pitchoune

Transcriptional Regulation

The almost completely superimposable pit and dmyc expression patterns as well as the similarities existing between the Pit sequence and that of MrDb, a target of mammalian c-myc strongly support the hypothesis that Drosophila pit might also be a target for the transcriptional factor d-Myc. d-Myc is encoded by the diminutive locus (Gallant, 1996; Schreiber-Agus, 1997). The expression of pit is not, however, noticeably affected in ovaries of females homozygous for the hypomorphic allele of diminutive: dm1. This result might indicate that dmyc is not required for the expression of pit. However, the low level of d-Myc in the mutants might be sufficient to promote high enough levels of pit, leading to an apparently normal expression. In the same line, no difference in the embryonic expression of pit is observed in dm1 homozygous mutants. As is the case for pit, dmyc is maternally expressed; it is not known whether the maternal protein is stable throughout embryogenesis. As yet, no complete loss-of-function allele of diminutive is known. Attempts to demonstrate a possible interaction between pit and d-myc have therefore been turned toward an ectopic d-myc expression by using a UAS-d-myc cDNA driven by a variety of tissue-specific GAL4-expressing lines. Since d-myc RNA is present neither in the nervous system nor in differentiating muscles, the 1407 and 24B lines were used: these lines, respectively, express GAL4 in the central and peripheral nervous system and in all muscles. pit is expressed in the central nervous system in embryos derived from the 1407 GAL4 line, suggesting that d-Myc can behave, at least in that tissue, as a transcriptional activator of pit. In contrast, no evident ectopic expression of pit could be demonstrated in muscle precursors when the d-myc driver was 24B. There are several likely reasons for a lack of induction of pit in muscle. For example, the Myc protein is known to dimerize with Max to make a heterodimer that activates transcription. d-Max expression in muscle has not been clearly established (Gallant, 1996) and a too low concentration in this tissue might impair the transcriptional activation of the Myc targets. In conclusion, these results strongly support the hypothesis that pit is a target for Myc transcriptional activation. Of course, it is not possible to anticipate from these experiments whether or not pit is a direct target of d-Myc (Zaffran, 1998).

DEVELOPMENTAL BIOLOGY

Embryonic

Northern blot analysis has shown that PIT mRNA is present both in preblastoderm (0-2 hour) and 8-12 hour embryos, indicating a maternal as well as a zygotic expression. The PIT transcript is uniformly distributed in preblastoderm embryos, also suggesting a maternal expression. This was confirmed by in situ hybridization on ovaries in which pit is ubiquitously expressed in late egg chambers. The transcript reproducibly displays only very low, if any, expression in stage 2 egg chambers. At gastrulation, pit is expressed in the invaginating mesoderm. At early stage 11, pit expression is visible in the mesoderm (from which it rapidly disappears during germband extension), in the anterior and posterior precursors of the midgut and in the precursors of the salivary glands in parasegment 2. At this stage, all these territories are mitotically active and are programmed to enter into endoreplicative cycles. A strong expression is also observed in the anal plate. In late stages, expression becomes restricted to the differentiating midgut, the Malpighian tubules and the mesodermal sheath of gonads. However, this late expression is at a much lower level than that observed in the anterior and posterior midgut from stage 10-13 embryos. Weak expression is also detectable in the epidermis at the end of embryogenesis. Finally, pit is ubiquitously expressed in the imaginal discs. Overall during development, but especially in the early stages following gastrulation, pit expression closely parallels that of dmyc (Gallant, 1996). This is also true in oogenesis, during which dmyc is present in large amounts except in stage 2 egg chambers (Zaffran, 1998).

Polyclonal antibodies directed against the C-terminal half of Pitchoune do not resolve its subcellular localization. To that end, Pit was c-Myc-tagged at its N terminus and this cDNA was expressed from heat shock promoter. Transfected S2 Drosophila cells were heat shocked and processed for immunodetection. The tagged protein was detected with anti-c-Myc antibodies and the nucleolus was detected by an AJ1 signal using anti-AJ1 antibodies. The complete overlapping of the expression of c-Myc-Pit and AJ1 suggests a nucleolar localization for Pit. The predominant distribution of c-Myc-Pit within the nucleolus, in the specialized compartment for ribosome synthesis, could point toward its participation in rRNA maturation or in another step in the biogenesis of ribosomal subunits. Since the function of Pit seemed to be required in the nucleolus, an eventual perturbation of this organelle was investigated in pit¹⁰ mutant larvae. As a marker for the nucleolus, an antibody directed against the Modulo protein was chosen, rather than AJ1, because Modulo exhibits a differential localization within the nucleolus as a function of the state of replication of the nucleus. Modulo is perinucleolar in the polyploid nuclei of the principal midgut epithelial cells whereas in diploid cells, which are able to divide, the expression of Modulo covers the whole volume of the nucleolus. This differential localization is not affected in pit mutant midgut cells, suggesting that the nucleolus is normal in the mutant with no alteration in its overall structure, at least as judged by the criterion that has been used (Zaffran, 1998).

Effects of Mutation or Deletion

The larvae of holometabolous insects are composed of two types of tissues: the larval cells, which do not proliferate but grow by enlargement and polytenization, and the imaginal cells, consisting of diploid cells that have a very high proliferative activity and that give rise to most of the adult structures. Histological examination of various tissues taken from 5-day-old pit¹⁰ mutant larvae (compared to third instar wild-type larvae of the same age) reveals profound alterations in the growth of larval tissues and imaginal progenitor cells. For instance, midgut progenitor cells or salivary glands can still be recognized but they do not significantly increase in size. Their total number remains identical to that present in wild-type larvae and they are been subjected to (at most) 1-2 cycles of DNA endoreplication. The imaginal cells and, in general, the adult precursor cells (which are diploid and divide during larval stages) do not proliferate in a pit¹⁰ mutant. Similarly, the imaginal discs are the same size as those in young first instar larvae, suggesting a lack of proliferation in this tissue as well. The same types of modifications probably prevail in other mutant tissues, especially in the epidermis, since the larvae remain small although perfectly shaped and identical to wild-type first instar larvae. Finally, the mutant larvae do not incorporate BrdU in the nuclei of their cells, indicating a failure in DNA replication. In conclusion, the loss of function of pit seems to lead to a general arrest in cell growth of larval cells and in cell proliferation of adult precursor cells in a precisely coordinated manner (Zaffran, 1998).

pit is strongly expressed during oogenesis and a maternal contribution to its expression during embryogenesis might explain an apparent lack of embryonic pit function. In order to investigate this issue, germline clones free of pit were generated. Oogenesis in pit- ovaries never proceeds through stage 6. This result is interpreted as an early requirement for pit activity in the germline during oogenesis. However, due to the incapacity of the females to lay eggs, pit¹⁰ embryos devoid of maternal contribution could not be observed (Zaffran, 1998).

Somatic homozygous mutant pit- clones, which are recognized by their hair and bristles phenotypes, were produced with the FLP/FRT technique. Clones are yellow and do not carry the Sb⁶³ marker (short and thick hair) in an otherwise yellow + and Sb⁶³ environment. Only very small clones carrying the associated yellow marker are observed and only when the recombinase is induced late in development (end of third instar larval stage). These clones are in consequence easily distinguished among the thorax chaetes, being thinner and even smaller than the surrounding Sb⁶³ chaetes. This is reminiscent of the phenotype encountered in the case of Minute and also of diminutive mutations, which both affect cell growth and proliferation. Under the same conditions, large wild-type clones with long hair, lacking the Sb⁶³ mutation are obtained in a control cross. The cells within homozygous mutant clones are also observed in imaginal wing discs of late third instar larvae. Here again the mutant clones, which are apparent because they do not possess the clonal cellular marker myc (a short sequence of c-Myc recognized by the 9E10 antibody), are very small and composed of only a few cells. They are at least two orders of magnitude smaller than wild-type clones, which are easily recognized due to the important staining of the myc marker. All these observations certainly illustrate the poor ability of pit¹⁰ mutant cells to grow and proliferate and also suggest that mutant cells can be overtaken by their wild-type sister cells and therefore eliminated and replaced as it has been previously shown in the case of Minute mutant cells. As a matter of fact, when homozygous pit¹⁰ clones are induced in Minute flies, large mutant clones are obtained indicating that the pit¹⁰ mutation is not cell lethal and does not directly interfere with the cell cycle machinery, but rather autonomously affects cell growth (Zaffran, 1998).

pit overexpression into the posterior compartment of otherwise wild-type imaginal discs was analyzed in larvae resulting from a cross between UAS-pit flies and engrailed-GAL4 flies. Larval development was allowed to proceed at 29°C. An increase in the number of mitoses, as judged from the expression of Phosphohistone H3 is repeatedly observed and is accompanied by a higher percentage of cell death. The number of mitoses in the posterior compartment is, however, never greater than 2 to 3 times that in the anterior compartment. Similar results are obtained by incorporating BrdU in living third instar larvae. Due to the high replicative activity prevailing in the whole disc at this developmental stage, the increase in the number of replicating nuclei in the posterior compartment is somewhat smaller than in the previous estimate but is, nevertheless significant. In rare instances (in a small percentage of the examined discs), a clear hyperproliferative phenotype specifically affecting the posterior compartment is observed. A normal proportion of adults emerge from larvae that have overexpressed pit and they do not present any evident mutant phenotype, with the reservation that a few individuals might have died and thus escaped scrutiny. This result is consistent with the idea that pit overexpression does not induce a permanent hyperproliferative phenotype in the imaginal discs and that some kind of compensatory mechanism (cell death etc.) may have been at work in this tissue (Zaffran, 1998).

The B1-93F line (also called B1-3-12) has a P-element insertion in the 93F region located in close proximity to pit. B1-93F is homozygous viable and no differences are observed when the pattern of expression of pit is compared in wild-type embryos and in homozygous B1-93F embryos. A P-element mobilization screen with B1-93F led to two complementation groups of lethal mutations. A mutation in one of these complementation groups, pit¹⁰, results from a small deletion of a 3.5 kb long genomic region starting from the initial site of insertion and extending toward the pit transcription unit. A part of the transposon, including the lacZ gene, still resides at the site of insertion. The proximal breakpoint falls within the second exon of pit leading to an almost complete deletion of the helicase domain. In contrast, no gross molecular alterations could be observed in the held out wings gene, which is in close proximity (Zaffran, 1997). The mutant alleles that specifically modify the how function are able to complement the pit¹⁰ mutation. Because this latter mutation removes most of the helicase domain, it is considered to be a loss-of-function allele and probably even a null mutation. Genetically, the phenotype of pit¹⁰ in trans to the deficiency Df(3R)e-BS2 is indistinguishable from that of homozygous pit¹⁰ animals. The pattern of the cDNA expression is not altered in pit¹⁰ homozygous embryos nor in embryos bearing pit¹⁰ in trans to the deficiency Df(3R)e-BS2, suggesting the presence of a truncated mRNA unable to produce a functional protein. Two other mutations in the pit gene were obtained by mobilization of the B1-93F P-transposon. pit⁴ in trans to pit¹⁰ produces the same phenotype as homozygous pit¹⁰ animals. Contrary to pit¹⁰, this mutation does not complement loss-of-function mutations of how (Zaffran, 1997). Finally, homozygous pit⁵ animals are completely viable but are lethal in trans to Df(3R)e-BS2 or to pit¹⁰. In this latter case, however, larvae developed normally but with a delay and, although they eventually are able to pupariate, they never produce adults. Homozygous pit¹⁰ animals display important growth defects. Larvae hatch normally and initially show a healthy behaviour, although with a constant delay in their timetable, when compared to wild-type larvae. In a pit¹⁰/+ cross, 75% of embryos hatch 24±2 hours after egg laying while 25% of the embryos that correspond to homozygous pit¹⁰ embryos hatch later with a delay of 7±4 hours. The pit¹⁰ mutants fail to grow beyond the first instar larval stage and they never accomplish their metamorphosis. Heterozygous larvae as well as wild-type controls continue their growth throughout each of the larval stages and develop normally. In contrast, the mutants are arrested at the first larval stage, although they can survive longer than 7 days. The mutant larvae appear normal and all of the tissues that could be examined have a wild-type morphology indistinguishable from that of a first instar wild-type larva (Zaffran, 1998).

Eberl, D. F., et al. (1997). A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle. Genetics 146(3):951-63.

Gallant, P., et al. (1996). Myc and Max homologs in Drosophila. Science 274: 1523-1526.

Grandori, C., et al. (1996). Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo. EMBO J. 15(16): 4344-57.

Schreiber-Argus, N., Stein, D., Chen, K., Goltz, J. S., Stevens, L. and DePinho, R. A. (1997). Drosophila Myc is oncogenic in mammalian cells and plays a role in the diminutive phenotype. Proc. Natl. Acad. Sci. USA 94, 1235-1240.

Zaffran, S., et al. (1997). The held out wings (how) Drosophila gene encodes a putative RNA-binding protein involved in the control of muscular and cardiac activity. Development 124: 2087-2098.

Zaffran, S., et al. (1998). A Drosophila RNA helicase gene, pitchoune, is required for cell growth and proliferation and is a potential target of d-Myc. Development 125(18): 3571-3584.

date revised: 4 November 98

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