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Spatial and temporal influence on the specification of migratory somite cells [Elektronische Ressource] : experimental studies with avian embryos / vorgelegt von Liwen He

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71 pages
Aus dem Institut für Anatomie und Zellbiologie (Lehrstuhl II) der Albert-Ludwigs-Universität Freiburg i. Br. (Direktor: Professor Dr. med. Dr. h.c. B. Christ) Spatial and Temporal Influence on the Specification of Migratory Somite Cells Experimental Studies with Avian Embryos INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt im Jahre 2004 von Liwen He geboren in Guangzhou, PR China Dekan Professor Dr. med. J. Zentner 1. Gutachter Privatdozent Dr. med. R. Huang 2. Gutachter Professor Dr. med. R. Korinthenberg Jahr der Promotion 2005 Contents Introduction ................................................................................................................................................... 1 Aims and experimental approaches ............................................................................................................... 9 Methods and Materials ............................................................................................................................... 10 Embryos....................................................................................................................................................... 10 Generation of quail-chick chimeras.........................................................................
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Aus dem Institut für Anatomie und Zellbiologie (Lehrstuhl II)  der Albert-Ludwigs-Universität Freiburg i. Br. (Direktor: Professor Dr. med. Dr. h.c. B. Christ)
Spatial and Temporal Influence on the Specification of Migratory Somite Cells Experimental Studies with Avian Embryos INAUGURAL-DISSERTATIONzur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt im Jahre 2004 vonLiwen Hegeboren in Guangzhou, PR China
Dekan
1. Gutachter
2. Gutachter
Professor Dr. med. J. Zentner
Privatdozent Dr. med. R. Huang
Professor Dr. med. R. Korinthenberg
Jahr der Promotion 2005
Contents Introduction................................1................................................................................................................... Aims and experimental approaches ............................................................................................................... 9Methods and Materials.............................................................................................................................01.. Embryos....................................................................................................................................................... 10 Generation of quail-chick chimeras............................................................................................................. 10 1. .............................................................................. 10Preparation of tools and solutions for microsurgery 2. 12Counting somites ................................................................................................................................... 3.Microsurgical procedures ...................................................................................................................... 12 4. 14Investigation on the fate of migratory somite cells in the limb.............................................................. 5.on the specification of cells migrating from different somites at different stages............ 14Investigation  Identification of quail originated cells and their differentiation .................................................................. 16 1.Treating slides with adhesive................................................................................................................. 17 2. 17Fixation and microtomy......................................................................................................................... 3.Immunohistochemistry andinsituhybridization .................................................................................. 17 4.Microscopy and photography ................................................................................................................ 18 5.Protocols of immunohistochemistry andinsituhybridization .............................................................. 19 Materials ...................................................................................................................................................... 29 Equipment.................................................................................................................................................... 31Results........23.................................................................................................................................................. Fate of migratory somite cells in the limb ................................................................................................... 32 Specification of migratory cells from different somites at different stages ................................................. 38 1.Specification of cells migrating from somite 15 at early stages ............................................................ 38 2.Specification of cells migrating from somite 16 at early stages ............................................................ 40 3. 41Specification of cells migrating from somite 21 at late stages............................................................... 4. 43Specification of cells migrating from intermediate brachial somites.....................................................
Discussion................................................................................................................................6.4.................... Three fates of migratory somite cells .......................................................................................................... 46 1. 46Contribution of somitic cells to limb blood vessels involves both angiogenesis and vasculogenesis ... 2. 47Cells migrating from somites give rise to superficial lymphatics in the limb........................................ Specification of migratory cells in the limb depends on the somite level from which they emigrate ......... 48 Specification of migratory cells in the limb depends on the time at which they leave the somite .............. 50 Conclusion and open questions ................................................................................................................... 52Abstract53........................................................................................................................................................Zusammenfassung..................................................................................................................5.....4................References....................................................................................................................................55................Curriculum Vitae................................................................36.........................................................................Acknowledgments....65....................................................................................................................................Presentations and Publications.................................................................................................................66.Erklärung..................................................................67...................................................................................
1
Introduction
Introduction During gastrulation, migration of cells is highly coordinated with other tissue movements to rearrange the single-layered epiblast blastoderm into three germ layers  dorsal ectoderm, ventral endoderm, and mesoderm in between. After gastrulation, the mesoderm can be divided into five compartments: the head mesoderm, axial mesoderm, paraxial mesoderm, intermediate mesoderm and lateral plate mesoderm. The paraxial mesoderm is situated in two thick bands lying on either side of the neural tube and the notochord. The unsegmented band of the paraxial mesoderm is referred to as presomitic mesoderm or segmental plate in birds. The segmental plate undergoes segmentation and epithelialization to form cellular blocks called somites. Since segmentation takes place from cranial to caudal at regular intervals, somites form and mature in a craniocaudal sequence. A newly formed somite consists of an epithelial ball of columnar cells enveloping mesenchymal cells within a central cavity, the somitocoele (Figure 1). When the somite matures, its ventromedial epithelium undergoes an epithelio-mesenchymal transition to form the mesenchymal sclerotome. The dorsal epithelium of the somite becomes more tightly organized, increasing in height and rotating laterally, and is now referred to as the dermomyotome. The dermomyotome gives rise to the dermis of the back and skeletal muscle (reviewed by Christ and Ordahl 1995). The lateral plate mesoderm resides in the periphery next to the intermediate mesoderm. It consists of two layers: the dorsal somatic mesoderm (somatopleure) and the ventral splanchnic mesoderm (splanchnopleure). The somatopleure is associated with the ectoderm and the splanchnopleure with the endoderm (Figure 1). Limb buds arise as local thickenings of the somatopleure. The limb fields of the chick embryo are located at somite level 16  21 for the forelimb bud (Beresford 1983; Zhi et al. 1996) and at somite level 26  33 for the hindlimb bud (Lance-Jones 1988; Rees et al. 2003). The limb bud is made up of mesenchymal cells covered by ectoderm. It grows outwards along the proximodistal axis. The proximal portion of the limb, the stylopod (humerus/femur), forms first, followed by the zeugopod (radius-ulna/tibia-fibula) in the middle region. The distal portion of the limb, the autopod (carpals-fingers/tarsals-toes), arises last (Figure 2).
2
Introduction
Figure 1.electron micrograph of a transverse fracture of a 2-day chick embryo.Scanning The somite is located between the neural tube and the intermediate mesoderm (courtesy of Dr. H. J. Jacob, Bochum). ao: aorta; co: coelom; ect: ectoderm; end: endoderm; im: intermediate mesoderm; nc: notochord; nt: neural tube; sm: somatopleure; so: somite; sp: splanchnopleure; W: Wolffian duct
Figure 2.Schematic drawing of the skeletal structures of the chick forelimb (originated from Gilbert 2000). The basic skeletal structures consist of a proximal stylopod (H: humerus) with a single skeletal element, a zeugopod (R-U: radius-ulna) with two elements in the middle region, and a distal autopod (C-MC-D: carpals-metacarpals-digits).
3 Introduction The contribution of somites to the development of the limb has been studied extensively. Early histological studies on avian embryos had led to the suggestion that cells detach from the somite, migrate out into the somatopleure of the limb bud where they may participate in limb myogenesis (Fischel 1895). However, the contribution of somites to the limb muscle remained to be an assumption because a permanent cell labelling technique was not available. The undifferentiated somitic cells could histologically not be distinguished from the surrounding mesenchymal cells, neither at the time when they detach from somites nor after they have immigrated into the limb bud. One of the most popular methods for permanent cell labelling is the quail-chick chimerization technique that has been introduced by Le Douarin (1969, 1973). The grafted quail cells are integrated into the chick embryo and participate in normal development and can be distinguished from that of the chick in two ways. (1) The quail heterochromatin in the nucleus is concentrated around the nucleoli, making the quail nuclei distinguishable from chick ones by using appropriate staining methods, such as Feulgen-Rossenbecks technique for DNA staining (Feulgen and Rossenbeck 1924). (2) Quail specific antibodies can be used to identify individual quail cells, even if they are in a large population of chick cells. The assumption that somites contribute to limb muscle was experimentally confirmed by quail-chick chimerization. After grafting a row of somites at limb level between quail and chick, it was shown that the limb musculature exclusively originates from somites (Christ et al. 1974, 1977; Chevallier et al. 1977). Furthermore, single somite transplatations at limb level from quail to chick have defined the contribution of each somite to individual limb muscles as well as the origin of each limb muscle from different somites. In general, one somite contributes at least to three limb muscles, while one limb muscle is derived from more than one somite (Beresford 1983; Lance-Jones 1988; Zhi et al. 1996). Even though the limb musculature originates from the somite, not all portions of the somite give rise to limb muscles. As first described by Fischel (1895), only the ventral edge of the myotome releases single cells that subsequently participate in limb myogenesis. Fischels view was confirmed by later experimental and electron microscopic studies (Christ et al. 1977, 1978). Further experiments performed with quail-chick chimeras indicate that the lateral domain of the somite gives rise to hypaxial muscles of the limb and body wall, whereas the
4 Introduction medial domain gives rise to epaxial muscle of the back (Ordahl and Le Douarin 1992). These two myogenic lineages are derived from two precursor populations which originate during gastrulation. The medial half of the somite originates from Hensens node, and the lateral half comes from the cranial part of the primitive streak (Selleck and Stern 1991; Schoenwolf et al. 1992; Psychoyos and Stern 1996). Recent investigations on somite and limb development at molecular level have shown that many genes and signal molecules are involved in the process of limb myogenesis. Since all of the limb muscles originate from somites, the first step in limb myogenesis is the specification of the precursor cells in the dermomyotome.Pax3 isthought to be involved in this process since it is expressed at a high level in the lateral dermomyotome where the limb muscle precursors generate from (Goulding et al. 1991, 1994; Bober et al. 1994; Williams and Ordahl 1994). In addition,Pax3mutant mice (Splotch) (Epstein et al. 1991; Goulding et at. 1993) fail to develop limb muscles but make apparently normal trunk muscles (Franz 1993; Franz et al. 1993; Bober et al. 1994; Goulding et al. 1994; Daston et al. 1996) indicating thatPax3plays a role in the establishing and maintaining the muscle precursor pool in the lateral dermomyotome. The second step in limb myogenesis is the migration of the precursor cells from the dermomyotome into the limb bud. An interaction between the transmembrane tyrosine kinase receptorc-met and its ligand SF/HGF (Scatter Factor/Hepatocyte Growth Factor) controls the release of dermomyotomal cells (Bladt et al. 1995; Maina et al. 1996; Dietrich et al. 1999). Thoughc-metis expressed in the lateral dermomyotome at all axial levels, its role is defined bySF/HGF, which is expressed only in the proximal limb mesoderm (Bladt et al. 1995; Dietrich et al. 1999). SF/HGF also has a role in guiding the proximodistal migration of the precursor cells by keeping them in an undifferentiated state (Scaal et al. 1999). Another homeobox geneLbx1is thought to be required for muscle precursor cells migrating along appropriate pathways (Schäfer and Braun 1999; Gross et al. 2000; Brohmann et al. 2000). Lbx1 expressed in the dermomyotomal lips of those somites that are destined to generate is migratory cells and in the migratory somite cells (Jagla et al. 1995; Dietrich et al. 1998; Mennerich et al. 1998;Schäfer and Braun 1999). The third step of limb myogenesis is the proliferation of myogenic cells and the differentiation of muscle cells after migratory precursor cells have reached the limb bud. The myogenic program is triggered by the expression of the
5 Introduction MRFs (Myogenic Regulatory Factors), includingMyf5,MyoD,myogenin andMRF4(Weintraub et al. 1991).Myf5 andMyoD mark the initial onset to myogenic commitment (Ontell et al. 1995; Delfini et al. 2000), therefore are also referred to as myogenic determination genes.Myogenin is expressed later and has a role in activating terminal differentiation of muscle cells (Hasty et al. 1993; Nabeshima et al. 1993). AlthoughPax3, c-met andLbx1 co-expressed in the lateral dermomyotome where the are precursor cells migrate from, and in the migratory somite cells, it is not yet determined whether the muscle fate of these cells is already committed in the somite before their migration or is adopted in the limb bud after their migration. The migratory cells do not express myogenic determination factors when they are located in the somite or during their migration (Ott et al. 1991; Pownall and Emerson 1992), indicating that they are undifferentiated before they reach the limb bud. This undifferentiated state of muscle precursor cells is even maintained for two days after their settling in the limb bud (reviewed by Christ et al. 1986). In addition to myogenesis, somites also contribute to the vasculature of the limb. Endothelial precursor cells are detected in all parts of the somites (Pardanaud et al. 1996). Quail-chick chimera analysis has revealed that all regions of the somite, including dorsal half, ventral half and somitocoele cells, are capable of contributing to the developing vasculature (Huang et al. 1994; Wilting et al. 1995). Endothelial cells of somitic origin participate in the formation of limb blood vessels (Lance-Jones 1988; Beddington and Martin 1989; Wilting et al. 1995; Pardanaud et al. 1996; Zhi et al. 1996; Ambler et al. 2001; Huang et al. 2003). Not only the dorsolateral somite quadrant but also the dorsomedial somite quadrant gives rise to endothelial cells of the limb (Wilting et al. 1995). Moreover, somites give rise to lymphatic endothelial cells of the limb as well. The lymphangiogenic potency of somites has been proved by quail-chick chimerization studies (Schneider et al. 1999; Wilting et al. 2000). Somite-derived endothelial cells were detected in the lymphatic vessels of the forelimb after grating the dorsal half of somites (Wilting et al. 2000). Till now, evidence from numerous experimental studies indicates that in addition to muscle cells, somites give rise to endothelial cells of both vascular and lymphatic vessels in the limb as well. Two questions arise as somites have the potential to derive multiple cell populations in
6 Introduction the limb. The first question is how somitic cells contribute to limb blood vessels and lymphatics. The second question is which factors determine the angiogenic and myogenic specification of somitic cells in the limb. Embryonic vasculature is formed by two processes: vasculogenesis and angiogenesis (Risau et al. 1988). In vasculogenesis, precursor cells originatede novofrom mesodermal mesenchyme (endothelial precursor recruitment, reviewed by Risau and Flamme 1995). Dependent on the site where the mesodermal endothelial precursor cells form blood vessels, vasculogenesis can be divided into two types (Poole and Coffin 1989; Coffin and Poole 1991). Vasculogenesis type I is characterized byin situ and differentiation of mesodermal endothelial proliferation precursor cells that form blood vessels. During vasculogenesis type II mesodermal endothelial precursor cells migrate from the site of origin to their destination where they proliferate and differentiate to form blood vessels. On the other hand, angiogenesis refers to the process of vessel formation by centrifugal growth of pre-existing vessels (blood vessel sprouting, Risau 1997). By labelling the whole somite or large portions of the somite, previous studies did not answer the question how somitic cells participate in the formation of limb blood vessels (Lance-Jones 1988; Beddington and Martin 1989; Wilting et al. 1995; Pardanaud et al. 1996; Zhi et al. 1996; Ambler et al. 2001; Huang et al. 2003)  via vasculogenesis or / and angiogenesis. The somitic endothelial precursor cells might first form blood vessels within the somite, from which the pre-formed vessels sprout into the limb bud (angiogenesis). On the other hand, if somites contribute to limb blood vessels in the same way they contribute to limb muscle, the process may also take place in three steps. Precursors of endothelial cells may first arise from the somite, from where they migrate into the limb bud, as the myogenic precursor cells do, and then differentiate into endothelial cells to form limb blood vessels (vasculogenesis type II). Similar to the formation of vasculature, embryonic lymphatic system is formed by endothelial precursor recruitment and vessel sprouting processes as well (reviewed by Wilting et al. 1999). By grafting the dorsal half of somites, the previous study did not answer the question in which way somitic cells contribute to limb lymphatics. Limb lymphatic vessels may develop from the somite via three steps as limb myogenesis occurs. Precursors of lymphatic endothelial cells might first arise from the somite, from where they migrate into the limb bud, and then
7 Introduction differentiate into lymphatic endothelial cells that form lymphatic vessels (endothelial precursor recruitment). Alternatively, the limb lymphatics may arise by spouting of lymphatic endothelial cells which have a somitic origin (vessel sprouting). The hypothesis that somites contribute to endothelial cells of both vascular and lymphatic vessels in the limb via somitic cells migration is based on the observation that the expression domain ofVEGFR2(Vascular Endothelial Growth Factor Receptor) is similar to that ofc met. -VEGFR2 is expressed in both blood vascular and lymphatic endothelial cells (Wilting et al. 1996, 1997), and is considered to be a marker of endothelial precursor cells in early development (Eichmann et al. 1993, 1996). During somitogenesis,VEGFR2is expressed in the dorsolateral region of the segmental plate and the newly formed somites. After somite maturation, expression ofVEGFR2 persists in the lateral dermomyotome and sclerotome (Nimmagadda et al. 2004).Asc-met is a marker of somite migratory cells, the spatial co-expression ofc-metandVEGFR2in the lateral dermomyotome leads to the first question addressed in our study whether cells migrating from the lateral dermomyotome include endothelial precursors. Controversy on the factors that determine the specification of somitic cells in the limb has lasted for a long time.In vivolineage analysis with retroviral vectors indicates local extrinsic signals in the limb determine the muscle and endothelial fate of the uncommitted somitic cells (Kardon et al. 2002).This view is in agreement with earlier chimerization studies: flank somites give rise to limb muscle when they are grafted into the position of limb level (Chevallier et al. 1977); likewise, when limb somatopleure is grafted into the flank, it gives rise to an ectopic limb, to which the flank somites provide the muscle (Hayashi and Ozawa 1995). On the other hand, based on grafting and gene overexpressing experiments, it is suggested that intrinsic,Hoxgene-dependent cues determine the fate of skeletal muscle precursors (Alvares et al. 2003). SincePax3 a  ismarker for myogenic precursor cells (Goulding et al. 1991, 1994; Bober et al. 1994; Williams and Ordahl 1994; Franz et al. 1993; Daston et al. 1996) and VEGFR2for endothelial precursor cells (Eichmann et al. 1993, 1996), the spatial co-expression of these two genes in the lateral dermomyotome may support the view that angiogenic and myogenic specification occurs in the somite. Evidence from chimerization studies shows that somite-derived endothelial and muscle cells
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