Phenotypic consequences and genetic interactions of a null mutation in the Drosophila posterior Sex Combs gene

The Posterior Sex Combs (Psc) gene of Drosophila is a member of the Polycomb (Pc) group of transregulatory genes. Previous analyses of the function of this gene in Drosophila em-bryogenesis have been hampered by the lack of a null mutation. We recently isolated a mutation that deletes the 5′ end of the Psc gene. This allele appears to be a null mutation, and we have used it to determine the Psc zygotic null phenotype and to look at the interactions of a null allele of Psc with five other Pc group mutations. We find evidence for transformations along both the anterior-posterior and dorsal-ventral axes in embryos of a variety of genotypes that include a null mutation in Psc. The phenotypes of embryos that are doubly mutant for a null allele of Psc and a mutation in a second Pc group gene show dramatic synergistic effects, but in their specifics they are dependent on the identify of the second Pc group gene. This is different from the relatively uniform phenotypes seen among double mutants that contained the allele Psc¹, which has both gain and loss of function properties. The differences in the phenotypes of the doubly mutant embryos allow us to eliminate one class of molecular models to explain the dramatic synergism seen with mutations in this group of genes.     

Genetic interactions of the suppressor 2 of zeste region genes

A wide variety of gain of function mutations have been induced in the Posterior Sex Comb (Psc)--Aristapedioid (Arp)--Suppressor 2 of zeste (Su(z)2) region of the second chromosome of Drosophila. This region contains at least three apparently related genes, two of which we have been studying. Psc1 has previously been used to identify Psc as a Pc group gene; however, it is a complex mutation with both gain and loss of function character. We report here that the Pc group character of Psc is not due to a gain of function and presumably reflects the function of the wild-type gene. We also provide evidence for a maternal function for Psc, as well as the neighboring Su(z)2 gene. Su(z)2 does not appear to be a Pc group gene as it does not act in a synergistic fashion with other Pc group genes in promoting posteriorly directed transformations. However, we have found that mutations in Su(z)2 do interact in a variety of interesting ways with mutations in Pc group genes.     

Interactions of Polyhomeotic with Polycomb Group Genes of Drosophila Melanogaster

The Polycomb (Pc) group genes of Drosophila are negative regulators of homeotic genes, but individual loci have pleiotropic phenotypes. It has been suggested that Pc group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic (ph) immunoprecipitates in a multimeric complex that includes Pc. Here, we show that duplications of ph suppress homeotic transformations of Pc and Pcl, supporting a mass-action model for Pc group function. We crossed ph alleles to all members of the Polycomb group, and to E(Pc) and Su(z)2 to look for synergistic effects. We observed extragenic noncomplementation between ph(503) and Pc, Psc(1) and Su(z)2(1) in females, and between ph(409) and Sce(1), Scm(D1) and E(z)(1) mutations in males, suggesting that these gene products might interact directly with ph. Males hemizygous for a temperature-sensitive allele, ph(2), are lethal when heterozygous with mutants in Asx, Pc, Pcl, Psc, Sce and Scm, and with E(Pc) and Su(z)2. Mutations in trithorax group genes were not able to suppress the lethality of ph(2)/Y; Psc(1)/+ males. ph(2) was not lethal with extra sex combs, E(z), super sex combs (sxc) or l(4)102EFc heterozygotes, but did cause earlier lethality in embryos homozygous for E(z), sxc and l(4)102EFc. However, ph(503) did not enhance homeotic phenotypes of esc heterozygotes derived from homozygous esc(-) mothers. We examined the embryonic phenotypes of ph(2) embryos that were lethal when heterozygous or homozygous for other mutations. Based on this phenotypic analysis, we suggest that ph may perform different functions in conjunction with differing subsets of Pc group genes.     

The Drosophila pleiohomeotic mutation enhances the Polycomblike and Polycomb mutant phenotypes during embryogenesis and in the adult

In Drosophila, the spatially restricted expression of the homeotic genes is controlled by Polycomb group (PcG) repression. PcG proteins appear to form different complexes to repress this gene expression. Although the pleiohomeotic gene (pho) shares mutational phenotypes with other PcG mutations, which demonstrates that PHO binds directly with a Polycomb (Pc)-containing complex, the genetic interactions of pho with other PcG genes have not been examined in detail. Here we investigated whether pho interacts with Polycomblike (Pcl) and Polycomb (Pc) during embryonic and adult development using developmental and genetic approaches. Pcl and Pc strongly enhanced pho phenotypes in the legs and tergite of the adult fly. Embryonic cuticle transformation was also greatly enhanced in Pcl; pho or Pc; pho double mutant embryos. The double mutant phenotypes were more severely affected by the pho maternal effect mutation than in zygotic mutant background, suggesting dosage-dependent processes. Taken together, these results provide genetic evidence of an interaction between PHO with other Polycomb group proteins at the embryonic and adult stages, and of the functioning of PHO as a component of the PcG complex.     

Comparison of Germline Mosaics of Genes in the Polycomb Group of Drosophila Melanogaster

The genes of the Polycomb group (PcG) repress the genes of the bithorax and Antennapedia complexes, among others. To observe a null phenotype for a PcG gene, one must remove its maternal as well as zygotic contribution to the embryo. Five members of the PcG group are compared here: Enhancer of Polycomb [E(Pc)], Additional sex combs (Asx), Posterior sex combs (Psc), Suppressor of zeste 2 [Su(z)2] and Polycomblike (Pcl). The yeast recombinase (FLP) system was used to induce mitotic recombination in the maternal germline. Mutant embryos were analyzed by staining with antibodies against six target genes of the PcG. The loss of the maternal component leads to enhanced homeotic phenotypes and to unique patterns of misexpression. E(Pc) and Su(z)2 mutations had only subtle effects on the target genes, even when the maternal contributions were removed. Asx and Pcl mutants show derepression of the targets only in specific cell types. Psc shows unusual effects on two of the targets, Ultrabithorax and abdominal-A. These results show that the PcG genes do not act only in a common complex or pathway; they must have some independent functions.     

Transvection in the Drosophila Ultrabithorax Gene: A Cbx(1) Mutant Allele Induces Ectopic Expression of a Normal Allele in Trans

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.     

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.     

A Genetic Analysis of the Suppressor 2 of Zeste Complex of Drosophila Melanogaster

The zeste(1) (z(1)) mutation of Drosophila melanogaster produces a mutant yellow eye color instead of the wild-type red. Genetic and molecular data suggest that z(1) achieves this change by altering expression of the wild-type white gene in a manner that exhibits transvection effects. There exist suppressor and enhancer mutations that modify the z(1) eye color, and this paper summarizes our studies of those belonging to the Suppressor 2 of zeste complex [Su(z)2-C]. The Su(z)2-C consists of at least three subregions called Psc (Posterior sex combs), Su(z)2 and Su(z)2D (Distal). The products of these subregions are proposed to act at the level of chromatin. Complementation analyses predict that the products are functionally similar and interacting. The alleles of Psc define two overlapping phenotypic classes, the hopeful and hapless. The distinctions between these two classes and the intragenic complementation seen among some of the Psc alleles are consistent with a multidomain structure for the product of Psc. Psc is a member of the homeotic Polycomb group of genes. A general discussion of the Polycomb and trithorax group of genes, position-effect variegation, transvection, chromosome pairing and chromatin structure is presented.     

DSP1, an HMG-like protein, is involved in the regulation of homeotic genes

The Drosophila dsp1 gene, which encodes an HMG-like protein, was originally identified in a screen for corepressors of Dorsal. Here we report that loss of dsp1 function causes homeotic transformations resembling those associated with loss of function in the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Abdominal-B. The expression pattern of Scr is altered in dsp1 mutant imaginal discs, indicating that dsp1 is required for normal expression of this gene. Genetic interaction studies reveal that a null allele of dsp1 enhances trithorax-group gene (trx-G) mutations and partially suppresses Polycomb-group gene (Pc-G) mutations. On the contrary, overexpression of dsp1 induces an enhancement of the transformation of wings into halteres and of the extra sex comb phenotype of Pc. In addition, dsp1 male mutants exhibit a mild transformation of A4 into A5. Comparison of the chromatin structure at the Mcp locus in wild-type and dsp1 mutant embryos reveals that the 300-bp DNase I hypersensitive region is absent in a dsp1 mutant context. We propose that DSP1 protein is a chromatin remodeling factor, acting as a trx-G or a Pc-G protein depending on the considered function.     

Enhancer of garnet/deltaAP-3 is a cryptic allele of the white gene and identifies the intracellular transport system for the white protein.

The white gene encodes an ABC-type transmembrane transporter that has a role in normal eye pigment deposition. In addition, overexpression in Drosophila leads to homosexual male courtship. Its human homologue has been implicated in cholesterol transport in macrophages and in mood disorders in human males. The garnet gene is a member of a group of other Drosophila eye colour genes that have been shown, or proposed, to function in intracellular protein transport. Recent molecular analysis indicates that it encodes the delta subunit of the AP-3 adaptin complex involved in vesicle transport from the trans-Golgi network to lysosomes and related organelles, such as pigment granules. This identification revealed a novel role for intracellular vesicular transport in Drosophila pigmentation. To further analyze this intracellular transport system, we examined the genetic interactions between garnet and a second site enhancer mutation, enhancer of garnet (e(g)). We show here that e(g) is a cryptic allele of the white gene. The white-garnet interaction is highly sensitive to the levels of both gene products but also shows some allele specificity for the white gene. The additive effect on pigmentation and the predicted protein products of these genes suggest that the garnet/AP-3 transport system ensures the correct intracellular localization of the white gene product. This model is further supported by the observation of homosexual male courtship behavior in garnet mutants, similar to that seen in flies overexpressing, and presumably mis-sorting, the white gene product. The w(e(g)) allele also enhances mutations in the subset of other eye-color genes with phenotypes similar to garnet. This observation supports a role for these genes in intracellular transport and leads to a model whereby incorrect sorting of the white gene product can explain the pigmentation phenotypes of an entire group of eye-color genes.     

Mutations in Polycombeotic, a Drosophila Polycomb-Group Gene, Cause a Wide Range of Maternal and Zygotic Phenotypes

The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycomb-group mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function.     

Functional analysis of Drosophila beta1,4-N-acetlygalactosaminyltransferases

Members of the mammalian beta1,4-galactosyltransferase family are among the best studied glycosyltransferases, but the requirements for all members of this family within an animal have not previously been determined. Here, we describe analysis of two Drosophila genes, beta4GalNAcTA (CG8536) and beta4GalNAcTB (CG14517), that are homologous to mammalian beta1,4-galactosyltransferases. Like their mammalian homologs, these glycosyltransferases use N-acetylglucosamine as an acceptor substrate. However, they transfer N-acetylgalactosamine rather than galactose. This activity, together with amino acid sequence similarity, places them among a group of recently identified invertebrate beta1,4-N-acetylgalactosaminyltransferases. To investigate the biological functions of these genes, null mutations were generated by imprecise excision of a transposable element (beta4GalNAcTA) or by gene-targeted homologous recombination (beta4GalNAcTB). Flies mutant for beta4GalNAcTA are viable and fertile but display behavioral phenotypes suggestive of essential roles for GalNAc-beta1,4-GlcNAc containing glycoconjugates in neuronal and/or muscular function. beta4GalNAcTB mutants are viable and display no evident morphological or behavioral phenotypes. Flies doubly mutant for both genes display only the behavioral phenotypes associated with mutation of beta4GalNAcTA. Thus Drosophila homologs of the mammalian beta4GalT family are essential for neuromuscular physiology or development but are not otherwise required for viability, fertility, or external morphology.     

Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo.

Seven zygotically active genes are required for normal patterning of the dorsal 40% of the Drosophila embryo. Among these genes, decapentaplegic (dpp) has the strongest mutant phenotype: in the absence of dpp, all cells in the dorsal and dorsolateral regions of the embryo adopt fates characteristic of more ventrally derived cells (Irish and Gelbart (1987) Genes Dev. 1, 868-879). Here we describe the phenotypes caused by alleles of another of this set of genes, tolloid, and show that tolloid is required for dorsal, but not dorsolateral, pattern. Extragenic suppressors of tolloid mutations were isolated that proved to be mutations that elevate dpp activity. We studied the relationship between tolloid and dpp by analyzing the phenotypes of tolloid embryos with elevated numbers of the dpp gene and found that doubling the dpp+ gene dosage completely suppressed weak tolloid mutants and partially suppressed the phenotypes of tolloid null mutants. We conclude that the function of tolloid is to increase dpp activity. We also examined the effect of doubling dpp+ gene dosage on the phenotypes caused by other mutations affecting dorsal development. Like tolloid, the phenotypes of mutant embryos lacking shrew gene function were suppressed by elevated dpp, indicating that shrew also acts upstream of dpp to increase dpp activity. In contrast, increasing the number of copies of the dpp gene enhanced the short gastrulation (sog) mutant phenotype, causing ventrolateral cells to adopt dorsal fates. This indicates that sog gene product normally blocks dpp activity ventrally. We propose that the tolloid, shrew and sog genes are required to generate a gradient of dpp activity, which directly specifies the pattern of the dorsal 40% of the embryo.     

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.     

Maternal and zygotic activity of the zebrafish ogon locus antagonizes BMP signaling.

The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mercedes(tm305), ogon(m60), and short tail(b180) mutations produce ventralized phenotypes, including expanded bmp2b/4 expression domains. We find that the three mutations are allelic and that the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo(m60) and ogo(b180) mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo(tm305) retains partial function. Aspects of the ogo(m60) and ogo(tm305) mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl(tc300), a null mutation in bmp2b, is epistatic to ogo(m60) mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. We have ruled out zebrafish noggin homologues as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity.     

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.     

Vertebrate homeobox genes

Recent highlights in vertebrate homeobox gene research include the discovery of new genes with novel expression patterns, observations that peptide growth factors and retinoic acid influence homeobox gene expression, and the generation of mutant phenotypes of embryos homozygous for null mutations. These combined studies reinforce the idea that homeobox genes function near the top of the gene hierarchies controlling vertebrate embryogenesis.