I believe that everything I have learned as a graduate student in the Stathopoulos lab will be invaluable to me during my postdoctoral research and beyond. I would like to thank the current and past members of the Stathopoulos lab for all their support and encouragement over the years. I would especially like to thank Louisa Liberman and Amy McMahon for sharing their graduate experience with me in and out of the lab.
To my friends outside the lab who made graduate school a fun time, I would like to express my gratitude for keeping me sane during the hard times and for helping me celebrate all the events. Finally, I would like to thank my husband, Sean Tulin, for his support, patience and reliability. My graduate work focused on addressing this challenge in the model system of Drosophila melanogastor, the vinegar fly.
By examining functional domains of Thisbe and Pyramus, FGF ligands in the fly, we have begun to understand the properties of Drosophila FGFs and how they may contribute to the regulation of FGF signaling.
INTRODUCTION TO FIBROBLAST GROWTH FACTOR SIGNALING
FGF9 was shown to be able to stimulate the growth of glial cells (MIYAMOTO et al. 1993). FGFR1 is expressed almost exclusively in the mesoderm and is essential for mediating early developmental functions and organogenesis (DENG et al. 1994). A loop phosphorylation results in phosphorylation of tyrosines in the C-tail, kinase insert and juxtamembrane regions (Figure 1) (MOHAMMADI et al. 1996).
Loss-of-function studies confirmed the relative importance of the FGF8 splice forms, and FGF8a mutants had no appreciable defect in the midbrain and cerebellum (GUO et al. 2010). The study of FGF signaling in Drosophila began with the discovery of the breathless (btl) FGFR gene in 1992 (KLÄMBT et al. 1992). Double mutants for thisbe (ths) and pyramus (pyr) [Df(2R)BSC25] phenocopy the htl mutant phenotype (STATHOPOULOS et al. 2004).
In vertebrates, FGF9 plays a similar role in the development of the male testis (COLVIN et al. 2001). FGF signaling has also been shown to play a major role in maintaining vascular integrity in the existing adult vasculature (MURAKAMI et al. 2008). Cleavage in the D3 domain profoundly alters ligand-binding specificity (MIKI et al. 1992; YAYON et al. 1992).
INTRODUCTION TO PROTEOLYTIC PROCESSING IN DEVELOPMENTAL SIGNALING
The proproteins are then cleaved by proprotein convertase enzymes of the Furin family to generate the active ligand (AONO et al. 1995; CUI et al. 1998). BMP signaling is required for proper development and one of its earliest and best documented roles is in establishing the dorsoventral axis, reviewed in (GRAFF. 1997). The propeptide of Furin itself is excised at a Furin site, but remains non-covalently associated and functions as an autoinhibitor of the active zymogen until a secondary cleavage releases the active enzyme (ANDERSON et al. 1997).
Unlike Dpp, however, one of the two resulting forms has a long N-terminal extension that is capable of signaling. Processing of the Notch receptor is crucial for the functional roles it plays in development. Rhomboid is a novel serine protease that cleaves Spi in its transmembrane domain (URBAN et al. 2001).
To demonstrate that Spi is cleaved in an equivalent position in its transmembrane domain to that of the Rhomboid active site, truncated forms of Spi were compared with Rhomboid-mediated cleaved Spi. Different regions along the D/V embryonic axis are specified by graded nuclear localization of the Dorsal transcription factor. The translocation of Dorsaal to the nucleus is a consequence of the activation of the Toll receptor by the ligand Spätzle.
Although the Toll receptor is uniformly distributed, the Spätzle ligand is converted to its active form only on the ventral side of the embryo, where Pipe activates the proteolytic cascade. The tube is inhibited on the dorsal side of the embryo by torpedo signaling activated by the Gurken protein. Gurken protein is synthesized from gurken mRNA transcripts produced by the nucleus after it has traveled to the dorsal-anterior angle of the oocyte.
Clearly, the correct timing and location of activation of the Spätzle ligand is essential for a normal D/V axis to develop. The cleavage reaction is also thought to produce a diffusible inhibitor of Spätzle, which contributes to the design of the dorsal gradient (MORISATO 2001).
ANALYSIS OF THISBE AND PYRAMUS FUNCTIONAL DOMAINS REVEALS EVIDENCE FOR CLEAVAGE OF
DROSOPHILA FGFS
In addition to understanding Ths and Pyr processing, we sought to link the structural domains to the function of the ligands. In this work, we used these excess Eve-positive cells as a functional readout of Ths and Pyr activity. If only the N-terminus is able to activate the receptor and enable downstream signaling, what is the role of the long C-termini of Ths and Pyr.
Results from additional genetic experiments were consistent with the hypothesis that Ths and Pyr are two FGF ligands for the Htl FGF receptor (GRYZIK and MÜLLER 2004b; STATHOPOULOS et al. 2004). To confirm the ability of full-length ths and pyr cDNAs to support the production of. Def(2R)BSC25 STATHOPOULOS et al. 2004) phenocopy the htl mutant phenotype, but in the early Drosophila embryo, Ths and Pyr proteins are expressed in the ectoderm, while Htl is restricted to the adjacent mesoderm cells.
Using anti-HA antibodies, we were able to immunoprecipitate N-terminally tagged Ths and Pyr from the culture medium. Separate forms of Ths and Pyr were detected in these samples, at ∼35kD for Ths and ∼45kD for Pyr ( Fig. 2C ). Therefore, we also examined cell pellet fractions and found that the cleaved N-terminal domains of Ths and Pyr are also present inside the cell ( Fig. 2B , lanes 5 and 6).
First, the addition of HA and Myc tags to Ths and Pyr did not affect the ability of Ths and Pyr to induce expansion of the Eve-positive cluster (Fig. 4B,F). -I) pUASt-HA-Ths and pUASt-HA-Pyr full-length and truncated constructs; the shedding assay was performed in S2 cell culture. Ths and Pyr give a graded output of Evo-positive cells with the most cells in the origin domain.
ThsN-PyrC supported more Eve-positive cells than HA-Ths and also had the same altered profile as HA-Ths1-158 (Fig. 7 C,E). Our present analysis is the first evidence for cleavage of Ths and Pyr FGF ligands for Htl FGFR. Finally, these new molecular data on Ths and Pyr raise questions about the evolutionary history of the FGF family.
In the present study, we have provided evidence for the proteolytic processing of Drosophila FGF ligands Ths and Pyr in both S2 cell culture and the embryo.
EVOLUTIONARY PERSPECTIVE OF THE FGF SUPERFAMILY
In the anthozoan cnidarian Nematostella vectensis, there are 4 ligands and 2 receptors: NvFGF8A, NvFGF8B, NvFGF1A, NvFGFa2, NvFGFRa and NvFGFRb (MATUS et al. Two FGFRs, Dj-FGFRG1 and Dj-inges have been found in planar .japonica, which rounds out representatives from all the major metazoan phyla (OGAWA et al. 2002) Nematostella vectensis, a sea anemone, is considered to be a representative of basal cnidarians and to have retained much of the genetic complexity contained in the cnidarian-bilaterian ancestor (BRIDGE et al.
The two FGFRs identified in Nematostella, NvFGFRa and NvFGFRb are thought to have arisen from a lineage-specific duplication, and therefore, there is thought to have been only 1 FGFR in the cnidarian-bilaterian ancestor (RENTZSCH et al. 2008). Morpholino knockdown of NvFGF1A and NvFGFRa showed that they are required for apical organ formation (RENTZSCH et al. 2008). Nematostella Sprouty, Nv-Sprouty, is expressed in the same domain as NvFGF8A, NvFGF8B, and NvFGFRa at the apical pole (MATUS et al. 2007).
It has been proposed that the 7 FGF subfamilies present in vertebrates (A-G) plus 1 additional subfamily lost in deuterostomes (H) represent what were once 8 proto-FGF genes in the protostome deuterosome ancestor (POPOVICI et al. 2005) (Figure 2) ). Ci-9/16/20 has been shown to be involved in the induction of notochord, induction of mesenchyme and heart specification (DAVIDSON et al. FGF9 and FGF16 are also known to be involved in heart development in the mouse.
In Xenopus neurula, FGF signaling has also been implicated in axial elongation, and possibly a similar mechanism is at play, but the details are still unclear (SIVAK et al. 2005). Ciona FGF8/17/18 is expressed in the nervous system of ascidian embryos and is thought to play a similar role in the patterning of brain territories as FGF8/FGF17/FGF18 plays in vertebrates (see the brain-hindbrain section in the introduction). In vertebrates, FGF17 and FGF18 are also expressed in the mid/hindbrain in a broader domain than FGF8 that includes the posterior midbrain (MARUOKA et al. 1998).
Ectopic FGF8 studies in the chick showed that only ectopic FGF8 leads to the expression of Engrailed-2, an early marker of mes/rhombencephalic development, Wnt1 and Fgf8 (CROSSLEY et al. 1996). The variability in the amino acid sequence of Ig domains relates to the high degree of variability in the amino acid composition of FGF ligands (POPOVICI et al. 2005).
DISCUSSION AND FUTURE DIRECTIONS
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