed, the highest frequency of defects occurs in larvae heteroallelic for the Su(var)2-5²⁰⁵ allele, which is carried on a chromosome marked with a hypomorphic rl allele. Therefore, reduced expression of rolled caused by loss of HP1 could contribute to mitotic defects in HP1 mutant larval brains (Lu, 2000).

How can HP1 be required both for activation of heterochromatic genes and silencing of euchromatic genes? It has been proposed that certain heterochromatin-associated proteins function to support normal transcription of heterochromatic genes when those genes are at their normal chromosomal sites and that position effects result when heterochromatic genes are deprived of such essential heterochromatic proteins by displacement away from heterochromatin 'compartments' where such proteins are in high concentration. Such context-dependent regulatory activity has also been described for yeast RAP1 (repressor/activator protein 1); RAP1 is required for high-level expression of many ribosomal protein and glycolytic enzyme genes, but it promotes position-effect silencing at the HM silent mating type cassettes and telomeres. Genetic evidence suggests that RAP1 has distinct activator and silencing domains that could recruit or stabilize distinct chromosomal complexes at distinct chromosomal sites. Similarly, HP1 could interact with different proteins or protein complexes to promote silencing or activation in different chromosomal contexts. Another possibility is that HP1 may contribute to the formation of a particular chromatin structure that interferes with activation of euchromatic genes but to which heterochromatic genes have become adapted and dependent. Loss of HP1 would deplete the nucleus of this particular chromatin conformation, releasing silenced genes from repression while simultaneously depriving the resident heterochromatin genes of their functional context (Lu, 2000).

Line HS-2 of Drosophila, carrying a silenced transgene in the pericentric heterochromatin, was used to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is about 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing (Sun, 2001).

Over 30 genetic functions reside within D. melanogaster heterochromatin: those characterized require a heterochromatic environment for their proper expression, exhibiting a variegating phenotype or reduced expression when rearrangements place them adjacent to a breakpoint in euchromatin. Particularly striking is the observation that expression of the heterochromatic genes rolled and light in their endogenous heterochromatic position is reduced in larvae mutant for HP1, suggesting that proper maintenance of heterochromatin structure is required for expression of these genes. Thus, it was somewhat surprising to observe that light and rolled have nucleosome arrays similar to those observed for the euchromatic transgene, rather than the heterochromatic transgene. This suggests that the regulation of these heterochromatic genes by HP1 may not be based on the impact of HP1 on heterochromatin structure in general (which is correlated with silencing of transgenes such as hsp70-white) but may be the consequence of a context-dependent (positive or negative) activity, similar to that displayed by RAP1 in S. cerevisiae. Alternatively, the impact of HP1 on a heterochromatic gene may reflect packaging of the surrounding area, rather than the transcribed region. A more detailed analysis of the chromatin structure encompassing these genes and their regulatory regions will be required to resolve this question (Sun, 2001)

Many different intercellular signaling pathways are known but, for most, it is unclear whether they can generate oscillating cell behaviors. Time-lapse analysis of Drosophila embryogenesis has been used to show that oenocytes delaminate from the ectoderm in discrete bursts of three. This pulsatile process has a 1 hour period, occurs without cell division, and requires a localized EGF receptor (EGFR) response. High-threshold EGFR targets are sequentially activated in rings of three cells, prefiguring the temporal pattern of delamination. Surprisingly, widespread misexpression of the relevant activating ligand, Spitz, is compatible with robust delamination pulses. A single chordotonal organ precursor (called C1) and its progeny provide the source of secreted Spi relevant for oenocyte induction. Although Spitz ligand becomes limiting after only two pulses, artificially prolonging its secretion generates up to six additional cycles, revealing a rhythmic underlying mechanism. These findings illustrate how intercellular signaling and cell movements can generate multiple cycles of a cell behavior, despite individual cells experiencing only one cycle of receptor activation (Brodu, 2004).

The induction of larval oenocytes in Drosophila has been used as a simple model system for investigating the developmental regulation of EGFR signaling. Oenocytes are induced from the dorsal ectoderm of abdominal segments by a fixed and highly restricted source of Spi. This triggers a local EGFR response within a ring of overlying dorsal ectodermal cells, termed a whorl, leading to the upregulation of numerous oenocyte-specification genes and subsequent cell delamination. The simple cell geometry of the oenocyte whorl, together with time-lapse microscopy, was used to explore the timing of Spi secretion, EGFR-target activation, early cell induction, and later cell delamination. These studies reveal that oenocytes delaminate in bursts of three and identify the cell-counting mechanism as an EGFR-dependent pulse generator converting the time window of Spi secretion into final oenocyte number. This represents the first example of a rhythmic cell behavior other than the cell cycle to be reported in the Drosophila embryo (Brodu, 2004).
Rather than delaminating from the ectoderm in a continuous stream, oenocyte precursors segregate in discrete well-separated bursts of three cells. Genetic backgrounds affecting the pattern of cell segregation but not early fate specification were used to show how these pulses are regulated by EGFR signaling. The signaling parameters regulating the time of onset, time of cessation, and in particular, the cyclical nature of cell delamination of oenocytes are discussed (Brodu, 2004).

Using a panel of markers for double- and single-ring stages, it was possible to place gene expression 'snapshots' in temporal order with the cell movements recorded in movies. Three generic EGFR targets (activated Rolled/ERK, Yan, and argos) and three oenocyte-specific EGFR targets (Sal, svp^lacZ, and svp^lacZΔ18) were analyzed. In wild-type embryos, the high-threshold EGFR outputs of argos and svp^lacZ expression, detectable Rolled activation, and strong Yan downregulation are all inner ring specific, whereas lower-threshold outputs such as Sal upregulation and svp^lacZΔ18 expression are present in both precursor rings. Delamination itself also appears to be a high-threshold EGFR response and is thus confined to the inner ring (Brodu, 2004).