https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Functional analysis of a parasitic nematode GATA transcription factor using C. elegans as a heterologous expression system Annabelle Couthier Supervisor: John Gilleard Department of Veterinary Parasitology University of Glasgow 2004 ProQuest Number: 10390855 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10390855 Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 Gla sg o w UNIVEkaiTY LIBRARY: Declaration I, Annabelle Couthier, declare that the following thesis embodies the results of my own special work, that it has been composed by myself and that it does not include work forming part of a thesis presented successfully for a degree in this or another university. Publications Couthier, A., J. Smith, P. McGarr, B. Craig and J. S. Gilleard (2004). "Ectopic expression of a. Haemonchus contortus GATA transcription factor in Caenorhabditis elegans reveals conserved function in spite of extensive sequence divergence." Molecular and Biochemical Parasitologv. 133: 241-253. Summary In recent years there has been a growing interest in the use of C. elegans in parasitic nematode research both as a model and as a suiTogate expression system. However, relatively little is know about the extent to which different aspects of C elegans biology is conserved in particular parasitic nematode species. Also, there are as yet very few examples of the use C. elegans as an expression system to study parasite gene function. The aim of this work was to exploit the in depth understanding of C. elegans GATA transcription factor function in order to explore conservation with parasitic nematode species and to develop C. elegans as a heterologous expression system for the study of parasite gene function. The endoderm is a relatively simple tissue which is present in all nematodes. In C. elegans, the endoderm has been extensively studied and its specification and development have been well described. Several GATA transcription factors, namely med-1/2, end-1/3 and elt-2/7, are involved in the regulation of endodermal development and maintenance of endodermal function. The first objective of this project was to isolate homologues of C. elegans GATA transcription factors from parasitic nematodes by searching available DNA sequence databases and by using moleculai' biology approaches. The next objectives were then to examine the conservation of both the function and regulation of these using C. elegans as a surrogate expression system. A search of the Brugia malayi genome sequence database revealed five potential GATA transcription factors. Interestingly, sequence analysis suggested that three of these were potential homologues of the three C. elegans GATA factors with essential functions {elt-1, elt-2 and elt~5). A search of the parasitic nematode EST databases identified a single GATA transcription factor from the species Ancylostoma ceylanicum, Strongyloides ratti and Meloidogyne arenaria. The relationship of these genes to individual members of the C. elegans GATA transcription factor family was not clear. A GATA transcription factor was also isolated from the strongylid parasitic nematode Haemonchus contortus. This was achieved by PCR using degenerate primers corresponding to the consensus sequence encoding the conserved GATA zinc finger of several C. elegans family members. A full-length cDNA A,ZAPII clone corTesponding to this gene was isolated and sequence analysis suggested this was homologue of the C. elegans elt-2 gene. Consequently this gene was named Hc-elt-2. Fusion proteins corresponding to two different regions of the Hc-ELT-2 polypeptide were used to generate specific rabbit polyclonal antisera. Immunofluorescence experiments with the specific antisera demonstrated that the Hc-ELT-2 was expressed in the H. contortus endoderm and the overall expression pattern was very similai' to that of C. elegans ELT-2. However one difference between the C. elegans and H. contortus ELT-2 expression patterns was that additional expression was seen in two (probably neuronal) cells either side of the H. contortus pharynx. These Hc- ELT-2 immunolocalisation experiments also revealed that development of the endoderm lineage is extremely similar' in C. elegans and H. contortus. The H. contortus elt-2 gene was ectopically expressed in transgenic C. elegans using the C. elegans hspl6-2 promoter. A series of experiments showed that expression of the Hc-ELT-2 polypeptide could be induced by heat shock in transgenic C. elegans. Expression of Hc-ELT-2 in early C. elegans embryos resulted in embryonic arrest with the absence of any signs of morphogenesis. Arrested embryos consisted of largely uniform balls of 200-300 endoderm-like cells. The ectopic experiments showed that the H. contortus elt-2 gene was capable of activating a program of endodermal differentiation in a very similar' manner to the C. elegans elt-2 gene itself. Hence the function and specificity of the elt-2 gene appears to be highly conserved between C. elegans and H. contortus. The genomic locus of the Hc-elt-2 gene, including 3.3kb of 5'Hanking sequence was isolated and the putative Hc-elt-2 promoter region was able to direct the expression of a GFP reporter construct in transgenic C. elegans. The highest levels of GFP expression were in the endoderm and the spatial expression pattern directed by the Hc-elt-2 regulatory elements was broadly similar to that of the endogenous H. contortus and C. elegans elt-2 genes. Although in addition to the endodermal expression, the GFP reporter was expressed in some neuronal cells: possibly amphids and phasmids. Also the temporal pattern of GFP expression pattern directed by the Hcelt-2 promoter in transgenic C. elegans was quite different to the endogenous expression of Hc-ELT-2 polypeptide in H. contortus: the transgene expression was only initiated during late embryogenesis compared to the eai'ly expression of the Hc-ELT-2 polypeptide in the 2E cells of the early H. contortus embryo. A transgenic C. elegans line was established which carried the Hc-elt-2 genomic locus on an extrachi'omosomal array. This line was used to show that the H. contortus elt-2 gene could at least partially rescue the C. elegans RNAi phenotype. Finally, an attempt was made to “knock down” H-celt-2 function during H. contortus LI to L3 development using RNA-mediated interference applied by feeding. However these attempts were unsuccessful. In summary, this work has shown that the function and regulation of the endodermal GATA factor elt-2 is highly conserved between C. elegans and H. contortus. Function was sufficiently conserved for the H. contortus elt-2 gene to function appropriately when expressed in transgenic C. elegans. Hence, for strongylid nematodes at least, C elegans should be a valuable tool for the direct in vivo study parasite gene regulation and function. Table of contents 1 Introduction............................................ 1 1.1 Caenorhabditis elegans as a model nematode........................ 2 1.1.1 What is C elegans?..........................................................................................2 1.1.2 Nematode Phytogeny........................... 5 1.1.3 To what extent is biology conserved across the phylum nematoda? 6 i) General features of embryogenesis in C. elegans and other nematodes 7 (ii) Endoderm specification in C. elegans and other nematodes ..............9 iii) Vulva development in C. elegans and other nematodes.............................. 11 1.1.4 Use of C. elegans to study parasite gene function..................................... 13 1.2 Haemonchus contortus: an important veterinary parasite and a close relative of C. elegans ...................................................................... 16 1.2.1 Description and life cycle.................................................................... 16 1.2.2 The veterinary importance of H contortus ............................... 17 1.2.3 Anthelminthic resistance in Haemonchus contortus and other trichostrongylid nematodes .................................................. 18 1.2.4 H. contortus as a ^‘model parasite”. .................................... 19 1.3 Overview of GATA transcription factors in C. elegans and other organisms