Organogensis

VI.1 COORDINATORS
VI.2 PARTICIPANTS
VI.3 SUMMARY
VI.4 INTRODUCTION
VI.5 SPECIFIC AIMS
VI.6 BACKGROUND AND SIGNIFICANCE

• VI.6.i General Background to Organogenesis
• VI.6.ii Interplay of Physical and Genetic Mechanisms in Organogenesis
  • VI.6.ii.a Cell Adhesion
  • VI.6.ii.b Chemotaxis and Haptotaxis
  • VI.6.ii.c Epithelial and Mesenchymal Tissues
  • VI.6.ii.d Epithelia and Cell Polarity
  • VI.6.ii.e Mesenchyme and Extracellular Matrix
  • VI.6.ii.f Mesenchymal Condensation
  • VI.6.ii.g Cell Excitability
  • VI.6.ii.h Reaction-Diffusion Mechanisms
  • VI.6.ii.i Differentiation
  • VI.6.ii.j Summary of Basic Mechanisms of Pattern Formation and Morphogenesis
  • VI.6.ii.k Neuronal Guidance, Fasciculation and Defasciculation
• VI.6.iii Background to Specific Experimental Systems
  • VI.6.iii.a Gastrulation
  • VI.6.iii.b Limb Development
  • VI.6.iii.c Innervation
    • VI.6.iii.c.1 Skeleto-muscular innervation
    • VI.6.iii.c.2 Retinotectal innervation
  • VI.6.iii.d Cardiovascular Development
    • VI.6.iii.d.1 Early vertebrate heart development
    • VI.6.iii.d.2 Trabeculation

VI.7 THEORETICAL FRAMEWORK
VI.8 PRELIMINARY RESULTS
VI.9 RESEARCH DESIGN AND METHODS
VI.10 RELATIONSHIP TO CYTOSKELETON (PROJECT 2) AND BIOLOGICAL NETWORKS (PROJECT 1)
VI.11 TIMELINE

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VI.6.iii Background to Specific Experimental Systems:

We have selected four developmental processes in vertebrates, which illustrate in unusually clear ways particular generic behaviors and their relation to underlying physical genetic-control mechanisms: gastrulation, limb skeletal development, innervation and fasciculation of nerves, and cardiovascular development. Related experimental systems under investigation by Consortium participants will be referred to more briefly.

VI.6.iii.a Gastrulation:

Gastrulation, the process by which germ layers are established and take up their characteristic positions in triploblastic (three-layered) embryos, is absolutely critical to the development of vertebrate and most invertebrate organisms. Coherent and highly patterned motion of cells, over distances which are large compared to cell dimensions, characterizes gastrulation in vertebrate development (Wolpert, 1998). Such large-scale rearrangements are necessary because early cell differentiations leave cells of a given type in the wrong location for their ultimate function. Invagination, radial intercalation, convergent extension, and epiboly are frequent motions that all occur in the gastrulation of chicken and the frog Xenopus laevis (Keller and Shih, 1992 and references therein).

Several mechanisms may affect cell movement during gastrulation. Reaction-diffusion establishes large-scale dynamic distributions of activator and inhibitor. Under the influence of the activator-inhibitor field, some cells are specified and form an organizer, which directs cell differentiation and movement in other regions (Meinhardt, 2001). Cells in an organizer, such as the posterior marginal zone (PMZ), secrete diffusible chemicals or growth factors), which may guide cell movement. Cells in other regions may relay and help to establish and stabilize such a chemotactic field (Painter and Maini, 2000). Our experiments provide evidence that differential cell division in various regions of the area pellucida may assist pattern-forming cell movement before and during primitive streak formation.

Gastrulation in vertebrates is a characteristic involution of blastomeres that migrate below the surface layer of cells to form the three germ layers; following gastrulation, the remaining surface layer is ectoderm, the internal cell layer is endoderm, and the cells between these layers are mesoderm. Blastomeres continue to converge and extend to elongate the body axis. During this critical period of embryogenesis, cell-fate determination, cell movements and tissue assembly events coordinate to produce a series of inductive signals that pattern the body axes and body shape to form primordial tissues for all future organ development (Myers et al., 2002). Therefore, detailed analysis of cell movements during gastrulation should reveal novel control points that determine body form and axis orientation. Sophisticated methods are available to label cells and observe cell migration during gastrulation (Jessen et al., 2002).

We will study gastrulation in the chick and zebrafish. Advantages of the chick include the large size of the gastrulating region and the ease with it can be maintained in culture, the embryo can be locallcaly transfected using local DNA injection and electroporation and the embryo is flat and translucent which makes it very accessible to investigation with modern microscopic techniques. Advantages of the zebrafish include its well-characterized and easy-to-manipulate genetics.

In chick interactions between wnt and vg1 signaling pathways in the posterior marginal zone (PMZ) at the boundary between the area pellucida and the area opaca, initiate primitive streak formation (Skromne and Stern, 2001, Heisenberg, 2000), visible as a thickening of the epiblast along the midline in the posterior region. Latero-posterior cells in the area pellucida migrate towards and ingress through the primitive streak and differentiate into individual mesoderm and endoderm cells. The anteriormost part of the streak, Hensen's node, acts as an organizer. The primitive streak elongates anteriorly along the midline; and epiblast cells in the streak undergo an epithelial to mesenchymal transition (EMT), starting to move as individual cells into the space between the epiblast and the hypoblast to form axial and lateral mesoderm as well as definitive endoderm ( Lawson and Schoenwolf, 2001). After the primitive streak reaches 60-75% of the length of the area pellucida, it starts to regress. Progression and regression establish the main body plan. Reaction-diffusion may play a key role in creating the organizers and the developmental axes (Meinhardt, 2001). Differential cell adhesion and division may cause the latero-posterior cells in the area pellucida to move first towards the PMZ then towards the primitive streak. During the EMT, adhesion among cells weakens and the originally attached cells migrate as individuals through the primitive streak into the blastocoel. Organizers may secrete repellents and attractants, causing mesoderm cells to move out from the midline after ingression and causing some of them to rejoin the main body where they will later form the somites (Yang et al., 2002).

While the chick embryo is ideal for identifying the mechanical processes at work during gastrulation, the zebrafish allows much more detailed genetic investigation. Large-scale mutagenesis screens have identified the zebrafish genes that control early morphogenetic movements. Mutations allow us to assign genes that regulate gastrulation cell movements into three groups: genes that regulate dorsal mesoderm formation and central nervous system patterning; genes that control ventral signaling pathways that regulate ventro-posterior structure formation; and genes that control convergence and extension associated with axis definition (Myers et al., 2002). Zebrafish mutants with defects in gastrulation permit a detailed dissection of the signaling pathways which control these cell movements.