Mailing Address:
University of California, Berkeley
Dept. of Integrative Biology
3060 VLSB #3140
Berkeley, CA 94720-3140
Lab phone: 510/643-6227
Fax: 510/643-5022
Email: parchem@uclink.berkeley.edu
University of California, Berkeley
Dept. of Integrative Biology
3060 VLSB #3140
Berkeley, CA 94720-3140
Lab phone: 510/643-6227
Fax: 510/643-5022
Email: parchem@uclink.berkeley.edu
Research Summary
Studies looking at the expression and function of segment polarity genes have shown this level of the Drosophila segmentation hierarchy to be highly conserved within arthropods. Despite this conservation, comparisons of pair-rule gene expression in insects have highlighted divergent mechanisms to establish the parasegment. The data gathered thus far indicates a possible conserved role for the pair-rule class of genes during segmentation, albeit not in the traditional pair-rule manner. To determine how pair-rule orthologs pattern the germband prior to parasegment formation and to uncover which aspects of this patterning may be ancestral to insects I have chosen to study a member of the sister group to the insects, the amphipod crustacean Parhyale hawaiensis.
The ectoderm of Parhyale condenses from an unorganized group of cells into the well-organized grid of columns and rows common to amphipod crustaceans. Once organized, each transverse row (Parasegment Precursor Row or PSPR) will undergo two rounds of division along the antero-posterior axis. The first division generates two rows termed “a/b” and “c/d”, which in turn become rows “a”,“b”,“c”, and “d” following the second division. Upon reaching the four row stage, the segment polarity gene engrailed is expressed in the most anterior "a" row. Thus, the initial rows of the germband and their four progeny rows generate a well-ordered set of genealogical units which we refer to as parasegments since they appear to be identical to the parasegment units found in Drosophila (although Drosophila parasegments lack the genealogical lineage relationship seen in Parhyale).
To obtain orthologs of Drosophila pair-rule genes from Parhyale I have used degenerate PCR followed by RACE to acquire full-length cDNA sequences. Thus far I have cloned three Pax3/7 family genes, two even-skipped family genes, two sloppy-paired family genes, five odd-skipped family genes, four Hairy/Enhancer of split family genes, and two odd-paired family gene. In cases where multiple genes were cloned, preliminary phylogenetic analysis yielded ambiguous results in assigning orthology. Therefore, I analyzed the expression of these genes using in situ hybridization to identify potential candidates expressed prior to parasegment formation (defined here as prior to the intitial expression of engrailed). Using this approach I have found at least one gene from each group that is expressed within a parasegment or its precursors prior to the onset of engrailed expression.
Currently, I am using siRNAs to knockdown gene function and characterize the role of pair-rule orthologs during Parhyale development. Thus far, I have found that the phenotypes of these knockdowns can be grouped into different functional classes. One group, when knocked-down, affects the migration and formation of the ectoderm-derived germband. Another, allows for germband formation, but causes a block in the initiation of PSPR divisions. Finally, the knockdown of some genes allows for both the organization of the germband and divisions of the PSPRs, with as yet uncharacterized downstream effects. Further investigation of these latter phenotypes is currently underway.
Another approach to understanding the role of pair-rule orthologs in Parhyale is the use of transgenic technology, which is currently being used and further developed in our lab. To this end, I have created transgenic Parhyale lines capable of overexpressing several of the pair-rule genes I have characterized. Thus far, I have found that I can cause significant segmentation defects through misexpression. Currently, I am beginning to characterize the nature of these defects by specifically trying to describe the role of these genes in controlling the proper timing of PSPR division, and the subsequent patterning of their progeny.
Education
PhD candidate
University of California, Berkeley
Department of Molecular and Cell Biology
2003-present
University of Chicago
Committee on Genetics
2002-2003
M.S. in Biology
University of Illinois, Urbana-Champaign
1999-2002
B.S. in Biology
University of Illinois, Urbana-Champaign
Minor in Chemistry
1995-1999
Studies looking at the expression and function of segment polarity genes have shown this level of the Drosophila segmentation hierarchy to be highly conserved within arthropods. Despite this conservation, comparisons of pair-rule gene expression in insects have highlighted divergent mechanisms to establish the parasegment. The data gathered thus far indicates a possible conserved role for the pair-rule class of genes during segmentation, albeit not in the traditional pair-rule manner. To determine how pair-rule orthologs pattern the germband prior to parasegment formation and to uncover which aspects of this patterning may be ancestral to insects I have chosen to study a member of the sister group to the insects, the amphipod crustacean Parhyale hawaiensis.
The ectoderm of Parhyale condenses from an unorganized group of cells into the well-organized grid of columns and rows common to amphipod crustaceans. Once organized, each transverse row (Parasegment Precursor Row or PSPR) will undergo two rounds of division along the antero-posterior axis. The first division generates two rows termed “a/b” and “c/d”, which in turn become rows “a”,“b”,“c”, and “d” following the second division. Upon reaching the four row stage, the segment polarity gene engrailed is expressed in the most anterior "a" row. Thus, the initial rows of the germband and their four progeny rows generate a well-ordered set of genealogical units which we refer to as parasegments since they appear to be identical to the parasegment units found in Drosophila (although Drosophila parasegments lack the genealogical lineage relationship seen in Parhyale).
To obtain orthologs of Drosophila pair-rule genes from Parhyale I have used degenerate PCR followed by RACE to acquire full-length cDNA sequences. Thus far I have cloned three Pax3/7 family genes, two even-skipped family genes, two sloppy-paired family genes, five odd-skipped family genes, four Hairy/Enhancer of split family genes, and two odd-paired family gene. In cases where multiple genes were cloned, preliminary phylogenetic analysis yielded ambiguous results in assigning orthology. Therefore, I analyzed the expression of these genes using in situ hybridization to identify potential candidates expressed prior to parasegment formation (defined here as prior to the intitial expression of engrailed). Using this approach I have found at least one gene from each group that is expressed within a parasegment or its precursors prior to the onset of engrailed expression.
Currently, I am using siRNAs to knockdown gene function and characterize the role of pair-rule orthologs during Parhyale development. Thus far, I have found that the phenotypes of these knockdowns can be grouped into different functional classes. One group, when knocked-down, affects the migration and formation of the ectoderm-derived germband. Another, allows for germband formation, but causes a block in the initiation of PSPR divisions. Finally, the knockdown of some genes allows for both the organization of the germband and divisions of the PSPRs, with as yet uncharacterized downstream effects. Further investigation of these latter phenotypes is currently underway.
Another approach to understanding the role of pair-rule orthologs in Parhyale is the use of transgenic technology, which is currently being used and further developed in our lab. To this end, I have created transgenic Parhyale lines capable of overexpressing several of the pair-rule genes I have characterized. Thus far, I have found that I can cause significant segmentation defects through misexpression. Currently, I am beginning to characterize the nature of these defects by specifically trying to describe the role of these genes in controlling the proper timing of PSPR division, and the subsequent patterning of their progeny.
Education
PhD candidate
University of California, Berkeley
Department of Molecular and Cell Biology
2003-present
University of Chicago
Committee on Genetics
2002-2003
M.S. in Biology
University of Illinois, Urbana-Champaign
1999-2002
B.S. in Biology
University of Illinois, Urbana-Champaign
Minor in Chemistry
1995-1999
NIPAM H. PATEL
PHOTO ABOVE
Larval disc of a butterfly showing the expression of Engrailed (red), p-SMAD (yellow/green), nuclei (blue).
Larval disc of a butterfly showing the expression of Engrailed (red), p-SMAD (yellow/green), nuclei (blue).
PHOTO ON LEFT
Shown here in this false colored image is the RNA expression of an even-skipped ortholog (RNA expression in red, nuclei in blue) in the posterior ectoderm of a developing Parhyale embryo.
Shown here in this false colored image is the RNA expression of an even-skipped ortholog (RNA expression in red, nuclei in blue) in the posterior ectoderm of a developing Parhyale embryo.
PHOTO ABOVE
Larval disc of Drosophila melanogaster showing the expression of Engrailed (red), p-SMAD (yellow/green), nuclei (blue).
Larval disc of Drosophila melanogaster showing the expression of Engrailed (red), p-SMAD (yellow/green), nuclei (blue).