Smith+Project

= = = Fate Specification in a Primitive Chordate = = = Efrat Oron, Idse Heemskerk and Bill Smith = = = Biological problem = As an experimental model we will use tunicates, which are invertebrate Chordates. Tunicates occupy the unique position of being the closest extant relative of the Vertebrates (Figure 1). Although there are several thousand tunicate species, only a handful are used experimentally. The most widely studied tunicate is the ascidian //Ciona//. In this course we will also use the species //Phallusia mammillata// and //Ascidiella aspersa//.


 * // Figure 1 //**// . Representative Animals. Genomic data show that tunicates are more closely related to vertebrates than are cephalochordates (indicated by the lines linking the animals). The sister group to the Chordates are the Echinoderms, such as the sea urchin shown here. //

While tunicates and vertebrates have highly similar embryology, anatomy and physiology, as would be expected due to their close evolutionary relationship, the tunicates have several unique features that make them ideal experimental animals. Most significantly, the tunicates are much less complex than vertebrates, which is evident at scales ranging from anatomical to genomic. For example, the end-product of tunicate embryonic development is the larva, which in //Ciona// is composed of only 2,000 cells. Moreover, the development of the larva follows a fixed and well-characterized cellular lineage. The larval organs and tissues typically contain only dozens to hundreds of cells. Equivalently-aged vertebrate embryos would have more than an order of magnitude more cells. This reduced embryonic complexity allows us to image, manipulate, and model morphogenesis at a whole-organ or whole-embryo level. For example, the //Ciona// neural plate and notochord each consist of only 40 cells (Figure 2A and B). The //Ciona// embryo is also considerably smaller than other model Chordates (e.g., // Xenopus // and Zebrafish; Figure 2C), allowing us to capture time-lapse movies of whole developing embryos at high resolution.


 * // Figure 2. //**// (A) Ciona notochord precursor cells (red) at the onset of convergent extension. (B) Ciona neural plate cells (expressing GFP) at the onset of neural tube closure. (C) Relative sizes of // Xenopus//, zebrafish and// Ciona //embryos at equivalent stages (tailbud). Red box indicates an approximately 50// // m //// m x 50 //// m //// m field of view. //

The //Ciona// neural plate is laid out in a simple and invariant grid of 40 cells with rows of cells corresponding to precursors of the forebrain, hindbrain, etc. (Figure 2 B). The tunicate CNS follows two distinct developmental pathways. The spinal cord and hindbrain are specified autonomously by maternally-determined regional expression of transcription factors, while the anterior parts of the CNS (corresponding to the vertebrate fore- and midbrains) require FGF signaling from neighboring cells to be specified (the a6.5 and a6.7 lineage in Figure 3). We have recently uncovered a requirement for gap junction communication and Ca2+ transients in the induction of the anterior CNS (right panel Figure 3).



// Figure 3. Lineage in the tunicate CNS. The three lineages (a4.2, b4.2, and A4.1) at the 8-cell stage are indicated. The “small-a” lineage (red), which gives rise to the anterior CNS, is induced starting at the 32-cell stage. When Ca2+ transients are blocked the small-a lineage defaults to epidermis. //

The precise roles of Ca2+ transients in the development of the anterior CNS are not known. In this workshop we will visualize and quantify these Ca2+ transients spatially and temporally.

= Microscopy =

- Single Plane Illumination Microscopy - Spinning Disk Microscopy = Data Analysis =

- Segment cells in neural plate - Quantify duration, intensity and spatial/temporal coordinates of Ca2+ transients = Skills that students acquire =

Tunicate fertilization, embryo staging, transient transfection, live embryo imaging and image analysis.