Characterization of a Negative Regulatory Domain in Fushi tarazu, a Pair-rule Protein in Drosophila melanogaster

Fushi tarazu (FTZ) is a pair-rule protein important for the development of the anterior-posterior axis during embryogenesis. Low level ectopic expression of FTZΔ148-206 from a Tubulin α1 promoter, but not FTZ1-410 (full length FTZ), results in the anti-ftz phenotype in developing Drosophila melanogaster larvae, indicating that FTZΔ148-206 is a hyperactive FTZ protein. Through deletion analysis, using a high level ectopic expression system and assaying survivorship, I narrowed the location of the negative regulatory domain (NRD) to the 178-206 amino acid region of FTZ. Mutations that mimic both constitutive phosphorylation and dephosphorylation in the NRD revealed that phosphorylation doesn’t control the activity of the NRD. When the NRD was deleted along with three important functional domains, the homeodomain, terminal tyrosines and the FTZ-F1 binding site, I found that both the FTZ-F1 binding site and terminal tyrosines were required for hyperactive activity and that the NRD may regulate homeodomain activity

A Genetic Test of a Model for Two Activities of Fushi Tarazu Protein in Drosophila Melanogaster

The important pair-rule segmentation gene fushi tarazu (ftz) encodes a homeodomain (HD)-containing protein involved in the establishment of even-numbered parasegments during embryonic development. The D. melanogaster ftz is a derived homeotic selector (Hox) gene which lost its homeotic function during the evolution of arthropods. Genetic analyses have shown that FTZ has two distinct activities required during development: HD-dependent and HD-independent FTZ activities. The aim of this study was to test the interaction of the two FTZ activities proposed by Hyduk and Percival-Smith (1996), by generating site-specific mutant ftz alleles for intragenic complementation. CRISPR-mediated homology directed repair (HDR) was used to introduce engineered ftz alleles into the ftz locus. Subsequently, four ftz engineered alleles were constructed in vectors for reintroduction by Recombinase-mediated cassette exchange (RMCE). Despite using multiple approaches no CRISPR mediated HDR events were detected, and therefore, the model could not be tested

Evolution of the Hox gene fushi tarazu in arthropods

Homeotic (Hox) genes are important in determining regional identity in virtually all metazoans,and are conserved throughout the animal kingdom. In Drosophila melanogaster, fushi tarazu (ftz) is located within the Hox complex and contains a Hox-like DNA-binding homeodomain, but functions as a pair-rule segmentation gene. At some point(s) during evolution, ftz has undergone three specific changes thought to contribute to its new segmentation function in Drosophila: 1) The gain of an LXXLL motif allowed for interaction with a new co-factor, Ftz-F1; 2) The degeneration of the YPWM motif decreased the ability to interact with the homeotic co-factor Exd; 3) ftz expression switched from Hox-like to seven stripes in Drosophila. Here I isolated ftz sequences and examined expression from arthropods spanning 550 million years of evolutionary time to track these changes in ftz. I found that while the LXXLL motif required for segmentation was stably acquired at the base of the holometabolous insects, the YPWM motif degenerated independently many times in arthropod lineages, and these `degen-YPWMs' vary in their homeotic potential. Additionally, ftz expression in a crustacean is in a weak Hox-like pattern, suggesting a model in which different ftz variants could arise in nature and not be detrimental to organismal development. Given my findings that ftz sequence and expression is so dynamic, I investigated the features that may be preventing ftz fossilization in arthropod genomes. I tested the hypothesis that a broadly conserved role of ftz in the developing central nervous system (CNS) retains ftz in arthropod genomes. This model predicts that the homeodomain, but not variable co-factor interaction motifs, is required for Ftz CNS function. Evidence supporting this model was obtained from CNS-specific rescue experiments in Drosophila. Additionally I examined the expression and function of ftz and ftz-f1 in the short-germ beetle Tribolium castaneum. I found that both genes are expressed in pair-rule patterns, and preliminary results suggest that ftz-f1 is important for proper segmentation and cuticle deposition, and ftz function may be partially redundant with ftz-f1. Taken together, these findings show that variation of a pleiotropic transcription factor is more extensive than previously imagined, and suggest that evolutionary plasticity may be widespread among regulatory genes

A comprehensive catalog of post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced

During formation of the anterior-posterior axis, Homeotic selector (HOX) proteins determine the identity of Drosophila body segments. HOX proteins are transcription factors that regulate gene expression during development. Besides a highly conserved DNA-binding homeodomain (HD), HOX proteins also contain functionally important, evolutionarily conserved small motifs. These short motifs found in HOX proteins may be Short Linear Motifs (SLiMs). SLiMs are proposed to be sites of phosphorylation and this may regulate the activity of HOX proteins. The primary aim of this work was to develop a comprehensive catalogue of the sites of phosphorylation and other post-translational modifications (PTMs) for Fushi tarazu (FTZ) and 8 HOX proteins extracted from developing Drosophila melanogaster embryos. Drosophila were transformed with constructs that express FTZ or HOX proteins fused to a triple tag (TT) from a heat-shock promoter. The HOXTT proteins are biologically active during embryogenesis. Triple tagged Sex combs reduced (SCRTT) protein was extracted from developing embryos and purified using Ni-NTA beads under denaturing conditions. Multiple sites of PTMs were identified in purified SCRTT by tandem mass spectrometry (MS/MS). The identified PTMs include phosphorylation at S185, S201 and T324, acetylation at K218, K434 and K439, formylation at K218, K309, K325, K369, K434 and K439, methylation at S19, S166, K168 and T364, carboxylation at W307, K309 and E323 and hydroxylation at P22, P107, D108, D111, P269 and P306. In testing the hypothesis that HOX SLiMs are preferential sites of phosphorylation, I found that the distribution of phosphorylatable residues, S, T and Y was biased to SLiMs, but there was no support for the hypothesis that SLiMs are preferentially phosphorylated

Evolution of Pair-rule genes

All insects have a segmented body. The genes controlling segment development have been well characterized in the fruit fly, Drosophila melanogaster. These genes were divided into three categories: gap genes specify several continuous segments over a broad region of the embryo; Pair-Rule Genes (PRG) are responsible for segment formation and are the first set of genes to be expressed in repetitive patterns in the embryo; Segment polarity genes define anterior and posterior polarities within each segment.To understand how PRGs evolve, I took a comparative approach in this thesis. First, I compared the function of the Drosophila PRG ftz-f1 to that of its mammalian orthologs by expressing them all in Drosophila embryos. I found that the molecular function of this family of nuclear receptors has been highly conserved during evolution. Next, I set out to establish new insect model systems to study PRG function. While, some PRGs have been studied in other insects, most of these studies focused on holometabolous insects. My work focused on the sister group to the holometabolous insects, the Hemipteroid Assemblage. I participated in the genome annotation of a hemipteras insect, Oncopeltus fasciatus. I annotated nuclear receptor super family, Hox and PRGs in Oncopeltus. I further studied the expression and function of four PRGs in Oncopeltus. Using in situ hybridization and RNAi, I found that, Of-ftz and Of-hairy do not have segmentation function, while Of-ftz-f1 has function in oogenesis and segmentation. Of-runt was found to induce cell death in oocytes, but its function in segmentation needs further analysis. Using the knowledge and expertise I gained from Oncopeltus, I successfully set up in situ hybridization, antibody staining and parental RNAi in an invasive hemipteran insect pest, the Brown Marmorated Stink Bug (BMSB) Halyomorpha halys. These studies show that the expression and function of PRGs varies extensively in diverse insects, despite the overall conservation of a segmented body plan

The isolation and characterization of a P. Angulosus homeobox

Bibliography: pages 93-108.The aim of this thesis was to isolate and characterize a homeobox-containing gene of the South African sea urchin Parechinus angulosus. This was achieved by constructing a genomic library of several individuals and screening this library using a probe containing the Antennapedia homeobox. Eight clones were isolated and shown to represent different alleles of the same gene. One clone was sequenced, revealing a homeobox which was termed PaHboxl. This homeobox was compared to published homeobox sequences and shown to be a member of the Antp (Hoxl.l) subclass (table 1.1). A splice donor site was identified 23 bp upstream of the homeobox and the observation confirmed by RNAase mapping. PaHboxl is situated in a genomic area showing a significantly higher degree of restriction fragment polymorphism than expected. This was shown by a statistical analysis which should be of general value in the interpretation of such polymorphisms. The expression of PaHboxl was examined by RNAase protection assays and Northern blotting. Two distinct phases of expression were observed - during embryogenesis PaHboxl is expressed transiently at low levels in 11,5 hr mesenchyme blastula stage embryos (44 ± 8 transcripts per embryo) with levels 3-5 fold lower 2,5 hr before and after this stage. Expression is observed again at up to 160 fold higher levels in the adult with maximal expression in testis (11 transcripts per 10 pg total RNA), and increasingly lower levels in intestines, ovary and Aristotle's lantern. Two transcripts of size 5,2 and 5,7 kbp were observed. Expression in Aristotle's lantern and embryonic stages could not be detected by Northern analysis

Investigating the functional significance of evolutionarily conserved protein motifs of the Drosophila melanogaster HOX protein, Sex combs reduced

Bilaterans share a common anterior-posterior (A-P) axis that is patterned by the Homeotic selector (Hox) genes. In Drosophila melanogaster, Hox gene expression in spatially restricted domains along the A-P axis of the embryo determines segmental identity. Identifying the genetic mechanisms of HOX control of development is essential for understanding body patterning in animals. I identified and characterized the role of evolutionarily conserved protein domains of the HOX protein, Sex combs reduced (SCR), in protein function. SCR is required for establishing the identity of both the labial and prothoracic segments. To identify regions of functional importance, 15 Scr point mutant alleles were sequenced and grouped into three allelic classes: null, hypomorphic and hypomorphic-antimorph. Null alleles were nonsense mutations resulting in truncation and loss of highly conserved protein domains. Hypomorphic alleles were missense and small deletion mutations in highly conserved protein domains, including the DYTQL motif, YPWM motif and C-terminal domain (CTD). Examination of the affect of changes in conserved domains on three SCR dependent phenotypes revealed multiple examples of differential pleiotropy: the observation that HOX proteins are made up of small independently acting peptide motifs that alone make small contributions to activity. The third class is the hypomorphic-antimorphic allele, Scr14, which is a missense mutation in the octapeptide motif. The mechanism of Scr14 antimorphy may be the acquisition of a leucine zipper motif in the octapeptide and LASCY motifs. This leucine zipper motif confers oligomerization potential in vitro, and allows inhibition of Scr activity by SCR14 in vivo in a reciprocal manner. Lastly, I tested the genetic model that SCR and the HOX protein, Proboscipedia (PB), form a complex to determine proboscis identity in the labial segment. Co-immunoprecipitation assays were unsuccessful at detecting a biochemical interaction between PB and SCR, indicating that the mechanism for proboscis determination may not involve complex formation between these two proteins. Together, these results demonstrate that the contribution of evolutionarily conserved HOX protein domains to HOX control of development is complex

Drawing lines in the sand: even skipped et al. and parasegment boundaries.

The pair-rule segmentation gene even skipped (eve) is required to activate engrailed stripes and to organize odd-numbered parasegments (PSs). The protein product Eve has been shown to be an active repressor of transcription, and recent models for Eve function suggest that activation of engrailed is indirect, but these models have not been fully tested. Here we identify the forkhead domain transcription factor Sloppy-paired as the key intermediate in the initial activation of engrailed by Eve in odd-numbered parasegments. We also analyze the roles of the transcription factors Runt and Odd-skipped in this process. Detailed analysis of engrailed and pair-rule gene expression in various mutant combinations shows how eve activates engrailed by repressing these engrailed repressors, and further indicates that mutual repression among pair-rule genes plays an important role in establishing parasegment boundaries. We present a new model of pair-rule gene function that explains the response of these boundaries to the relative levels of Eve and Fushi Tarazu

Combinatorial Activity of Pair-Rule Proteins on the Drosophila gooseberry Early Enhancer

The early expression of the Drosophila segment polarity gene gooseberry (gsb) is under the control of the pair-rule genes. We have identified a 514-bp enhancer which reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired- and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, we have identified sites critical for Prd activity. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organisation of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both paired- and homeodomains, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites