Download Midgut and muscle development in Drosophila melanogaster

Umeå University Medical Dissertations
New Series No 1264 ISSN 0346-6612 ISBN 978-91-7264-782- 4
Midgut and muscle development in
Drosophila melanogaster
Margret Shirinian
Department of Molecular Biology
Umeå University
Umeå 2009
Copyright © 2009 by Margret Shirinian
ISBN: 978-91-7264-782- 4
Print by Arkitektkopia
Umeå, Sweden 2009
To my Family
“I prefer to be a dreamer among the humblest, with visions to be realized, than
lord among those without dreams and desires.”
Khalil Gibran
TABLE OF CONTENTS ABSTRACT …………………………………………………………………………………..4
PAPERS IN THIS THESIS ………………………………………………………….............6
ABBREVIATIONS …………………………………………………………………………..7
INTRODUCTION …………………………………………………………………………..10
Drosophila as a model system to study midgut development ……………………………….10
Genetic tools in Drosophila …………………………………………………………............10
How it all starts - Drosophila gastrulation …………………………………………………12
Endoderm development in Drosophila………………………………………………............14
Formation of the midgut endoderm…………………………………………….14
Molecular signals driving the midgut endoderm formation ……………...........16
Formation of the midgut visceral muscles……………………………………………..........18
The Receptor Tyrosine kinase Alk and its role in cell specification in the visceral
Mesoderm ……………………………………………………………………...22
Molecular targets of the RTK ALK in the embryonic midgut ………………...24
Alk in human disease ………………………………………………………….25
Development of the somatic muscles in Drosophila……………………………………….25
Architecture of the larval somatic muscle and muscle attachment sites……...25
The sarcomeric structure of muscles …………………………………………29
Guanine exchange factors and their role as regulators of small GTPases ………………31
Rap GTPases and their effectors ……………………………………………..32
TABLE OF CONTENTS AIMS ………………………………………………………………………………………...35
RESULTS AND DISCUSSION…………………………………………………………….36
Drosophila Anaplastic Lymphoma Kinase regulates Dpp signalling in the
developing embryonic gut (Paper I)……………………………………………36
Fusion of circular and longitudinal visceral muscles in Drosophila is
independent of the endoderm but further visceral muscle differentiation requires
a close contact between mesoderm and endoderm (Paper II).............................41
Mutational analysis of the Guanine Nucleotide Exchange Factor C3G in
Drosophila melanogaster reveals a role for C3G in larval muscle development
(Paper III)………………………………………………………………………45
CONCLUSIONS ……………………………………………………………………………48
ACKNOWLEDGEMENTS ………………………………………………………………..49
REFERENCES ......................................................................................................................52
ABSTRACT The fully developed and functional Drosophila midgut comprises two layers, the visceral
mesoderm and the endoderm. The visceral muscle of the midgut is formed by the fusion of
founder cells with fusion competent cells to form the muscle syncytia. The specification of
these cells and thus the fusion and the formation of the midgut muscle is dependent on the
Receptor tyrosine kinase (RTK) Alk (Loren et al., 2003). The endoderm underlies the visceral
muscle and is formed from cells that originate from the anterior and the posterior parts of the
embryo. These cells use the visceral mesoderm as a substrate for their migration. Using Alk
mutant animals, we have studied endoderm migration during embryonic development. While
the initial migration of the endoderm is not affected in the absence of the visceral mesoderm,
we observe that the later dorsal-ventral endodermal migration does not take place.
The development of the visceral muscle and its dependence on the endoderm is poorly
understood. We have analysed gürtelchen (gurt) mutant animals, originally identified in a
genetic screen for mutations affecting visceral muscle formation. Gurt mutants are so named
due to their belt-like phenotype of the visceral muscle (gürtelchen is German for belt).
Mapping of the genomic locus identified gurt as a mutation in a previously described gene huckebein (hkb) which is known to have an important function in endoderm development.
Gurt (hkb) mutants were used to further study the interaction between the endoderm and the
visceral muscle during development. The initial specification of founder cells and fusion
competent myoblasts as well as fusion events are unaffected in gurt (hkb) mutants, however,
the elongation and stretching of the visceral muscle does not proceed as normal. Moreover,
ablation of the visceral mesoderm disrupts endoderm migration, while ablation of the
endoderm results in a delayed disruption of visceral muscle formation.
Signaling between the two tissues was investigated in detail. Since Alk is a critical player
in visceral muscle development, we employed Alk mutant embryos for this task. In addition to
the role of Alk in specifying the founder cells and initiating the visceral muscle fusion, we
have shown that Alk mediated signaling has a role in the induction of the midgut constriction
process by regulating dpp expression in the developing embryonic gut.
4 ABSTRACT Finally, we wished to identify genes in the founder cells/fusion competent myoblasts that
might be regulated by Alk. C3G is a gunaine nucleotide exchange factor expressed in the
visceral muscle founder cells. Deletion of the Drosophila C3G locus resulted in the
generation of null mutants in C3G which are viable, but display decreased longevity, fitness
and are semi-lethal. Further analysis of C3G mutants indicated that C3G is essential for
normal larval musculature development, in part by regulating integrin localization at muscle
attachment sites.
5 PAPERS IN THIS THESIS This thesis is based on the following articles and manuscript, which will be referred to in the
text by their Roman numerals:
Paper I
Margret Shrinian, Gaurav K. Varshney, Christina Loren, Caroline Grabbe and Ruth Palmer.
(2007): Drosophila Anaplastic Lymphoma Kinase regulates Dpp signalling in the developing
embryonic gut. Differentiation Jun; 75(5):418-26).
Paper II
Georg Wolfstetter *, Margret Shirinian *, Christiana Stute, Caroline Grabbe, Thomas
Hummel, Stefan Baumgartner, Ruth H. Palmer and Anne Holz. (2009): Fusion of circular and
longitudinal visceral muscles in Drosophila is independent of the endoderm but further
visceral muscle differentiation requires a close contact between mesoderm and endoderm.
Mechanisms of Development (Under revision). *Joint first authors.
Paper III
Margret Shirinian, Gaurav Varshney and Ruth H. Palmer. (2009): Mutational analysis of the
Guanine Nucleotide Exchange Factor C3G in Drosophila melanogaster reveals a role for
C3G in larval muscle development. Manuscript.
6 ABBREVIATIONS Alk
Anaplastic Lymphoma Kinase
ALCL
Anaplastic Large Cell Lymphoma
AMG
Anterior midgut primodium
Ant
Antennapedia
Abd A
Abdominal A
AMP
Anterior midgut precursor
ADD
Adducin
Bap
Bagpipe
Bin
Binou
Byn
Brachyenteron
CNS
Central Nervous System
Duf
Dumbfounded
DO
Dorsal Oblique
DAD
Daughters against Dpp
DBCL
Diffuse B-cell Lymphoma
ECM
Extracellular Matrix
EXD
Extradenticle
ERK
Extracellular signal regulated kinase
FC
Founder Cell
FCM
Fusion Competent Myoblast
FAS II/III
Fasciclin II/III
Fkh
Fork head
GAP
GTPase activating protein
GEF
Guanine Nucleotide Exchange Factor
Gurt
Gürtelchen
7 ABBREVIATIONS GFP
Green Fluorescent Protein
GTP
Guanosine Triphosphate
GDP
Guanosine Diphosphate
Hbs
Hibris
Hkb
Huckebein
HAJ
Hemiadherence
HVM
Hindgut visceral mesoderm
ISH
In situ hybridization
Jeb
Jelly Belly
Kirre
Kin of irregular chaism-C
LO
Lateral Oblique
LL
Lateral Longitudinal
LBC
Large Basophilic Cells
Mad
Mothers against Dpp
Mbc
Myoblast city
MAPK
Mitogen activated protein kinase
Mef-2
Myocyte specific enhancer factor-2
NDG
Nidogen
NGS
Normal goat serum
PMG
Posterior midgut primodium
PS
Position Specific
PMEC
Principle midgut epithelial cells
Pros
Prospero
R-Smad
Receptor-regulated Smad
RTK
Receptor Tyrosine Kinase
8 ABBREVIATIONS SCC
Non-small cell lung cancer
Scr
Sex combs reduced
SBM
Segment Border Muscle
Sns
Sticks and stones
Srp
Serpent
TGFβ
Transforming growth factor β
Tor
Torso
Tll
Tailless
Twi
Twist
Tin
Tinman
TUNEL
Terminal deoxynucleotidyl transferase dUTP nick end labeling
Ubx
Ultrabithorax
UAS
Upstream activating sequence
VA
Ventral acute
VT
Ventral Transverse
VL
Ventral Lateral
VO
Ventral Oblique
VM
Visceral mesoderm
Wg
Wingless
ps
Parasegment
DAPI
4’,6-diamidino-2-phenylindole
9 INTRODUCTION Drosophila as a model system to study midgut development
The little insect 3mm long, living on spoiled food first used as an experimental model by
Thomas Hunt Morgan in 1910 has become a very important model organism. Initially,
Drosophila melanogaster was used to study the rules of genetic inheritance and later
thousands of researchers around the world used this model organism to study rules of
development from a simple fertilized egg into an adult. This process of development, which is
similar to the one taking place in us, has made the fruitfly a popular model to study complex
biological processes and answer difficult developmental questions. Development of the gut
and its two components - the muscle and the epithelium - which I will describe below is very
complex and involves tightly regulated tissue-tissue interactions, which are demanding to
study in higher organisms. The short life cycle, the ability to perform genetic manipulations
and the large collection of mutants has together with other advantages made Drosophila an
attractive system with which to study gut development.
Genetic tools in Drosophila
The constant advancement of genetic tools and techniques has facilitated the study of
developmental processes and cell fate determination in Drosophila. The introduction of the
UAS/GAL4 system by Brand and Perrimon (Brand and Perrimon, 1993) made it possible to
express genes in a directed fashion. This is achieved by overexpressing the gene of interest in
a desired tissue or cell type and analyzing its effects at different developmental stages. This is
done by generating transgenic flies expressing a yeast transcriptional activator (GAL4) under
the control of a tissue specific promoter regulatory region. This transgenic animal is
subsequently crossed to another transgenic fly that contains binding sites for GAL4, known as
UAS (Upstream Activating Sequence) sites. UAS sites are coupled to the gene of interest,
which will then be expressed in a specific tissue (Figure 1). During the course of my studies, I
have utilized this system for two main purposes. Firstly, to allow expression of my gene of
interest in specific tissues, and more specifically in the visceral mesoderm, endoderm and the
somatic muscles. The second purpose has been to specifically ablate tissues of interest, and
study the resultant effect on neighboring tissues. To accomplish that, I have used a method
10 INTRODUCTION developed by Hidalgo and coworkers (Hidalgo et al., 1995). This method is based on the
ectopic expression of a cytotoxin (RicinA) in a tissue specific manner. Ricin consists of two
polypeptide chains; the B chain binds to the cell surface and allows the toxin to be
internalized, while the A chain inactivates eukaryotic ribosomes by disrupting the 28S
ribosomal RNA, which irreversibly inhibits protein translation (Moffat et al., 1992). By
employing only the Ricin A chain in this experimental strategy we are able to kill cells, while
preventing Ricin from entering neighbouring cells. In addition to the above mentioned genetic
methods, I have also used the enhancer trap strategy to follow the endogenous expression of
specific genes. This method utilizes a P element which contains a reporter gene inserted
nearby the enhancer of a gene (Bellen et al., 1989).
In order to appreciate the function of one of my favorite genes (C3G), creation of a
deletion mutant allele was one way of providing clues about its physiological role. There are
many different ways of creating mutants in Drosophila (Golic et al., 1997; Gong and Golic,
2003; Ryder et al., 2004; Ryder and Russell, 2003). One recently developed method which is
efficient and less time consuming compared to other P-element based methods, is the FRT
based deletion approach provided by the PiggyBac insertions made by the Exelixis
consortium (Thibault et al., 2004). A large number of PiggyBac insertions have been
generated and mapped within the Drosophila genome and these are available upon request.
Figure 1: Schematic representation of the UAS/GAL4 system in Drosophila. 11 INTRODUCTION How it all starts - Drosophila gastrulation
Before gastrulation, the embryo consists of a single epithelial sheet, the cellular blastoderm
which consists of about 6000 columnar cells enclosing the central yolk, and a group of
arround 30 germ line cells known as the pole cells lying on the surface of this sheet at the
posterior pole of the embryo. (Campos-Ortega, 1997; Turner and Mahowald, 1977) (Figure
2). Cells in the blastoderm are created by invagination of plasma membranes from the surface
of the egg (Foe and Alberts, 1983). Gastrulation begins as soon as the ventral cells are
formed, although the cellularization process is still taking place in the dorsal region of the
embryo. The term ‘gastrulation’ encompasses all of the morphogenetic changes that take
place between the blastoderm stage and the time when the three germ layers are
distinguishable (Figure 2). First the ventral furrow starts to invaginate and when this
invagination is complete, it gives rise to the mesoderm which in turn will develop into the
muscles, gonads and fat body. Next, the posterior pole of the embryo begins to shift dorsally,
carrying with it the pole cells, initiating the process of germ band extension.The posterior end
then folds inward to form the proctodeal invagination. The proctodeal invagination
internalizes the primodia for the posterior midgut (PMG) and the hindgut. At the anterior
pole, part of the anterior midgut primodia (AMG) invaginates together with the ventral
furrow. The more anterior region invaginates much later together with parts of the ectodermal
prospective foregut. A number of transcription factors determine the boundaries and the germ
layer fates of the blastoderm domains. twist (twi) and snail (sna), a bHLH transcription factor
and zinc finger transcription factor respectively, are expressed on the ventral side of the
embryo (Thisse et al., 1988) (Figure 3), where they are involved in determination of
mesodermal fate (Simpson, 1983). huckebein (hkb) is a zinc finger transcription factor which
is expressed in the anterior and posterior tips of the embryo (Figure 3) (Bronner and Jackle,
1991). hkb specifies the endodermal primodium (Bronner et al., 1994). tailless (tll), a zinc
finger transcription factor is also expressed at the posterior tip, but its expression extends
anteriorly beyond hkb expression into the hindgut primodium (Figure 3) (Pignoni et al.,
1990). tll is required for the development of the hindgut and for the proctodeal invagination
(Strecker et al., 1986). zerknüllt (zen), a
12 INTRODUCTION homeodomain protein, expressed on the dorsal side of the embryo determines the fate of the
amnioserosa. One of the downstream target genes for the twi, sna, tll and hkb transcription
factors is folded gastrulation (fog) (Sweeton et al., 1991; Zusman and Wieschaus, 1985). fog
encodes a secreted molecule first expressed in the mesoderm and then in the PMG primodium
as well as part of the hindgut primodium (Costa et al., 1994) (Figure 3). Embryos mutant for
fog exhibit defects in ventral furrow formation and PMG invagination (Sweeton et al., 1991).
Figure 2: The fate map of Drosophila melanogaster. Schematic representation of the Drosophila fate map after gastrulation. AMG (Anterior midgut primodiun), PMG (Posterior midgut primodium), FG (Foregut), HG (Hindgut), PC (Pole cells). Adapted from (Hartenstein, 1993). 13 INTRODUCTION Figure 3: Gene expression patterns of the Drosophila melanogaster blastoderm Endoderm development in Drosophila
Formation of the midgut endoderm
In Drosophila, the midgut consists of two layers, the visceral mesoderm and the
endoderm. The endodermal midgut arises from two cell primodia, the anterior midgut
primodium (AMG) and the posterior midgut primodium (PMG), which are situated very close
to the ectodermally-derived primodia of the foregut and the hindgut (Figure 4A) (Hartenstein,
1993). The formation of the midgut endoderm is initiated by an epithelial-mesenchymal
transition. The underlying visceral mesoderm at this stage acts as a substratum for endodermal
cell migration and mutations in genes required for visceral mesoderm specification lead to a
failure of endodermal midgut migration (Reuter et al., 1993; Tepass and Hartenstein, 1994a;
Tepass and Hartenstein, 1994b). When the migrating primodia contact the underlying visceral
mesoderm, they undergo a mesenchymal to epithelial conversion, thereby forming two rows
of cells (Figure 4B). Together with the visceral mesoderm, the endoderm subsequently
14 INTRODUCTION extends dorsally-ventrally to enclose the yolk and form the midgut sac (Figure 4C). This
mesenchymal-epithelial conversion as well as endoderm migration depends on the visceral
mesoderm (Tepass and Hartenstein, 1994b). After the formation of the midgut sac, three
constrictions take place that generate four midgut lobes, The first of which will form the
anterior midgut, the second and the third will form the middle midgut, while the fourth lobe
will develop into the posterior midgut (Figure 4D).
Figure 4: Schematic representation of the midgut endoderm formation. The endodermal midgut primodia (magenta), and ectodermal foregut (fg) and hindgut (hg) primodia (blue) are located at the anterior and the posterior ends of the blastoderm embryo at stage 8 (A). (B) Endodermal cells migrate along the bands of the visceral mesoderm (green) at stage 12. (C) Midgut primodia meet in the middle and form a single gut sac at stage 13/14. (D) At stage 16 three constrictions divide the midgut into four lobes. (am= anterior midgut, pv = proventriculus, pm = posterior midgut). 15 INTRODUCTION Molecular signals driving midgut endoderm formation
During gastrulation, the germ layers in Drosophila are formed by three morphogenetic
movements, ventral furrow formation, PMG (posterior midgut primodium) invagination
(which is also called proctodeal invagination) and germ band extension. The invagination of
the ventral furrow will give rise to the anterior endoderm, whereas the invagination of the
proctodeum will form the posterior endoderm. The primodium of the endoderm is set up by a
key regulator of the terminal system in Drosophila - the maternal effect gene torso (tor)
(Klingler et al., 1988; Nusslein-Volhard et al., 1987; Strecker et al., 1989) which encodes a
RTK (Sprenger et al., 1989). In embryos lacking maternal tor activity, structures posterior to
the seventh abdominal segment fail to develop (Schupbach and Wieschaus, 1986). Torso
activates the expression of two zygotic transcription factors hkb and tll described earlier.
(Casanova, 1990; Pignoni et al., 1990; Weigel et al., 1990). Hkb is expressed at the posterior
and the anterior cap of the blastoderm (Bronner and Jackle, 1991). In embryos lacking torso
function, hkb and tll expression is completely abolished at the posterior tip, but small patches
remain at the anterior tip (Casanova, 1990; Pignoni et al., 1990; Weigel et al., 1990). The
expression in these patches is controlled by the maternal anterior morphogen bicoid (bcd).
Embryos lacking both tor and bcd show no expression of either hkb or tll (Reuter and Leptin,
1994). Hkb is required for the specification and the invagination of the endodermal PMG
(Reuter and Leptin, 1994; Weigel et al., 1990). hkb mutant embryos fail to form the
endodermal midgut and they lose the expression of Prospero which is a marker for
endodermal cells (Bronner and Jackle, 1996), thus indicating a crucial role for hkb in
endodermal midgut development. Once the AMG and the PMG are formed, the endodermal
cells migrate along the two bands of the visceral mesoderm. Mutants in genes required for
visceral mesoderm specification such as twist (twi), tinman (tin), bagpipe (bap) also exhibit
defective endoderm migration (Azpiazu and Frasch, 1993; Lee et al., 1997; Leptin, 1991;
Tepass and Hartenstein, 1994b; Yin et al., 1997).
Integrins are cell surface extracellular matrix (ECM) receptors which play an important
role in endoderm migration. All integrins are αβ heterodimers, and the Drosophila genome
encodes five α subunits (αPS1-5) and two β subunits, βPS and βv (described in more detail
later). The βPS subunit is expressed broadly in the embryo, whereas the βv subunit is
primarily expressed in the midgut endoderm. Removal of both β subunits results in complete
16 INTRODUCTION block of
midgut migration, indicating that integrin function is a requisite for midgut
migration (Devenport and Brown, 2004). In addition, four homeotic genes expressed in the
visceral mesoderm of the midgut are important for gut morphogenesis and the formation of
the midgut constrictions (Reuter and Scott, 1990; Tremml and Bienz, 1992). These are Sex
combs reduced (scr), Antennapedia (Ant), Ultrabithorax (Ubx) and Abdominal A (Abd
A)(Reuter and Scott, 1990; Tremml and Bienz, 1989). It has been shown that in the absence of
any of these genes, the midgut constriction is defective. However, the mechanisms by which
these four homeotic genes influence the formation of the constrictions are poorly understood.
Target genes that are regulated by these homeotic genes encode secreted signaling molecules
such as the Wingless (Wg) Wnt family of proteins and the TGFβ family ligand
Decapentaplegic (Dpp). Ubx expressed in the central region parasegment (ps7) of the visceral
mesoderm stimulates the expression of dpp in the same region (ps7) and wg in the adjacent
cells (ps8) (Figure 5). Dpp secreted from the visceral mesoderm will be detected by the apical
side of the endoderm (Panganiban et al., 1990; Reuter et al., 1990). Induction of Labial (Lab),
a homeotic protein expressed specifically in the endoderm is dependent on dpp and wg
expression in the visceral mesoderm (Panganiban et al., 1990; Reuter et al., 1990). The
upstream flanking sequence of lab contains both dpp and wg respose elements (Tremml and
Bienz, 1992). A simplified model of this signaling cascade concentrated at paragsegment
(ps6, ps7, and ps8) is represented in (Figure 5).
Ubx alone has been shown to be necessary but not sufficient for the full activation of Dpp
in the visceral mesoderm, indicating the requirement of another homeodomain protein (Sun et
al., 1995). This protein has been reported to be Extradenticle (Exd) (Stultz et al., 2006), which
belongs to the PBC family of homedomain proteins. In mammalians the Exd homologue is
known as Pbx (Burglin, 1997). To access the nucleus PBC family proteins associate with
members of another family of homeodomain proteins which belong to the MEIS family (AbuShaar et al., 1999). In Drosophila homothorax (hth) belongs to this family (Burglin, 1997).
hth and exd have identical mutant phenotypes with severe head defects, including a failure of
head involution and transformation of the thoracic abdominal segment into a segment with a
more posterior identity (Rieckhof et al., 1997).
17 INTRODUCTION Figure 5: Genetic interactions in the embryonic midgut at parasegments 6, 7and 8. Formation of the midgut visceral muscles
The Drosophila larval digestive tract is divided into the foregut, midgut and hindgut. The
midgut occupies the biggest portion of the digestive tract and consists of two tissue layers, the
visceral muscle layer and the epithelial layer. The visceral muscle is derived from the
mesoderm whereas the epithelial layer originates from the endoderm (Tepass and Hartenstein,
1994b). The midgut muscle is comprised of two types of muscles; the inner circular muscles
that are derived from the trunk mesoderm and the outer longitudinal muscles, derived from
the caudal mesoderm (Hartenstein, 1993). My focus in this thesis will be on the circular
muscles, but I will also briefly describe the longitudinal muscle, as well as the formation of
the hindgut and foregut in the following section.
One of the earliest known genes required for the formation of the trunk visceral mesoderm
is bagpipe (bap) which encodes a NK family homedomain containing transcription factor
(Azpiazu et al., 1996). Bap-expressing cells define the trunk
18 INTRODUCTION visceral mesoderm primodia which include the founder cells and fusion competent myoblasts
(Azpiazu and Frasch, 1993). The founder cells are columnar shaped cells that express
Dumbfounded (Duf)/ (Kin of irregular-chiasm-C) (Kirre), a transmembrane receptor that
belongs to the immunoglobulin superfamily (IgSF) (Ruiz-Gomez et al., 2000). They also
express Roughest (Rst)/ (Irregular-chiasm-C) (IrreC) (Strunkelnberg et al., 2001) which is a
paralogue of Duf/Kirre. Both Duf/Kirre and Rst have been shown to act as attractants for the
fusion competent myoblasts (Ruiz-Gomez, Coutts et al. 2000), (Strunkelnberg et al., 2001).
The fusion competent myoblasts exhibit a pebble-like morphology and are characterized by
the expression of another immunoglobulin superfamily protein called Sticks and stones (Sns)
(Bour et al., 2000). Hibris (Hbs) is a paralogue of Sns which is also expressed in the fusion
competent mysoblasts (Artero et al., 2003; Artero et al., 2001; Dworak et al., 2001). Both Sns
and Hbs are important for the fusion of the fusion competent myoblasts with the founder cells
(Artero et al., 2001; Bour et al., 2000; Dworak et al., 2001). This fusion of the visceral muscle
takes place during stage 12, and results in the formation of the circular visceral muscle
syncytium which is an elongated binucleated muscle (Martin et al., 2001) (see Tables 1 and 2
below).
19 INTRODUCTION Table 1: Genes expressed early in the visceral mesoderm primodium
Genes expressed early in the visceral mesoderm primodium Protein class Abbreviated phenotype in the visceral mesoderm Reference Twi (Twist) Transcription Factor
Mesodermal derivatives are abscent (Leptin, 1991), (Simpson, 1983) Tin (Tinman) NK‐family homeodomain containing transcription factor Disrupted visceral mesoderm, absence of midgut muscles (Azpiazu and Frasch, 1993)
Bap (Bagpipe) NK‐family homeodomain containing transcription factor Reduction/loss of midgut visceral muscles, loss of midgut constrictions (Azpiazu and Frasch, 1993),
Bin (Binou) FoxF forkhead domain protein
Disrupted visceral mesoderm, absence of midgut muscles (Zaffran et al., 2001)
Β3 Tub 60D ( Β3 Tubulin 60D) GTP binding/GTPase activity
Defects in midgut morphogenesis, failure of gut function (Dettman et al., 1996)
Fas III (Fasciclin III) Transmembrane protein of the Ig (Immunoglobulin) family No visceral muscle phenotype (Patel et al., 1987)
reported Con (Connectin) Adhesion molecule
No visceral phenotype reported (Bilder and Scott, 1998)
Hh (Hedghog) Secreted molecule No visceral phenptype reported (Bilder and Scott, 1998)
20 INTRODUCTION Table 2: Differential expression of genes in the founder cells and fusion competent
myoblasts in the visceral mesoderm
Genes expressed in Founder cells (FC) Genes expressed in Fusion competent myoblasts (FCM) References Alk Receptor Tyrosine kinase Alk Receptor Tyrosine kinase
(Englund et al., 2003), (Loren et al., 2003), (Lee et al., 2003a) Duf/Kirre (Ig family transmembrane protein) (Ruiz‐Gomez et al., 2000) Rst/IrreC (Ig family transmembrane protein) Rst/IrreC (Ig family transmembrane protein) (Strunkelnberg et al., 2001)
Sns (Ig family transmembrane
protein) (Bour et al., 2000) Hbs (Ig family transmembraneprotein) (Artero et al., 2001), (Dworak et al., 2001) Ants/rols (Multidomain protein) (adaptor protein) (Chen and Olson, 2001) Mbc (Multidomain protein) (SH3,GEF and CB) Mbc (Multidomain protein) (SH3,GEF and CB) (Erickson et al., 1997) C3G (GEF) (Ishimaru et al., 1999), (Shirinian, M. unpublished) D‐Titin ( N terminal Ig, PEVK domain, C terminal FN‐III and kinase activity), (muscle sarcomere binding protein) D‐Titin ( N terminal Ig, PEVK domain, C terminal FN‐III and kinase activity), (muscle sarcomere binding protein) (Machado and Andrew, 2000)
Blown fuse (PH domain containing protein) (Doberstein et al., 1997) Sltr/Wip/Vrp (Wasp associated protein) (Massarwa et al., 2007), (Kim et al., 2007), (Eriksson and Palmer unpublished) Dpp (TGF‐β family ligand) (Shirinian et al., 2007) Hand (bHLH transcription factor) (Varshney and Palmer, 2006), (Popichenko et al., 2007) Lameduck (zinc finger transcription factor) * also known as minc (Duan et al., 2001) 21 INTRODUCTION The longitudinal muscles of the visceral mesoderm are derived from the caudal visceral
mesoderm. At stage 11 of embryonic development, the caudal visceral muscle clusters
migrate towards the trunk mesoderm. Once they spread out over the visceral muscles the
longitudinal specific founders fuse with the remaining fusion competent myoblasts of the
visceral mesoderm to form the muscle syncytium (Klapper et al., 2002; Martin et al., 2001).
The caudal visceral mesoderm (CVM) can be visualized by reporter gene expression driven
by enhancer regions of crocodile (croc) and couch-potato (cpo). Brachyenteron (byn), a
Brachyury-related T-box gene, the transcription factor forkhead (fkh) and the Zn finger
homeodomain 1 (zfh-1) are required for caudal visceral mesoderm development. Mutations in
any of these genes result in disruption of caudal visceral mesoderm development (Kusch and
Reuter, 1999).
The hindgut primodium is situated at the posterior pole of the embryo. It is called the
proctodeal primodium (see Figure 2). The most ventral cells of this primodium will become
the hindgut visceral mesoderm (HVM) whereas the rest will form the hindgut epithelium.
During the invagination of the posterior midgut primodium (PMG), the hindgut primodium
simultaneously invaginates. After completion of germband extension, the malpighian tubules
evaginate from the proctodeal primodium. The hindgut visceral mesoderm remains associated
with the hindgut epithelium and begins to migrate around it. By the end of germband
retraction (stage 13) the hindgut elongates by cell rearrangement and is completely
surrounded by the hindgut visceral mesoderm (HVM) (reviewed in (Lengyel and Iwaki,
2002). The foregut primodium is located at the an.terior pole of the embryo which is also
called the stomodeum (see Figure 2). During gastrulation the stomodeum, containing the
foregut epithelium and the foregut visceral mesoderm invaginates and gives rise to the foregut
esophagus and proventriculus.
The Receptor Tyrosine kinase ALK and its role in early specification of visceral muscle
cell types
The signaling pathway regulating the specification of the founder cells and ultimately
controling the fusion process has been identified as the Jeb-Alk RTK signaling pathway, a
novel signaling pathway in Drosophila (Englund et al., 2003; Lee et al., 2003b; Stute et al.,
2004) (Figure 6). Jelly belly (Jeb) encodes a secreted molecule containing a LDL receptor
motif and is expressed in the somatic muscles adjacent to the visceral muscles (Weiss et al.,
22 INTRODUCTION 2001). Jeb secreted from the somatic muscles is taken up by the visceral muscle cells which
express Alk (Figure 6). Alk is the Drosophila homologue of mammalian Anaplastic
Lymphoma Kinase (Alk), a receptor tyrosine kinase which belongs to the Insulin Receptor
superfamily. In the Drosophila embryo, Alk is expressed in all cells of the visceral muscle
(both founder cells and fusion competent myoblasts) (Loren et al., 2001). In both Alk and Jeb
mutants the visceral muscle founder cells do not specify, therefore visceral muscle fusion
does not take place. Consequently, mutants fail to develop a functional midgut musculature
(Lee et al., 2003a; Loren et al., 2003; Weiss et al., 2001).
Figure 6: Schematic figure representing the fusion of visceral muscle cells. The secreted molecule Jelly Belly (Jeb) (green) produced by the somatic mesoderm binds to the Alk expressing visceral muscle cells (blue) and activates the receptor. This activation leads to the specification of the founder cells (A). (B) Fusion of the founder cells with the fusion competent myoblasts generates a muscle fiber with two nuclei. 23 INTRODUCTION Molecular targets of the RTK ALK in the embryonic midgut
Alk has been shown to activate the RAS/MAPK signaling cascade in vivo in the embryonic
visceral mesoderm (Englund et al., 2003; Loren et al., 2001). It has also been shown to
activate transcriptional expression of duf/kirre and the T-Box gene org1 (Englund, 2003;(Lee
et al., 2003a). Our knowledge of downstream signaling molecules activated by Alk is
expanding constantly, and recent work has demonstrated that Alk activates transcription of the
bHLH transcription factor hand (Varshney and Palmer, 2006) as well as the TGFβ family
homologue in Drosophila – dpp - (Shirinian et al., 2007) in the visceral mesoderm (Figure
7).
Figure 7: Schematic representation of the Jeb/Alk signaling pathway and its downstream targets. 24 INTRODUCTION Alk in human disease
Alk was originally identified in a chromosomal translocation resulting in an NPM-Alk
fusion protein, which is the causative agent of ALCL, Anaplastic Large Cell Lymphoma, a
subtype of non-Hodgkin’s lymphoma, arising in T-cells (Morris et al., 1994). Alk fusion
proteins have been also described in inflammatory myofibroblastic tumours (Griffin et al.,
1999), non-small cell lung cancer (SCC) (Soda et al., 2007), and diffuse B-cell lymphoma
(DBCL) (Arber et al., 1996). In a significant breakthough, a series of landmark articles in
2008 identifed gain-of function mutations in ALK in neuroblastoma (Caren et al., 2008; Chen
et al., 2008; George et al., 2008; Janoueix-Lerosey et al., 2008; Mosse et al., 2008). Studying
the function of this receptor in a simpler organism such as Drosophila will hopefully provide
some ground information which might explain its involvement in neuroblastoma and other
diseases.
Development of the somatic muscles in Drosophila
The architecture of larval somatic muscle
As soon as the mesodermal fate is specified after gastrulation, the mesoderm is divided
into two subdomains. A domain of low twist expression which will give rise to the visceral
mesoderm and the fat body, and a domain of high twist expression level which will give rise
to the somatic muscles and the heart. Low twist expression occurs in response to the
segmental gene even skipped (eve) whereas high twist expression is triggered by another
segmental gene, sloppy-paired (slp )(Azpiazu et al., 1996) (Riechmann et al., 1997). Within
the region of high expression there are clusters of cells which express Lethal-of-scute (LSc).
One cell in this population will be singled out by Notch and Delta-mediated lateral inhibition
(Baylies et al., 1998; Carmena et al., 1995). This cell will undergo asymmetric division to
form either two muscle founder cells or one muscle founder and one adult muscle precursor.
The remaining cells by default become fusion competent myoblasts (Carmena et al., 1995;
Ruiz Gomez and Bate, 1997). The development of the larval body wall musculature depends
on these two myoblast populations (Bate, 1990), which express a specific group of
transcriptional regulators that are called “identity genes” (Bourgouin et al., 1992; Dohrmann
et al., 1990; Jagla et al., 1998; Ruiz-Gomez et al., 1997). These transcription factors act
together to regulate specific target genes which in turn will control the unique morphology of
25 INTRODUCTION each muscle. For example founder cells differentially express apterous, even-skipped, kruppel
and ladybird (Dworak and Sink, 2002) whereas all fusion competent myoblasts express the
transcription factors lame duck, myoblast incompetent and gleeful (Duan et al., 2001; Furlong
et al., 2001; Ruiz-Gomez et al., 2002).
There are 30 larval body wall muscles in each abdominal hemisegment of Drosophila
melanogaster. All 30 mature muscles in each hemisegment share common physiological and
contractile properties. However, each muscle is distinct from the rest and can be recognized
by a unique set of morphological characteristics, including size, shape, orientation, attachment
within the epidermis and innervation by the central nervous system (reviewed in (Beckett and
Baylies, 2006). The 30 Drosophila larval muscles in each hemisegment are described below
(Figure 8). They consist of 3 DA (dorsal acute muscles), 1 LL (Lateral longitudinal), 1 LO
(lateral oblique), 4 VL (ventral longitudinal), 6 VO (ventral oblique), 4 DO (dorsal oblique),
4LT (lateral transverse), 1DT (Dorsal transverse), 2 SBM (segment border muscles), 3 VA
(ventral acute), and 1VT (ventral transverse) (Figure 8). In the larvae, the contractions of the
longitudinal muscles result in a shortening of the muscle fiber and forward movement of the
larvae. The musculature of the larvae is almost completely histolyzed during the
metamorphosis of the pupae. At this time a new set of adult muscles is formed from a pool of
myoblasts which remain undifferentiated during embryonic and larval life (Currie and Bate,
1991). The number of fusion events determines the size of an individual muscle, but the shape
is mostly controlled by muscle attachment. Muscles form attachments to specialized
epidermal cells called tendon cells (Figure 9), and also to each other (Schnorrer and Dickson,
2004). Where an individual muscle will attach is initially determined by the position of the
founder cell (FC). The FC determines the location of attachment while the fusion events
determine the muscle type whether longitudinal or transverse. As fusion proceeds, the
myotube extends filapodial extensions searching for a suitable attachment site. Once the
myotube reaches the tendon cell no more filapodia can be observed and the muscle becomes
smooth at both ends (Beckett and Baylies, 2006). This leads to the formation of (HAJs) hemiadherence junctions in both the myotube and the tendon cell. The HAJs are mediated by
heterodimeric transmembrane integrin receptors (described in detail in the following section)
which specifically bind to the intervening ECM. Within the cell, integrins recruit factors that
bind to and cause rearrangements of the cytoskeleton to form a strong
26 INTRODUCTION attachment between the inside and the outside of the cell (Brown, 2000; Brown et al., 2000).
Following integrin attachment to the ECM a large number of proteins become associated with
integrins. I will briefly describe some of best understood of these, such as Vinculin, Talin,
Paxillin and Tensin. Vinculin is a cytoskeletal protein which contains a central proline-rich
domain and a globular head domain that contains binding sites for Talin and α-actinin. The
tail region of vinculin contains binding sites for F-actin, Paxillin and lipids. In the nematode
Caenorhabditis elegans mutations in vinculin cause lethality (Barstead and Waterston, 1991).
However, in Drosophila, vinculin mutants are viable and fertile and have no vital importance
(Alatortsev et al., 1997). Talin is also recruited to the attachment sites by binding the
cytoplasmic tail of integrins (Horwitz et al., 1986; Patil et al., 1999). Talin deficient
Drosophila embryos exhibit failure in germ band retraction and muscle detachment, similar to
integrin null mutants (Brown et al., 2002). Paxillin is a LIM domain containing adaptor
protein which binds to integrin scaffolding proteins such as Src, Crk, Csk, FAK, vinculin etc
reviewed in (Turner, 1998). In Drosophila Paxillin has been shown to colocalize with
integrins at the muscle attachment sites (Wheeler and Hynes, 2001). To date, there is no
Drosophila paxillin mutant available to assess the functional importance of this protein.
Tensin is a multi-domain protein which binds the cytoplasmic tail of the integrin β-subunit via
its PTP domain (Lo, 2004). Drosophila tensin is encoded by the blistery locus and appears to
be required for the stabilization of adhesion in the wing since mutant animals exhibit a wing
blister phenotype (Lee et al., 2003b; Torgler et al., 2004).
27 INTRODUCTION Figure 8: The Drosophila melanogaster larval body wall muscles. Cartoon showing the internal (left) and external (right) muscle pattern of the Drosophila larvae (Reprinted from Karen Beckett and Mary Baylies, 2006, Int Rev Neurobiol) with permission from Elsivier). Muscle position (D, dorsal; L, lateral; V, ventral) followed by orientation (A, acute; L, longitudinal; O, oblique; T, transverse); SBM, segment border muscle. Motorneurons are also indicated in this picture. 28 INTRODUCTION Figure 9: Schematic representation of the organization of muscle attachment sites. Muscles attach to specialized cells called tendon cells, which express the transcription factor stripe. Tendon cells express αPS1/βPS integrins and muscles express αPS2/βPS integrins. At the attachment sites the extracellular domains of both integrin classes bind to the ECM (extracellular matrix). Within the cells integrins recruit factors that bind to and cause rearrangements of the cytoskeleton to form a strong connection between the outside and the inside of the cell (Reprinted from Karen Beckett and Mary Baylies, 2006, Int Rev Neurobiol) with permission from Elsivier). The Sarcomeric structure of muscles
The striated pattern of skeletal and cardiac muscles is due to sarcomeric protein
localization. Recent studies have shown that the localization of sarcomeric proteins is not
fixed. Proteins at the sarcomere and the cytosolic pool can dynamically move. There are for
instance proteins that localize at the Z band but are able to relocate to the A band or the
nucleus (Faul et al., 2007; Kadrmas and Beckerle, 2004; Wang et al., 2005) also reviewed in
(Kadrmas and Beckerle, 2004). The contractile units of the myofibrils are called sarcomeres.
The distribution of sarcomeres under light microscopy is visible as A, I and Z bands (Figure
29 INTRODUCTION 10). The A band comprises thick filaments of myosin and proteins that bind myosin. The
middle part of the A band is called the M line (Figure 10). The I band is composed of thin
filaments of actin and proteins that bind actin. The Z-line is located at the middle of the I
band, and is also known as the Z discs (Figure 10). The Z bands also anchor at the ends of
myofibrils in specialized junctions called costameres. Many myopathies have been linked to
proteins associated with the Z bands (Frank et al., 2006) For example, a mutation within the
kinase domain of the Z band protein Titin is linked to the human muscle disease hereditary
myopathy with early respiratory failure (HMERF) (Frank et al., 2006). Moreover, a Q9R
mutation in α-Actinin - a major component of the Z-disc - causes dilated cardiomyopathy.
This mutation disrupts the interaction of α-Actinin with the muscle LIM protein (MLP)
(Frank et al., 2006). In addition to Titin and α-Actinin, many proteins exist which are
associated with Z bands that are implicated in different myopathies such as
Ankyrin,
Myotilin, Zasp and PkD, CapZ, Nebulin, ϒ-Filamin, Myosin II, PDZ/LIM proteins,
Myopodin amoung others (Frank et al., 2006).
Figure 10: Schematic representation of the sarcomere. 30 INTRODUCTION Guanine nucleotide exchange factors (GEFs) and their role as
regulators of small GTPases
Small G proteins have a typical size of 20-25 kDa and are important regulators of cellular
functions. They cycle between an inactive GDP bound conformation and an active GTPbound conformation. When they are in their active conformation, G proteins interact with
effectors proteins, which will then induce downstream signaling events (Kooistra et al., 2007).
The importance of proper regulation of small GTPases is illustrated by the fact that 15% of all
human tumors posses a mutation in the Ras family of small GTPases (Wennerberg et al.,
2005). Critical elements for the regulation of small GTPases are the GEFs (Guanine
nucleotide exchange factors) and GAPs (GTPase-activating proteins). Through the activity of
their catalytic domain GEFs stimulate dissociation of the nucleotide from the G protein by
modifying the nucleotide-binding site so that the affinity to GDP is decreased and replaced by
GTP (reviewed in (Bos et al., 2007) (Figure 11). In contrast, GAPs stimulate the intrinsic
ability of small GTPases to hydrolyze GTP.
Figure 11: The mechanism of GEF ‐ induced nucleotide exchange. The nucleotide (yellow) interacts with the G protein (white) through its base and phosphate moieties. The GEF competes with the nucleotide for binding to the G protein thereby promoting nucleotide exchange. 31 INTRODUCTION Rap GTPases and their effectors
Rap proteins are members of the Ras-like small G-protein superfamily. The Rap family
includes Rap1 (A and B) and Rap2 (A, B and C). Rap1A was originally identified as a clone
in a revertent screen for cell transformation by oncogenic Ras (Bourne et al., 1990; Kitayama
et al., 1989). Rap1 is activated by various extracellular stimuli, such as RTKs, E-Cadherin and
GPCRs (G-protein coupled receptors), which induce the conversion of the inactive GDPbound form of Rap into the active GTP-bound form, via the stimulation of different GEFs. To
date six classes of GEFs are known to activate Rap1, including C3G, Epac, PDZ-GEFS,
RapGRPs and DOCK4 (Figure 12). Rap1A and Rap1B play important roles in cell polarity
(Schwamborn and Puschel, 2004), strengthening of cell attachments to the extracellular
matrix as well as to neighbouring cells (Bos, 2005), and cell-cell adhesion (Kooistra et al.,
2007). The above mentioned central functions of Rap1 explain its function as an inhibitor of
transformation induced by oncogenic Ras.
32 INTRODUCTION Figure 12: Summary of the Rap1 signalling network. Reproduced from (Kooistra et al., 2007) with permission from the publisher. In Drosophila, elegant work linked Rap1 to adherence junctions function in vivo (AJs)
(Knox and Brown, 2002). Rap1 localizes at adherence junctions, particularly between newly
divided sister cells. Rap1 mutant cell clones lose their normal cohesion as well as their normal
hexagonal shape and detach from the surrounding wild-type tissue. In these cells proteins
localized at the adherence junctions such as DE-cadherin, α-catenin and β-catenin lose their
even distribution and localize at clusters in one side of the cells (Knox and Brown, 2002). A
variety of proteins are described to be activated upon Rap activation, including Integrins,
Cadherins, Rho GTPases, the Cdc42 small GTPases, and the small GTPase Rac, which in turn
influence actin remodeling and organization. I will briefly mention Cadherins and Integrins
which are of most relevance to this thesis.
33 INTRODUCTION Cadherins are a large group of cell-cell adhesion molecules which mediate adhesion in a
calcium dependent manner. Classic Cadherins are by far the best understood in terms of
mechanism and function within a developing animal. The basic feature of this family is the
presence of a conserved intracellular domain that interacts with a set of cytoplasmic proteins
called catenins, which are concentrated at cell-cell adherence junctions. In Drosophila, there
are three classical Cadherins: DE-cadherin (DEcad), DN-cadherin (DNcad) and DN-cadherin
2 (DNcad2). In comparison, mammals have over 20 classic Cadherins (Nollet et al., 2000;
Tepass et al., 2000). DE-cadherin - Shotgun (shg) in Drosophila is a major epithelial cadherin
and is known to be required for maintaining epithelial cell integrity during cell
rearrangements (Tepass et al., 1996). DN-cadherin is the major Drosophila neural cadherin
and is required for axon patterning (Iwai et al., 1997) .
Integrins are a family of cell surface receptors that mediate cellular interactions with the
extracellular matrix. Each integrin is a heterodimer consisting of two transmembrane
subunits, α and β. Drosophila contains 2 β (βPS and βv) subunits and 5 α (αPS1-5) subunits
(Adams et al., 2000; Brown, 2000; Brown et al., 2000). Almost all mutations in PS integrin
genes cause lethality at late embryonic or early larval stages. Lethality is due to detachment of
somatic body wall muscles, failure of dorsal closure and defective germband retraction (Bokel
and Brown, 2002; Brown, 1994). Integrins bind their ligands in the extracellular matrix. The
αPS2βPS binds laminin and αPS1βPS bind RGD-containing ligands. In addition to the
integrin function in attaching the muscles to the epidermis, they have another role in linking
the actin-myosin contractile structures in the muscles. In both cases there is a requirement for
an ECM-dependent adhesion between two muscles or between the muscle and the epidermis.
34 AIMS Aims of this thesis:
•
To understand the interplay between the visceral mesoderm and the endoderm, which
together form the Drosophila midgut, during embryogenesis. This has been
approached as follows:
1. Analysing the importance of the visceral mesoderm for endodermal
development and migration during embryogenesis.
2. Genetic ablation of the visceral muscle and the endodermal epithelium
individually to analyse the function of each tissue.
3. Analysis of visceral muscle development in the absence of the
endoderm using mutants for the transcription factor huckebein .
•
To understand the signaling events between the visceral mesoderm and the endoderm.
•
To study the function of the founder cell specific Guanine nucleotide exchange factor
C3G in Drosophila melanogaster.
35 RESULTS AND DISCUSSION Drosophila Anaplastic Lymphoma Kinase regulates Dpp signaling in the developing
embryonic gut (Paper I).
Who is affecting whom and in what manner, when we consider the visceral muscle and the
endoderm during midgut development in Drosophila? What happens in the absence of either
tissue? How do these tissues communicate? In order to answer these questions we have taken
advantage of the simple model organism Drosophila to study gut development in different
aspects and conditions.
Migration of the endoderm is not complete in the absence of the visceral mesoderm.
Anaplastic lymphoma kinase Alk mutant embryos fail to specify founder cells in the
visceral mesoderm, and therefore do not form a midgut (Loren et al., 2003). Hence, these
mutant animals serve as a good model to examine the status of the endoderm in the absence of
the visceral mesoderm. Analysis of the endoderm in Alk mutants revealed that in the absence
of the visceral mesoderm, the initial anterior-posterior migration of the endoderm is not
affected but the later dorsal-ventral migration is blocked. This finding implies that founder
cells are not required to provide initial cues for the anterior-posterior migration of the
endoderm, and that the fusion competent myoblasts express those proteins required to provide
a ‘track’ such as the integrins that are essential for the migration of the midgut primodia
(Devenport and Brown, 2004; Martin-Bermudo et al., 1999; Roote and Zusman, 1995). This
interesting observation raised questions concerning the regulation of this event and the role of
the RTK Alk. Our investigations of both endodermal and visceral muscle markers have led us
to the following conclusions.
1- Dpp signaling in the midgut is impaired in Alk mutants.
Dpp belongs to the family of TGF-β related ligands and binds to a heteromeric receptor
complex of Type I and Type II transmembrane serine-threonine kinases (Figure 13). Ligand
binding to the type II receptor triggers association between the receptors leading to serine
phosphorylation of the type I receptor by the type II receptor. The activated Type I receptor
initiates pathway specific signaling by phosphorylating a distinct member of the receptorregulated Smad (R-Smad) family of cytosolic signal transduction proteins. Activated R-Smad
associates with a common Smad called (Co-Smad). The heterodimeric Smad/Co-Smad
36 RESULTS AND DISCUSSION complex subsequently translocates to the nucleus where it interacts with DNA-binding
cofactors to activate or repress expression of target genes. In Drosophila there are seven
TGF-β related ligands (including Dpp) (Doctor et al., 1992; Padgett et al., 1987), two RSmads (Mad and dSmad2) and one Co-Smad (Media) (Chen et al., 1996; Henderson and
Andrew, 1998; Newfeld et al., 1996; Raftery et al., 1995). There is also an inhibitory Smad
which inhibits the Smad/Co-smad complex known as Dad (Daughters against Dpp)
(Tsuneizumi et al., 1997) (Figure 13). Dpp is expressed in the founder cells of the visceral
mesoderm at early stages and in parasegment 7 of the developing midgut at late stages.
Analyzing dpp mRNA expression in Alk mutants by in situ hybridization revealed that the
expression of Dpp is dependent on Alk activity in the visceral mesoderm. Since the R-Smad
(Mad) becomes phosphorylated upon receptor activation, we employed phospho-Smad
specific antibodies that recognize activated Smad to analyse Mad activity. In Alk mutants
phospho-Mad staining was absent both in the visceral mesoderm and the endoderm
suggesting that normal Alk function is essential for Mad activation in the developing midgut
and endodermal cell population.
It has been previously shown that expression of the homeodomain protein Labial is
dependent on Dpp expression in the visceral mesoderm (Panganiban et al., 1990; Reuter et al.,
1990; Tremml and Bienz, 1992), and indeed in the absence of Alk no Labial expression could
be observed in the endoderm. Thus the expression of dpp in the visceral mesoderm and
consequently Labial in the endoderm is dependent on Alk in the visceral mesoderm. This
initial finding naturally led to further questions, such as how is the expression of dpp
regulated in the visceral mesoderm, as well as what role does Alk play in regulating this
expression?
37 RESULTS AND DISCUSSION Figure 13: Schematic of Dpp signaling pathway. The binding of Dpp to a type II receptor induces a structural rearrangement of the intracellular/cytoplasmic kinase domain, favoring a catalytically active conformation. Upon activating the type II receptor phosphorylates a serine residue on the type I receptor, leading to Mad recruitment and phosphorylation. Phosphorylated Mad subsequently associates with the co‐Smad Medea and the complex is translocated into the nucleus to regulate target gene expression. 2- Drosophila Alk regulates the nuclear accumulation of Exd
Stultz and coworkers (Stultz et al., 2006) described an analysis of the activation of dpp
expression in parasegment 7 of the developing visceral mesoderm, in which they reported that
the homeodomain protein Exd (Rauskolb et al., 1993) has the ability to activate dpp
expression. Drosophila Exd is a homeodomain protein that translocates to the nucleus in a
regulated manner (Abu-Shaar et al., 1999; Aspland and White, 1997). Moreover, this study
further suggested that the nuclear localization of Exd is regulated by phosphorylation of as yet
38 RESULTS AND DISCUSSION unidentified residues. Given that Alk drives a signal transduction pathway that regulates the
activation of downstream components such as the protein kinase ERK (Englund et al., 2003;
Lee et al., 2003a; Stute et al., 2004), we were interested in examining Exd in the context of
Alk-mediated regulation of dpp. Interestingly, Exd accumulation was abolished in the visceral
mesoderm of Alk mutant embryos (Figure 14A, A’). Using a genetic trick of conversion of all
visceral mesoderm cells into founder cells by ectopically overexpressing Jeb, we observed an
accumulation of Exd in all cells (Figure 14B, B’). This suggests that the Jeb/Alk signaling
pathway regulates Exd accumulation in founder cells leading to dpp transcription in the
visceral muscles.
39 RESULTS AND DISCUSSION Figure 14: Alk regulates the nuclear translocation of Exd. Expression of the homeodomain protein Exd (red), the visceral mesoderm is visualized by anti‐Alk (green (A­C)), DAPI (blue) marks the nuclei (A). (A­B), nuclear accumulation of Exd in wild type embryos (arrowheads in A‐A’ indicate the founder cells). In Alk mutant embryos, nuclear Exd is not observed (B). Conversion of FCMs to FCs by ectopically expressing the Alk ligand Jeb in the visceral mesoderm causes nuclear Exd accumulation in all the cells of the visceral mesoderm (C). Schematic demonstrating the conversion of visceral muscle cells into founder cells (D). 40 RESULTS AND DISCUSSION Fusion of circular and longitudinal visceral muscles in Drosophila is independent of the
endoderm but further visceral muscle differentiation requires a close contact between
mesoderm and endoderm (Paper II)
We wished to understand the visceral mesoderm-endoderm interaction more thoroughly. In
the previous article we used Alk mutants as a model system in which the visceral muscle is
not formed, allowing us to study the endoderm epithelium formation and migration. In this
article we continued to analyze the interaction between these tissues, with focus on the effect
of the absence of the endoderm on the formation of the visceral mesoderm. We have
performed this by the following methods.
1. Ablation of the endoderm
We have performed tissue specific expression of the toxin Ricin using the UAS-GAL4
system (Hidalgo et al., 1995). Ricin binds irreversibly to the ribosome and thereby inhibits
protein synthesis. By utilizing tissue-specific GAL4 drivers, we have thereby ablated one
tissue of the digestive system at a time (Figure 15 A). Ablation of the endoderm caused a
severe disruption of the visceral mesoderm architecture (Figure 15B). Careful examination
revealed that the visceral muscle attempts to migrate dorsally and ventrally to form an
enclosed gut tube, but it never succeeds.
41 RESULTS AND DISCUSSION A
Figure 15: The endoderm is required for visceral muscle stretching and gut tube formation. Schematic representing the endoderm ablation experiment (A). (B­E) Embryos stained with anti.Alk antibody to visualize the visceral mesoderm. (B) wild type embryo (arrowhead, indicates the visceral mesoderm). (C­E) Confocal sections through embryos expressing Ricin in the developing endoderm. Alk positive visceral mesoderm attempts to migrate dorsally and ventrally to creat a gut tube, but is unable to do so (arrowheads in E). 42 RESULTS AND DISCUSSION 2. Analyzing (gurt) gürtelchen mutants
The visceral muscle phenotype observed upon ablating the endoderm strengthened our
assumptions that there is a reciprocal need for both tissues in order to develop a normal
midgut. To genetically test the dependence of the visceral muscle on the underlying
endoderm, we wished to use a mutant that does not develop an endoderm. At this point, our
findings overlapped with those of our collaborators. They had identified a mutant from an
EMS screen for mutants with visceral muscle defects which exhibited a phenotype very
similar to that observed upon ablation of the endoderm with Ricin. This mutant was named
Gurt; due to the belt-like phenotype of the visceral muscle (gürtelchen is German for belt).
Gurt is a new allele of the huckebein (hkb) gene, a transcription factor known to be
required for endodermal midgut development (Bronner and Jackle, 1996). The endodermal
phenotype of the new huckbeingurt mutants phenocopies the endodermal ablation experiment
in that the visceral muscle is unable to stretch and form the midgut tube (Figure 16), instead
forming belt like phenotype.
Figure 16: Gurt mutant embryos display a belt like phenotype due to defects in visceral muscle stretching.Embryos are stained with Fas III to visualize visceral muscles. (B) Belt like gut morphology compared to properly developed gut compartments in wild type embryos (A). 43 RESULTS AND DISCUSSION Careful analysis of these mutants revealed that the phenotype observed is neither due to
visceral cell specification nor muscle fusion defects since founder cells and fusion competent
myoblasts are correctly specified and fused efficiently in huckbein
gurt
mutants. Furthermore,
our morphological analysis of genes required for hindgut development did not reveal any
influence of the ectoderm in visceral muscle stretching, indicating that stretching and
outgrowth of the visceral muscle is predominantly dependent on the endoderm.
44 RESULTS AND DISCUSSION Mutational analysis of the Guanine Nucleotide Exchange Factor C3G in Drosophila
melanogaster reveals a role for C3G in larval muscle development. (Paper III)
The guanine nucleotide exchange factor C3G was originally isolated as a binding partner
for the SH3 domain of the v-CRK adaptor molecule (Knudsen et al., 1994). C3G binds to Crk
through four proline rich regions situated within the central region of the molecule (Knudsen
et al., 1994; Tanaka et al., 1994). Further, the CDC25 catalytic region of C3G has been shown
in vitro to stimulate guanine nucleotide exchange on at least two Ras family members, RAP1
and R-RAS (Gotoh et al., 1995; Gotoh et al., 1997).
Identifying molecules that are downstream of Alk is one approach through which we
would like to explore the key players in this signaling cascade to better understand its
function. Given that Alk is expressed in both founder cells and fusion competent myoblasts,
we naturally wished to analyze genes that are expressed in these cells as potential targets.
C3G was shown previously to be expressed in the founder cells (Ishimaru et al., 1999) and
this expression was further verified by our in situ hybridization analysis, thus making C3G
one possible attractive candidate as an Alk target.
In this study we generated a Drosophila C3G null mutant in order to understand the C3G
function. ∆C3GMS flies are semi-lethal and have shorter life span suggesting that C3G clearly
has a role in the fitness and longevity of flies. C3G is expressed in embryonic CNS, somatic
and visceral muscles. Embryonic somatic and visceral muscles in ∆C3GMS mutants had no
obvious defects. However upon looking closely at the larval muscles we identified a
characteristic morphological defect specifically in the ventral longitudinal muscle 3 and 4.
Furthermore, monitoring of larval movements in vivo revealed defective propagation of
muscle movements (data not shown).The limiting factor in this analysis is the fact that C3G is
also expressed in the CNS, and therefore it is difficult to distinguish if the defect is due to the
function of C3G in the muscle or the brain. To overcome this problem rescue experiments
should be performed by overexpressing C3G specifically in the CNS or the muscles and
scoring for movement defects. More detailed analysis of larval muscle in ∆C3GMS mutant
animals revealed defects in integrin localization at the attachment sites in addition to
irregularities in the distribution of the Z-band specific protein Zasp. Zasp is a PDZ-LIM
domain containing protein that have been shown in Drosophila to genetically interact with
integrins (Jani and Schock, 2007). This suggests that the integrin phenotype in ∆C3GMS
45 RESULTS AND DISCUSSION mutant flies could be due to defective anchorage of the actin filaments to Z lines.
Overexpressing activated C3G in the somatic muscles caused severe defects in the actin
architecture of the larval muscle further strengthening the hypothesis that C3G is required for
the preservation of muscle integrity presumably by regulating integrins in larval muscles.
However, further studies will be required to better characterize these defects in the muscles
and the muscle attachment sites in relation to key proteins that regulate muscle attachment
integrity, contractility and muscle maintenance.
The muscle phenotype observed in ∆C3GMS mutants is also found in genes which are
required for maintaining muscle integrity and preventing muscle degeneration such as the
Dystrophin family of proteins (Blake et al., 2002; Hoffman et al., 1987). Strikingly,
Dystrophin RNAi knockdown has a similar phenotype in the muscles as ∆C3GMS mutants
including the VL3/VL4 muscles (van der Plas et al., 2007). To ask whether the similarity of
the phenotypes at the tissue level is maintained at the cellular level, we assayed for apoptosis
in ∆C3GMS mutants. We were not able to detect any apoptotic features in the larva of ∆C3GMS
mutants as it was the case also in Dystrophin RNAi knockdown. Muscle degeneration in
Dystrophin RNAi knockdown resulted in muscle necrosis characterized as (swollen
mitochondria) rather that apoptosis (van der Plas et al., 2007). Therefore, it is possible that a
similar phenomenon is taking place in ∆C3GMS mutants. A characteristic feature of muscular
dystrophy is that the disease progresses by time when the muscles are used and contracted
more often. A potential role for C3G in progressive muscle degeneration is plausible since the
phenotypes of ∆C3GMS and the RNAi knockdown of dystrophin in larval muscles is very
similar.
Mechanism of action of C3G is another branch of this project which will be very
interesting to address. In the mammalian system C3G has been shown to be mostly activating
the Rap1 GTPase (Balzac et al., 2005; Fukuyama et al., 2005). In Drosophila, Ishimaru and
coworkers (Ishimaru et al., 1999) have shown that Rap1 can rescue the rough eye phenotype
observed by overexpressing C3G, suggesting a genetic interaction between C3G and Rap1.
Double mutants between Rap1 and ∆C3GMS will be useful to test if there is any synergistic
effect. In Drosophila as second member of the Rap family of GTPases – Rap2L – has not
been characterized but is assumed to function in a similar way as Rap1, although there is no
data yet verifying this possibility. Double ∆C3GMS and Rap2L mutants will help us identify
46 RESULTS AND DISCUSSION any genetic interaction. Moreover, GEF activity assays should be performed in vitro to further
confirm C3G as a GEF for the Rap small GTPases in Drosophila.
47 CONCLUSIONS The development of the visceral mesoderm and the endoderm are interdependent:
•
Late endoderm migration and the formation of the endodermal midgut is dependent on
the visceral mesoderm (Paper I). •
The receptor tyrosine kinase Alk regulates the transcription of dpp in the visceral
mesoderm (Paper I). •
Ablation of the endoderm affects the stretching and the outgrowth of the visceral
mesoderm (Paper II) •
The visceral mesoderm and the endoderm are interdependent and the normal
development of both tissues is required to achieve a proper midgut (Paper I and II) Analysis of the Guanine Nucleotide Exchange Factor C3G in Drosophila
melanogaster:
•
∆C3GMS mutants are semi-lethal and exhibit a shortened life span. •
∆C3GMS mutants exhibit a characteristic larval somatic muscle phenotype,
specifically at VL 3, 4 (ventral lateral muscles 3 and 4). •
We suggest that C3G regulates the proper localization of integrins, thereby
regulating muscle integrity. 48 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all friends and colleagues that contributed in
one way or another to this thesis and made my years in Umeå to be fun and warm (As
opposed to cold Umeå’s nature).
My supervisor Ruth Palmer, thanks for introducing me to the world of scientific research and
most importantly to my dear friends (the flies). I will never forget your first funny comment
when you showed me the flies under the microscope (Margret, you should remember not to
sneeze when you are pushing the flies☺ ). Thanks for your continuous encouragement in my
projects, endless support, your caring attitude towards scientific or life related matters, your
constant excitement which was always successful in motivating me. I will always value you!
Camilla, thanks for listening to all my stories from Lebanon and daily life matters. You were
always generous when I needed help.
To members of Ruth Palmer’s laboratory, which I have shared a lot of time with.
Gaurav, thanks for sharing projects together, introducing me to the Indian culture and all the
fun barbeques, jokes and nice time. Therese thanks for awakening the painting hobby in me,
the most effective (in forcing) IKSU companion. I am sure you will finally run 3 Km in 20
minutes! And now I know that tables with lines are old fashion. Fredrik, it is so fun to have
you in the lab; thanks for letting me share your scientific enthusiasm and the happy
atmosphere you have created. Caroline, thanks for your help and most importantly for the fun
we had during these years (Dubrovnik, Concerts, and Parties), for reading the thesis and
thanks for introducing me to your wonderful parents Ann-Britt and Gunnar, for giving me
some parental touch in Umeå.Olga thanks for introducing me to the Samba Orchestra.
Dimitri, it was nice to talk with you about your hobbies and fishing experiences, good luck in
your project. Joel, you have been a nice “visitor” to our lab, good luck with your
studies.Yasuo, it is nice to meet you and looking forward to learn some Japanese, good luck!
Thanks to all the former members of the Ruth Palmer laboratory
Christina, working with you was fun during the start of my PhD. Thanks for introducing me
to the magic confocal! Jana, thanks for your positive attitude and good luck with your big
family.Anna, it was wonderful experience to teach genetics with you. Ludmilla, Therese
Norsken, Mattias, it was really great to share these years with you!
Bengt Hallberg, thanks for your support, encouragement and your nice suggestions
concerning my C3G project.
49 ACKNOWLEDGEMENTS Members of Bengt Hallbergs group are specially appreciated for the Friday Journal Clubs and
Project presentations and the FIKA! Christina, Björn, Cecelia and the former members
Emma, Lubna, Hai-Ling, Charlotte…
Group Jan Larsson, you create happy and nice atmosphere in the floor, Lina, thanks for
joining us to IKSU.Group Dan Hultmark, thanks for making everyday life enjoyable.
Group Åssa Rasmuson-Lestander, it was nice to have another fly group in our floor. Erik
squash was fun and I would like to do it again!
Maria Westling, your help is highly appreciated, big Thanks for you!
Anne Holz and Georg Wolfstetter, thanks for the fruitful collaboration in the endoderm
project.
Simon Tuck and Anna Berghard, thanks for following my progress every year.
Lars, Rahul, Ming, Agneta and Eva, it was fun to be around you also during my Masters
work.
Fly food facility, Media and Dishes, we are very grateful to you, thanks for making our life
easier. Kai, where did you disappear? It was always fun to chat with you.
Freddie, without you many things would not have happened! Thanks for your friendship and
the great times we spent in Umeå, Stockholm and traveling.
Tobias, they say real friends share not only the happiness but also the tough times. You have
been a real friend , thanks for your friendship, for the fun times, for endless laughter’s, for
listening even to my non sense, for the memorable trips, for making Umeå enjoyable, for all
the hobbies and scientific discussion.
Gautam thanks for your companion and for all sort of activities, cultural, Canary Islands
travelling (will you forget the sunset from the mountains and the Veniguera road?). Thanks
for your friendship and affection you always showed concerning all matters. I enjoyed
discussing science with you and thanks for reading my thesis.
The Lithuanian gang: Jurate and Darius thanks for the Skiing experience and the great
dinners at your place! Ignas, Rima, Karolis for the nice parties, ski trips and wonderful time.
Marija P, thanks for introducing me to snowboarding which did not end up to be so
successful but was fun.
Olena and Patrik, thanks for the nice FIKAs, Skiing trips, and special thanks to Patrik for
updating us with the Palestinian activities in Umeå.
Azade, Saeed and Fremi we met in the student corridor and shared many social activities
thanks for the great time.
50 ACKNOWLEDGEMENTS Andreas, thanks for not giving up in teaching me Swedish! Jag tror att nu är Jag redo att prata
svenska!
Aron, Tanushri and Jonathan, thanks for your company and good luck for the future.
The German gang: Chritina, Franziska, Alexandra, I enjoyed all the activities and parties
with you. Barbara, I was going to do a mistake and put you together with the German gang!
My Parents Robinson and Melvina: Em serelines, arants tser kachaleranken, arants tser
kourkouranken, arants tser anverch serov angareli er vor yes garenaye eraganatsenel yerazes.
Shad shenorhagalem tsez! Tsez shad ge serem, as kerke ge neverem tsez yev yeghpayrnerous!
Nazareth yev Garen, ankin, anneman yeghpayrneres, inch pakhdavor em yes tsez bes
tangakin yeghpayrner ounenlous, Shnorhagalem tsez.
Mazen, There is definitely not enough space in this book to thank you. Thanks for your
support and encouragement. What would one ever want more than love, care and someone to
share the dreams with? That’s all what I wanted and luckily have!
For all those whose name is not mentioned, you should forgive me and you are all
appreciated!
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