Figure 1.3 Mechanisms of axon guidance.
1.5 Molecular pathways involved in forebrain development.
Signalling mechanisms between cells specify regional identities within the developing brain (reviewed in Wilson et ah, 2002). Anterior-posterior (AP), dorso-ventral (DV) and left-right (LR) patterning events within the early neural tissue provide differentiation cues to cells dependent upon their position. A number of molecular signalling mechanisms are involved in forebrain development, including the BMP, FGF, Hedgehog, Nodal and Wnt signalling pathways. Descriptions o f two pathways with direct importance to the work presented in this thesis are given below (the Hedgehog and Nodal pathways). The Wnt pathway is discussed in section 6.1.3.
1.5.1 The Hedgehog pathway.
The Hedgehog (HH) pathway is typical o f molecular pathways identified in developmental biology in that it functions in a variety o f tissues at different times throughout development. It is able to direct cells to a variety o f states at different points in development (both temporal and spatial) primarily by the differential response in recipient cells, dependent upon their own status. Following is an introduction to the hedgehog genes of zebrafish and current thoughts on the molecular pathway in which they function.
1.5.1.1 Zebrafish hedgehog genes.
The first hedgehog gene identified was the D rosophila segment polarity gene hedgehog (hh) (Nusslein-Volhard & Wieschaus, 1980; Lee et al, 1992). All hh genes encode two peptides, an N-terminal product which initiates Hedgehog signalling (and is referred to as the HH protein herein), and a C-terminal product that has protease activity (Bumcrot et aL, 1995). The C-terminal product functions to modify the secreted HH protein with the addition o f cholesterol to the N-terminal. This is suggested to be critical for the long-range signalling activity of HH, facilitating its regulated secretion (Lewis et aL, 2001; Zeng et aL, 2001), although the exact mechanism is unclear (Ingham, 2001). The activity o f the secreted HH protein is further enhanced by the addition o f palmitoyl to the C-terminal, by an as yet unidentified enzyme (Kohtz et aL, 2001).
Three homologues of the Drosophila hedgehog gene have been identified in both zebrafish and mammals. The mammalian hedgehog genes, (identified in mouse) are called sonic, Indian and desert hedgehog (Echelard et a l, 1993). In zebrafish, the three hedgehog family genes are called sonic, echidna and tiggywinkle hedgehog (Krauss et al, 1993).
Hedgehog signalling is involved in a large number o f processes within the developing embryo, including floor plate and neuronal specification within the ventral neural tube, eye patterning and retinal cell specification, and the guidance o f certain axons
(reviewed by Marti & Bovolenta, 2002). The most detailed analysis o f vertebrate HH function is focused on its role in specification of ventral structures in the neural tube.
Underlying the ventral midline of the neural plate is the notochord, a rod o f axial mesoderm cells providing structural support to the early, boneless embryo. The notochord tissue o f chick was shown, by grafting experiments, to influence the identity o f cells within the neural tube. Notochord tissue is responsible for establishing a population o f cells in the ventral midline o f the neural tube, called floor plate cells. Floor plate cells in turn have a role in patterning the dorsoventral axis of the neural tube (Placzek et aL, 1990). The Sonic Hedgehog protein was found to be the responsible agent, (Roelink et aL, 1994), secreted from the notochord with the ability to induce both floor plate at the ventral extreme, and motor neurons more dorsally. Subsequently, Shh expression is induced in the floor plate itself. Many cell types (both neuronal and non-neuronal) are found to differentiate in a HH concentration dependent manner along the dorsoventral axis of the neural tube (Patten & Placzek, 2000).
The induction of the zebrafish floor plate however may be regulated by a different mechanism. Fish that lack both Sonic and Tiggy-winkle Hedgehog activity still form a subset of medial floor plate cells in the ventral neural tube (Etheridge et aL, 2001). Instead, Nodal signalling may be the key molecule in zebrafish, as various mutants in this pathway lack all floor plate (Gritsman et aL, 1999). The Nodal and Hedgehog pathways may also be interacting in the zebrafish axial mesoderm (Varga et aL, 1999; Rohr et aL, 2001).
Genes encoding homeoproteins of the Pax, Nkx, Dbx, and Irx families of transcription factors are all responsive to a HH activity in the neural tissue (Barth & Wilson, 1995; Rohr et aL, 2001; Marti & Bovolenta, 2002). The expression o f these genes influences cell identity. The mechanism by which secreted HH can influence gene expression is discussed in the next section.
1.5.1.2 Hedgehog signal transduction.
A summary of the interactions between some of the key proteins in the HH pathway is illustrated in figure 1.4. Hedgehog proteins across species signal by binding to membrane bound Patched (Ptc) proteins (Ingham et aL, 1991; Stone et aL, 1996; Goodrich et aL, 1996). Two Ptc proteins have been identified in both mouse and zebrafish (Lewis et aL, 1999). This Ptc-HH complex is then internalised by the cell, (Strutt et aL, 2001), where it alters the status of the Smoothened (Smu) protein.
In the resting state o f the pathway, without HH signalling, Ptc proteins repress the activity o f the Smu protein, a function described as tumour suppressor activity (Wicking & McGlinn, 2001). Activation of the pathway causes the Ptc protein to lift this repression. Although Ptc is in a complex with HH, there is no interaction between HH and Smu (Ingham & McMahon, 2001). Smoothened is a G-protein coupled receptor, related to the Frizzled family o f Wnt receptors (Dann et aL, 2001) that is essential for classical HH activity (Chen et aL, 2001).
Evidence is also emerging o f a Ptc-independent mechanism o f Hedgehog signal transduction (Ramirez-Weber et aL, 2000; Testaz et aL, 2001), but the molecules responsible for intracellular transduction are as yet elusive. Potential candidates exist such as Hedgehog Interacting Protein (HIP), which binds all vertebrate HH proteins, but it is likely to be involved in regulation of ligand secretion as opposed to activating a reception response (Chuang & McMahon, 1999; Ingham & McMahon, 2001).
The biochemical workings of Smu are unclear, but the transcriptional target of Smu in Drosophila is the cubitus interruptus {ci) protein (Forbes et aL, 1993). Ci is a transcription factor that mediates expression of HH target genes, although mutant analysis reveals that it does not control transcription o f all HH target genes (Gallet et aL, 2000). The ci gene has three vertebrate homologues: gli-1, gli-2 and gli-3. The gli-1 and gli-3 genes are themselves targets of Hedgehog signalling, gli-1 expression is activated in response to HH, gli-3 is repressed (Lee et aL, 1997). This concurs with data suggesting Gli3 is a repressor of HH signalling (Masuya et aL, 1995).