Progress 10/01/00 to 09/30/05
Outputs
During the tenure of this project we investigated the role of the partially redundant genes, disconnected (disco) and disco-related (disco-r) during Drosophila development. These genes encode zinc finger transcription factors that are required for normal development of the Drosophila larval head. Flies lacking both genes develop with defects similar to those lacking the two hox genes, Deformed and Sex combs reduced. Since the phenotypes were so similar, disco and disco-r are good candidates for Hox cofactors. hox genes encode homeodomain-containing transcription factors that control body patterning during development of all animals. Further, misexpression of hox genes has been linked to certain diseases and cancer. Therefore, understanding how they control gene expression and embryonic development is critical. All Hox proteins contain nearly identical DNA-binding homeodomains, and, therefore, they all interact with specific but nearly identical sequences of bases in
the genome. Yet developmentally, these genes are very specific. Their encoded proteins promote specific segmental identities by establishing segment-specific arrays of gene expression, which are required for proper identity within each segment. Because of this discrepancy in specificity between DNA recognition and developmental function, it is not clear how each protein can impart a highly specific segment identity. The answer is that interactions with other transcription factors must participate in establishing developmental specificity, but, currently, very few Hox cofactors are known. Therefore, understanding why disco and disco-r mutants have a phenotype resembling loss of hox genes is quite important, as these could be hox cofactors. Since these zinc finger-encoding genes appear to be extensively redundant, we concentrated our work on the disco gene since it is smaller and more easily manipulated. The work we undertook during this project has shown that the hox proteins Dfd and
Scr can only establish proper head segment identity when they are coexpressed with disco or disco-r. Our results indicate that the expression of disco establishes a developmental field in which these two Hox proteins can function in a head segment-specific mode. We used both genetic and molecular experiments to investigate this relationship. We determined that disco can bind to a specific DNA sequence, and that the zinc finger motifs and structure are necessary for disco function. We also found that disco and disco-r are members of an interactive network of zinc finger transcription factors that establish fields throughout the Drosophila larva permitting establishment proper of segment identity throughout the entire body. Finally, we obtained evidence that this developmental mechanism is conserved in many higher animals, including humans. These results provided a new look into the mechanisms of Hox control of embryonic development.
Impacts
Birth defects in livestock and humans cost our society millions of dollars each year. Because the hox genes and the parallel pathway of zinc-finger proteins described above are conserved in most animals, understanding how these factors interact to properly pattern animal forms is paramount to improving animal development and lowering the expense to society of developmental defects.
Publications
- Robertson, L.K., and Mahaffey, J.W. 2005. Insect homeotic complex genes and development, lessons from Drosophila and beyond. In Comprehensive Molecular Insect Science,Vol. 1, reproduction and development (eds., L.I. Gilbert, K. Iatrou, and S. Gill) Elsevier Limited, London, UK.
- Mahaffey, J.W. 2005. Assisting Hox proteins in controlling body form: are there new lessons from flies (and mammals)? Current Opinion in Genetics and Development 15, 422-429.
|
Progress 10/01/03 to 09/30/04
Outputs
All metazoans develop distinct morphological features at predictable positions along their anterior-posterior body axis. Development of these features is controlled by the spatially restricted expression of the homeotic or hox genes. hox genes encode conserved homeo domain-containing transcription factors, and their encoded proteins establish region-specific expression of realizator (or target) genes. In Drosophila, hox genes specify larval and adult segment identities. Lack of a particular hox gene disrupts development of the normal body pattern in a characteristic manner because the correct target genes cannot be activated. Exactly how the encoded HOX proteins control pattern formation is not well understood. The main dilemma is that, developmentally, HOX proteins have quite specific roles, yet they lack specificity in DNA recognition in vitro and perhaps in vivo, as well, so it is not clear how they can activate specific sets of target genes necessary for different
body pattern elements. Interactions with cofactors must contribute to HOX protein function, but currently, only a few are known. Previously, we reported that the partially redundant Drosophila genes, disconnected and disco-related, encode potential cofactors functioning in the post-oral head segments (gnathal segments) with the HOX proteins DEFORMED and SEX COMBS REDUCED. Disconnected and disco-related encode conserved zinc finger-containing transcription factors. Our data indicate that these and other zinc-finger proteins form a genetic hierarchy of factors that function with the HOX proteins to establish regional body patterning. We have demonstrated that these gnathal HOX proteins cannot function well in embryos lacking these cofactor genes. Further, we have shown using ectopic expression studies, expressing these gnathal genes in the trunk segments, that the DISCONNECTED protein can transform the trunk toward what appears to be a gnathal identity, and this ectopic DISCONNECTED
makes the trunk segments sensitive to the gnathal HOX proteins. The presence of DISCONNECTED and DEFORMED transforms the trunk segments toward a gnathal identity. We have begun studying the molecular basis of this process, examining the DNA-binding properties of the DISCONNECTED protein. We have identified a specific DNA recognition sequence for DISCONNECTED. In addition, we have begun several studies designed to determine whether DISCONNECTED and the gnathal HOX proteins interact directly.
Impacts
Birth defects in livestock and humans cost our society millions of dollars each year. Because the hox genes and the parallel pathway of zinc-finger proteins described above are conserved in all animals, understanding how these factors interact to properly pattern animal forms is paramount to improving animal development and lowering the expense to society of developmental defects.
Publications
- Robertson LK, Bowling DB, Mahaffey JP, Imiolczyk B, Mahaffey JW. (2004) An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domains of HOM-C protein function. Development. 131:2781-2789.
|
Progress 10/01/02 to 09/30/03
Outputs
All metazoans develop distinct morphological features at predictable positions along their anterior-posterior body axis. Development of these features is controlled by the spatially restricted expression of the homeotic or hox genes. hox genes encode conserved homeo domain-containing transcription factors that establish region-specific expression of realizator or target genes. In Drosophila hox genes specify larval and adult segment identities. Lack of a particular hox gene disrupts development of the normal body pattern in a characteristic way because the correct target genes cannot be activated in that region of the embryo. Though the hox genes are thought to be the master regulators of this process, exactly how they control pattern formation is perplexing. The main dilemma is that, in vivo, HOX proteins have quite specific roles, yet they lack specificity in DNA recognition in vitro and perhaps in vivo, too, so it is not clear how they can activate specific sets of
target genes necessary for different body pattern elements. Interactions with cofactors must contribute to HOX protein function, but currently, only a few are known. Previously, we reported that the Drosophila gene, disconnected, encodes one such cofactor functioning in the post-oral head segments (gnathal segments) with the HOX proteins DEFORMED and SEX COMBS REDUCED. Our work during the past year has further examined the role of disconnected using ectopic expression of the gene in the trunk segments, in places where it is normally not expressed. Our recent data indicate that the expression of disconnected establishes a domain within the Drosophila embryo in which the gnathal hox proteins can function. Further, we have found that there is a hierarchy of zinc finger proteins, including disconnected, teashirt, and spalt, that establishes domains within the Drosophila embryo, the trunk, and gnathal head domains. Spalt establishes the boundary between the trunk and gnathal by repressing
the expression of teashirt. Teashirt, in turn, represses disconnected in the trunk, thereby separating the trunk and gnathal domains. This pathway establishes domains in which the hox proteins can then establish individual segment identities. All of these factors are conserved in metazoans, and we suspect that this developmental role is also conserved.
Impacts
Birth defects in livestock and humans cost our society millions of dollars each year. Because the hox genes and the parallel pathway of zinc finger proteins described above are conserved in all animals, understanding how these factors interact to properly pattern animal forms is paramount to improving animal development and lowering the expense to society of developmental defects.
Publications
- No publications reported this period
|
Progress 10/01/01 to 09/30/02
Outputs
All metazoans develop distinct morphological features at predictable positions along their anterior-posterior body axis. Development of these features is controlled by the spatially restricted expression of the homeotic or hox genes. The hox genes encode conserved homeo domain-containing transcription factors that establish region-specific expression of 'realizator' or 'target' genes. In Drosophila, hox genes specify larval and adult segment identities. Lack of a particular hox gene disrupts development of the normal body pattern in a characteristic way because the correct target genes cannot be activated in that region of the embryo. Though the hox genes are thought to be the master regulators of this process, exactly how they control pattern formation is perplexing. The main dilemma is that, in vivo, HOX proteins have quite specific roles, yet they lack specificity in DNA recognition in vitro and perhaps in vivo, too, so it is not clear how they can activate
specific sets of target genes necessary for different body pattern elements. Interactions with cofactors must contribute to HOX protein function, but currently, only a few are known. Previously we reported that the Drosophila gene, disconnected, encodes one such cofactor functioning in the post-oral head segments (gnathal segments) with the HOX proteins DEFORMED and SEX COMBS REDUCED. Our work during the past year has further examined the role of disconnected using ectopic expression of the gene in the trunk segments, in places where it is normally not expressed. The results of these experiments indicate that disconnected may have multiple roles during specification of segment identity. These studies have helped us understand the role of disconnected and similar cofactors during Drosophila development.
Impacts
Birth defects in livestock and humans cost our society millions of dollars each year. Understanding the genetic regulation of animal development is paramount to improving animal development and lowering this cost.
Publications
- Robertson L.K., Dey B.K., Campos A.R., Mahaffey J.W. 2002. Expression of the Drosophila gene disconnected using the UAS/GAL4 system. Genesis 34:103-106.
|
Progress 10/01/00 to 09/30/01
Outputs
The goal of this project is to investigate the interplay between the hox protein, Deformed, and the zinc finger proteins, disco and disco-related during development of the maxillary and mandibular segments in Drosophila. We have used a yeast two-hybrid assay to investigate whether Dfd and Disco proteins interact directly and have an indication that they do. We have used a Polymerase Chain Reaction technique to identify the DNA sequence to which disco binds. We have obtained the binding fragments and soon will send the resulting clones to be sequenced. We are also examining DNA binding of Disco and Dfd on a 3 kb gnathal enhancer from the gene, Serrate. This enhance element requires both proteins for expression in the head. We have evidence that disco will bind to this DNA fragment, and we are continuing to examine the specific DNA bases to which disco binds. We will then examine Disco and Dfd interactions at this enhancer sequence to understand the arrangement of
binding sites for these proteins and to determine whether there is cooperative binding to the DNA. We have generated Drosophila lines with Disco, Disco and Deformed and Deformed genes under Gal4 control in order to determine the effect of ectopic expression of the proteins individually and in combination. We have examined over-expression in the embryo and in the developing adult eye. We have determined that overexpression of Dfd can only cause a transformation in the trunk in regions that are also expressing Disco and Disco-r. If overexpression is carried out in embryos lacking Disco and Disco-r, Dfd does not generate maxillary structures in the maxillary, labial or in the trunk segments. Using the uas overexpression of Disco and Dfd, we have found that overexpression of Disco alone has a more severe effect on development than does overexpression of any hox gene. This might be expected if the zinc finger can activate genes in the absence of the hox protein or if it can interact
(destructively) with other hox proteins. It is not clear at this time what is happening to the trunk segments, but normal development is severely disrupted. However, overexpression of both Disco and Dfd has a much more severe effect than either alone. This supports the proposal that the two proteins (or disco-r and Dfd) function together to determine maxillary identity, supporting the overall model that developmental fate is specified by a combinatorial process composed of at least hox and regionally expressed zinc finger proteins. We find that joint Disco and Dfd expression act synergistically such that a more severe transformation occurs with lower levels of Dfd if Disco is also overexpressed. We have created P- element constructs to express inverted repeat RNAs of disco and disco-r under Gal4 regulation, so that we can use RNA interference to inactivate either mRNA. The Gal4 system will allow us to interfere with gene expression at various times and tissues during Drosophila
development by using specific Gal4 expressing lines.
Impacts
Though it has been known for many years that the hox genes control anterior-posterior body pattern during animal development, how this is accomplished is unknown. Our studies indicate that regionally expressed zinc finger transcription factors are partners with the Hox proteins during this process. Understanding the mechanisms involved is critical to understanding the genetic control of development in all animals.
Publications
- Pederson, J.A., LaFollette, J.W., Gross, C., Veraksa, A., McGinnis,W. and Mahaffey, J.W. 2000. Regulation by homeoproteins: a comparison of Deformed-responsive elements. Genetics 156: 677-686.
|
|