Lecture 15 Notes on somitogenesis, neural crest cell migration and eph-eprhin signaling March 14, 2011.doc

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University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

1 Lecture 15 March 14, 2011 (1) Somitogenesis and intersomitic boundary formation –|Eph-Ephrin signaling (2) Neural crest cell migration Notes from Gilbert (2003) Developmental Biology, 7 Ed. Chapter 14. Pasquale (2005) Eph receptor signaling casts a wide net on cell behavior. Nature Cell Biology review 6:462 A subset of the text is verbatim Key embryological terms Commitment: signaling events that result in the commitment of a cell towards a particular cell fate. Commitment can be subdivided into two stages: specification and determination. 1) Specification: the first stage of commitment Specification is defined as signaling events that alter the cellular biochemistry and function of a cell such that, when cultured in a neural environment such as a Petri dish, the cell is capable of differentiating autonomously towards a particular developmental pathway. The key point is that while its fate has become more restricted, its fate can be reversed by placing the cell in a new environment, i.e. position in an early embryo. 2) Determination: the second stage of commitment. A cell is said to be determined when its fate cannot be altered by placing it in another region of an embryo. Hence, it differentiates according to its original fate in the embryo. In other words, determination is a term applied to the stage when a cell becomes irreversibly committed to a particular fate. Differentiation: the development of specialized cell types. Somitogenesis At the end of gastrulation, involuting mesoderm cells adopt a mesenchymal morphology. Somites are derived from the paraxial mesenchymal mesoderm cells starting, in most vertebrates, just below the otic vesicles (the anterior domain of the trunk) and form in a rostrocaudal direction on both sides of the neural tube and notochord to the caudal tip of the embryo. The somites pairs consist of a repetition of functionally equivalent units or segments (metamers). Somitogenesis can be subdivided into several stages: periodicity, epithelialization, specification and differentiation. Periodicity: Somitogenesis begins after the presomitic paraxial mesoderm has been transformed into two rods of mesenchyme tissue separated by the notochord and neural tube. The first overt sign of segmentation under high resolution electron microscopy occurs when paraxial mesoderm cells have organized themselves into compact whorls of cells called “somitomeres” in the anterior portion of the trunk. Somitomeres pairs are transformed into epithelial spheres that eventually separate from the PSM. Newly formed somites consist of an outer epithelial layer, surrounding a central cavity, referred to as the somitocoele. Epithelialization of somites and other embryonic tissues is dependent on the expression, secretion and assembly of basal laminae. Formation of paired epithelial spheres processed sequentially from the rostral extremity of the PSM. The rhythm of somite production is characteristic of a species at a specific temperature. For example, in chicken 2 embryos, new somites are formed every 90 minutes at 37 C, forming a total of 52 somites. Humans form 42 to 44 pairs of somites, mice 65 pairs, whereas some snake form up to 500 somite pairs. Gaining a better understanding of the molecular mechanisms regulating periodicity, boundary formation and somite number is the subject of intensive studies in several laboratories. Somite maturation Sclerotome: formed a few hours after the first somite is born. The medial ventral half of the somite de-epithelializes to become mesenchymal sclerotome cells, while the dermomyotome remains epithelial. Sclerotome formation is initiated in response to inductive signals from the notochord, such as sonic hedgehog. Before sclerotome cells can undergo an epithelial to mesenchyme transition, the underlying basal lamina must be broken down by matrix remodeling metalloproteinases (MMPs). Sclerotome cells differentiate into chondrocytes. They migrate on from both sides of the neural tube migrate ventrally towards the notochord (axially) to form the vertebral cartilage and ribs. Myotome: formed soon after sclerotome formation has been initiated. Myotome compartments forms by the delamination of cells from the dorsomedial and ventrolateral lips of the dermomyotome, giving rise to epaxial and hypaxial muscle cell precursors. In the next lecture, more details will be provided on how the caudal and rostral lips of the dermomyotome also contribute to epaxial and hypaxial muscle cell precursors. The dorsomedial lip is induced by signals from the dorsal neural tube (Wnts) and the ventral lateral bud (Wnts, BMP4, FGF5) is induced by the surface ectoderm. Dermotome: As the somite continues to mature, de-epithelialization of the medial lateral cells of the dermomyotome generate cells fated to form the skin of the back. Recent studies indicate that in addition to forming dermal fibroblast, this dermogenic compartment also serves as a precursor pool for other cell types, such as endothelial cells lining a subset of blood vessels. Anterior to posterior fate of somites Somites can be subdivided into five domains based on their locations along the anterior posterior axis: occipital, cervical, thoracic, lumbar, sacral, and caudal: i) occipital somites give rise to the occipital bones and muscles of the tongue, ii) cervical somites give rise to the atlas and axis of the vertebral column and the vertebrae anterior of the ribs, muscles of the neck, pectoral girdle, and forelimb. iii) thoracic somites give rise to the vertebrae, ribs, and trunk muscles iv) lumbar and lumbar/sacral somites give rise to vertebrae, hindlimb and pelvic girdle muscles v) caudal somites give rise to the vertebrae and muscles in the tail Eph receptor signaling This family of receptor tyrosine kinases has been receiving a great deal of attention due to their diverse biological functions ranging from axonal guidance, neural plasticity, angiogenesis, cell morphology, tissue patterning, cell migration, and cell-fate determination. This list is growing every year. Not surprisingly, this is a large family of receptors. In vertebrates there are 10 EphA (EphA1-A10) and EphB receptors (EphB1-B6). A schematic of an EphB receptor as a prototype and the basic design of the two classes of ligands: Ephrin-B and Ephrin-A. Both Ephrin B and Ephrin A have similar molecular designs except Ephrin B is a transmembrane proteins whereas Ephrin-A is anchored to plasma membranes via glycophosphotidylinositol (GPI) moiety at the C- terminus. In addition to Ephrin-binding sites, the N-terminal ectodomain of Eph receptors include a cysteine- 3 rich EGF-like motif and two fibronectin type-III repeats – structural moieties often found in the ectodomain of cell surface receptors. The endodomain of Eph receptors have a kinase domain and a sterile α-motif (SAM). Although binding between receptors and ligands occurs predominantly between members of the same class (EphAs with EphrinAs), numerous examples of strong interactions between classes interactions. An important point is the interactions require close cell-cell contact and the signals are propagated bidirectionally. Eph-ephrin dimers and tetratmers. The first step in Eph-ephrin signaling is the formation of signaling clusters is the monovalent high affinity interactions between receptor and ligand at the N-termini on juxtaposed cells surfaces (dimerization). This is followed by lateral tetramerization via low affinity sites (two receptors and two ligands). Eph-ephrin tetrameric complexes can progressively aggregate into large clusters that enhance signaling. Activation and signaling Tetramerization favors trans-phosphorylation of the cytoplasmic domains, resulting in extensive phosphorylation on tyrosine residues. Further phosphorylation occurs via kinases such as Src. Phosphorylation relieves the inhibitory interactions between the juxtamembrane segment and kinase domain. As you are aware, tyrosine phosphorylation creates SH2 docking sites for many different signaling factors, e.g., GEFs which activate Rho GTPases is one example of signaling molecules that bind to the endodomain of Eph receptors. Mechanisms of Eph signal attenuation and termination Because of the high affinity between Eph and ligands, attenuation and termination of signaling by the simple dissociation of membrane complexes does not appear to be an option. A series of slides illustrate four possible strategies for attenuating
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