Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.     

Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.     

The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth

Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf. Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia. In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression. The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

极性蛋白Crumbs胞内域功能保守性研究

跨膜蛋白Crumbs(Crb)是细胞顶部的决定因子,对上皮细胞顶-底极性的建立和维持起着关键的作用。其胞内域虽然仅有37个氨基酸,但对Crb的功能必不可少。在果蝇(Drosophila melanogaster)中,如果胞内域发生突变,将造成胚胎发育异常、上皮细胞顶底极性丧失等严重后果。Crb胞内域从果蝇到小鼠(Mus musculus)和人类(Homo sapiens)具有很高的同源性,但线虫(Caenorhabditis elegans)两个Crb蛋白的胞内域与果蝇和哺乳动物却较为不同。为验证线虫Crb蛋白胞内域是否功能保守,本文利用基因组工程法(Genomic engineering),将果蝇基因组中Crb基因编码胞内域的部分替换为一致性和相似性较远的线虫Crb2基因的相应区段。与其他Crb胞内域突变果蝇不同,替换突变体胚胎发育正常,Crb及其他极性蛋白的表达和定位正常,胚胎上皮细胞顶底极性能够正确的建立和维持。这些结果证实虽然线虫和果蝇Crb蛋白胞内域之间存在大量序列变异,但重要的氨基酸位点和功能模块则完全保守。     

Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae

Embryo segmentation has been studied extensively in the fruit fly, DROSOPHILA: These studies have demonstrated that a mechanism acting with dual segment periodicity is required for correct patterning of the body plan in this insect, but the evolutionary origin of the mechanism, the pair-rule system, is unclear. We have examined the expression of the homologues of two Drosophila pair-rule genes, runt and paired (Pax Group III), in segmenting embryos of the two-spotted spider mite (Tetranychus urticae Koch). Spider mites are chelicerates, a group of arthropods that diverged from the lineage leading to Drosophila at least 520 million years ago. In T. urticae, the Pax Group III gene Tu-pax3/7 was expressed during patterning of the prosoma, but not the opisthosoma, in a series of stripes which appear first in even numbered segments, and then in odd numbered segments. The mite runt homologue (Tu-run) in contrast was expressed early in a circular domains that resolved into a segmental pattern. The expression patterns of both of these genes also indicated they are regulated very differently from their Drosophila homologues. The expression pattern of Tu-pax3/7 lends support to the possibility that a pair-rule patterning mechanism is active in the segmentation pathways of chelicerates.     

Essential roles for the Dhr78 orphan nuclear receptor during molting of the Drosophila tracheal system

The Drosophila Dhr78 orphan nuclear receptor has been proposed to play a role in molting of the tracheal cuticle and regulate gene expression during the third larval instar, possibly in response to a novel systemic hormonal signal. Here, we show that there are no essential maternal functions for Dhr78 during development, and that mutants missing both maternal and zygotic Dhr78 function die primarily during second and third instar larval development. We show that defects in the tracheal system can be observed as early as the first instar, manifested as regions of fluid in the dorsal tracheal trunks. In addition, Dhr78 mutant tracheae show a highly penetrant defect in gas filling at the first-to-second instar larval molt. Dhr78 expression in only the tracheal system is sufficient to rescue the lethality of Dhr78 mutants, and selective inactivation of Dhr78 function in the tracheae by targeted RNAi is sufficient to result in tracheal defects. Finally, we see no evidence for widespread activation of the Dhr78 ligand binding domain in third instar larvae using the GAL4-LBD system, arguing against a systemic hormone for the receptor at this stage in development. Taken together, our results indicate that Dhr78 exerts its essential functions during molting of the tracheal cuticle in Drosophila.     

The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart

In an effort to isolate genes required for heart development and to further our understanding of cardiac specification at the molecular level, we screened PlacZ enhancer trap lines for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, was particularly interesting because of its early pattern of expression in cardiac precursor cells, which is dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. We isolated and characterized a gene in the vicinity of B2-2-15 that exhibits an identical expression pattern than the reporter gene of the enhancer trap. The product of his gene, apontic (apt; see also Gellon et al., 1997), does not appear to have any homology with known genes. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles, in that segments of heart cells (those of myocardial and pericardial identity) are often missing. Most strikingly, however, apt mutant embryos or larvae only develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. These cardiac defects may be the cause of death of these mutants during late embryonic or early larval stages.     

A screen for genes that interact with the Drosophila pair-rule segmentation gene fushi tarazu

The pair-rule gene fushi tarazu (ftz) of Drosophila is expressed at the blastoderm stage in seven stripes that serve to define the even-numbered parasegments. ftz encodes a DNA-binding homeodomain protein and is known to regulate genes of the segment polarity, homeotic, and pair-rule classes. Despite intensive analysis in a number of laboratories, how ftz is regulated and how it controls its targets are still poorly understood. To help understand these processes, we conducted a screen to identify dominant mutations that enhance the lethality of a ftz temperature-sensitive mutant. Twenty-six enhancers were isolated, which define 21 genes. All but one of the mutations recovered show a maternal effect in their interaction with ftz. Three of the enhancers proved to be alleles of the known ftz protein cofactor gene ftz-f1, demonstrating the efficacy of the screen. Four enhancers are alleles of Atrophin (Atro), the Drosophila homolog of the human gene responsible for the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Embryos from Atro mutant germ-line mothers lack the even-numbered (ftz-dependent) engrailed stripes and show strong ftz-like segmentation defects. These defects likely result from a reduction in Even-skipped (Eve) repression ability, as Atro has been shown to function as a corepressor for Eve. In this study, we present evidence that Atro is also a member of the trithorax group (trxG) of Hox gene regulators. Atro appears to be particularly closely related in function to the trxG gene osa, which encodes a component of the brahma chromatin remodeling complex. One additional gene was identified that causes pair-rule segmentation defects in embryos from homozygous mutant germ-line mothers. The single allele of this gene, called bek, also causes nuclear abnormalities similar to those caused by alleles of the Trithorax-like gene, which encodes the GAGA factor.     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.     

Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, we report here that r2d2¹ mutants have striking developmental defects. r2d2¹ mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. We show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2¹ mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. We conclude that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila.     

Production and evolution pattern of “fruity smell” aggregation pheromones in genus Drosophila

Insects produce pheromones to serve a range of ecological functions throughout their lifetime. The chemical composition, production pattern, and interspecies specificity provide information for carrying out their function and biological significance. Several species of Drosophila produce a class of volatile esters considered as “fruity smells”; however, the production pattern and ecological functions of these “fruity smell” volatiles in genus Drosophila are poorly understood. Here, using the headspace solid-phase microextraction (SPME) method, we tested the production pattern of volatile pheromones in Drosophila immigrans and factors that possibly affected pheromone production, including mating, feeding conditions, age of adult flies, and geographical distribution. We also explored the evolution and production pattern of volatile pheromones in 14 species of genus Drosophila. Our result showed that male D. immigrans adult flies produce three male-specific volatile ester pheromones, which are also considered as “fruity smell” chemicals, in a relatively stable pattern. In addition, a series of “fruity smell” ester pheromones with similar structure and chemical properties were found to appear in the species of D. virilis and D. immigrans species group, but not in the species of D. melanogaster species group. The ester volatile pheromone production of male flies has a correspondence with the female's demand for host plants. Integrating the production and evolution pattern of these volatile chemicals, we inferred the interaction between insects and host plants reflected in the Drosophila “fruity smell” pheromones.     

Non-periodic cues generate seven ftz stripes in the Drosophila embryo

We have examined the expression pattern of the segmentation gene fushi tarazu (ftz) by in situ hybridization to whole mount embryos using digoxygenin labeled probes. This method has revealed previously undetected stages in the development of the ftz RNA pattern. The ftz stripes arise individually in a distinct, non-linear order along the anterior-posterior axis of the embryo. In addition, the stripes develop differentially along the dorsal-ventral axis; most stripes emerge on the ventral side and then gradually spread dorsally until they surround the entire circumference of the embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy (h) stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the ftz stripes. The contribution of cis-acting regulatory elements to the ftz pattern was examined. The zebra and upstream elements interact to generate seven correctly positioned stripes at the end of cellularization. However, stripe establishement is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date. These observations suggest that the ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with previously identified ftz regulatory elements. This multi-level regulation provides a back-up system that ensures the development of seven stripes in the blastoderm.