Neel B. Randsholt

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.

polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs.

Polycomb group (PcG) genes maintain cell identities during development in insects and mammals and their products are required in many developmental pathways. These include limb morphogenesis in Drosophila melanogaster, since PcG genes interact with identity and pattern specifying genes in imaginal discs and clones of polyhomeotic (ph) null cells induce abnormal limb patterning. Such clones are associated with ectopic expression of engrailed, hedgehog, patched, cubitus interruptus and decapentaplegic, in a compartment specific manner. Our results also reveal negative engrailed regulation by ph in both disc compartments: ph silences engrailed in anterior cells and maintains the level of engrailed expression in posterior ones. We suggest that PcG targets are not exclusively regulated by an on/off mechanism, but that the PcG also exerts negative transcriptional control on active genes.

How Drosophila change their combs: the Hox gene Sex combs reduced and sex comb variation among Sophophora species

SUMMARY Identification of the events responsible for rapid morphological variation during evolution can help understand how developmental processes are changed by genetic modifications and thus produce diverse body features and shapes. Sex combs, a sexually dimorphic structure, show considerable variation in morphology and numbers among males from related species of Sophophora, a subgenus of Drosophila. To address which evolutionary changes in developmental processes underlie this diversity, we first analyzed the genetic network that controls morphogenesis of a single sex comb in the model D. melanogaster. We show that it depends on positive and negative regulatory inputs from proximo‐distal identity specifying genes, including dachshund, bric à brac, and sex combs distal. All contribute to spatial regulation of the Hox gene Sex combs reduced (Scr), which is crucial for comb formation. We next analyzed the expression of these genes in sexually dimorphic species with different comb numbers. Only Scr shows considerable expression plasticity, which is correlated with comb number variation in these species. We suggest that differences in comb numbers reflect changes of Scr expression in tarsus primordia, and discuss how initial comb formation could have occurred in an ancestral Sophophora fly following regulatory modifications of developmental programs both parallel to and downstream of Scr.

Drosophila melanogaster Cyclin G coordinates cell growth and cell proliferation

Mammalian Cyclins G1 and G2 are unconventional cyclins whose role in regulating the cell cycle is ambiguous. Cyclin G1 promotes G2/M cell cycle arrest in response to DNA damage whereas ectopic expression of CCNG2, that encodes Cyclin G2, induces G1/S cell cycle arrest. The only Drosophila Cyclin G was previously shown to be a transcriptional regulator that interacts with the chromatin factor Corto and controls expression of the homeotic gene Abdominal B. It is very close to mammalian Cyclin G1 and G2 except in its N-terminal region, that interacts with Corto, and that seems to have been acquired in dipterans. Ubiquitous misregulation of Cyclin G (CycG) using transgenic lines lengthens development and induces phenotypes suggesting growth or proliferation defects. Using tissue-specific misregulation of CycG and FACS, we show that overproduction of Cyclin G produces small cells whereas shortage produces large cells, suggesting that Cyclin G negatively regulates cell growth. Furthermore, overexpression of CycG lengthens the cell cycle, with a prominent effect on G1 and S phases. Genetic interactions with Cyclin E suggest that Cyclin G prevents G1 to S transition and delays S phase progression. Control of cell growth and cell cycle by Cyclin G might be achieved via interaction with a network of partners, notably the cyclin-dependent kinases CDK4 and CDK2.

Maternal and zygotic requirement for thepolyhomeotic complex genetic locus inDrosophila

SummaryThe complex genetic locuspolyhomeotic (ph) is a member of thePolycomb (Pc)-group of genes and as such is required for the normal expression of ANT-C and BX-C genes. It also has probably other functions since amorphicph alleles display a cell death phenotype in the ventral epidermis of 12-h-old embryos. Here it is shown that lethal alleles ofph (amorph and strong hypomorph) show transformation of most of their segments towards AB8. Theph+ product is required autonomously in imaginal cells. The total lack ofph+ function prevents viability of the cuticular derivatives of these cells.ph has a strong maternal effect on segmental identity and epidermal development that can not be rescued by one paternally supplied dose ofph+ in the zygote. These phenotypes differ substantially from those of previously describedPc-group genes. AmongPc-group genes,ph seems to be the only one that is strongly required both maternally and zygotically for normal embryonic development.

New Partners in Regulation of Gene Expression: The Enhancer of Trithorax and Polycomb Corto Interacts with Methylated Ribosomal Protein L12 Via Its Chromodomain

Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Cortos chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA–seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.

Pattern triplications following genetic ablation on the wing ofDrosophila

SummaryThe effects ofpolyhomeotic (ph) mutants in imaginal cells have been studied in a clonal analysis. Clones of cells, homozygous forph, sort-out after a few divisions, probably as a consequence of modified cell affinities. The dorso-ventral margin of the wing has special characteristics that retard this phenomenon. The formation and exclusion of a clone of 8–16 cells affect the polarity of the wild-type neighbour cells and can provoke pattern triplications. The results suggest that a defect in intercellular communication prevents the wild-type cells from maintaining coordinated positional information. The cells react by regenerative growth, and reorganize into a new pattern. The pleiotropic phenotypes ofph mutants are explained according to a common hypothesis aboutph+ function.

Transcription of dispersed repeated sequences during Pleurodeles waltl oogenesis

Summary— Several repeated DNA sequences were isolated from a partial genomic DNA library of the newt Pleurodeles waltl. These repeated DNA elements are dispersed over the 12 P. waltl bivalents, and some of them are transcribed in the oocyte. We describe the localization of label following in situ hybridization to nascent RNA attached to the lateral loops of lampbrush chromosomes. Variations in the number and the location of labelled loops were constantly found for several of the probes. The results are discussed in view of the “cotranscription model” of RNA synthesis on lampbrush chromosomes. We speculate on the possible origins of variation in transcription on lampbrush loops.

Cloning of the newt Pleurodeles waltlii chromosomal DNA.

Pleurodeles waltlii genomic DNA has been cloned using several phage lambda vectors. We have isolated approx. 600 000 clones, which correspond to about 20% of the total DNA sequences of this organism. This constitutes the first large gene library of a Urodele. The low yield of cloning was attributable to the abundance of highly repetitive sequences, since recombinations in the bacterial host could lead to the loss of clones. Indeed, the existence of highly repetitive sequences was directly demonstrated by hybridization between recombinants and the total genome, and some of the cloned DNA was found to be unstable. We suggest new methods for cloning the highly repetitive sequences.

Implication of the Drosophila beta-amyloid peptide binding-like protein AMX in Notch signaling during early neurogenesis

Lateral inhibition provides a mechanism to regulate neuroblast specification during early neurogenesis in Drososphila melanogaster embryos. This mechanism is mediated by the highly conserved Notch pathway. Defective lateral inhibition results in CNS hyperplasia at the expense of ectoderm development, hence genes causing this defect are called neurogenic. D. melanogaster almondex (amx) is a maternal neurogenic gene, crucially required for embryonic lateral inhibition. Genetic interaction studies previously revealed amx as a positive Notch pathway partner in several processes, acting potentially upstream of Notch. We show here that embryonic overexpression of Notch intracellular domain partially rescues maternal lack of amx, suggesting a role for AMX at the level of Notch processing. Our molecular data reveal that amx is ubiquitously expressed and encodes a conserved putative transmembrane protein, composed of several domains that are differently required for amx function in the fly. Sequence comparisons identify AMX as a Drosophila Beta-amyloid peptide Binding Protein (BBP) family member, a BBP-like protein or dBLP. Based on these data, we discuss the potential molecular function of AMX in early neurogenesis.