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Fused H h - N S u ( f u ) Patched secretory pathway C o s 2 PKA Gli c o m p l e x S lim b Hh precursor + H h - H h C h o l e s t e r o l G li m ediated transcrition G li m ediated rep ression hedgehog e .g . p tc, wnf, b m p , nk e .g . hh, b m p ^Hedgehog receiving cell Hedgehog sending cell

Gli-2 appears to have both activation and repression activity over the expression of HH target genes (Altaba, 1998; Sasaki et aL, 1999). A zebrafish line, you-too, carries a mutation in the gli-2 gene, which interestingly lacks lateral floor plate (Karlstrom et aL, 1999). As discussed in the previous section, HH signalling plays an important role in floor plate specification (though not definitive in zebrafish). A suggested target gene of HH signalling in the floor plate is the wnt4b gene, which is not expressed m. you-too mutants (Liu et aL, 2000). Furthermore, the developing anterior pituitary in you-too mutants is trans-fated and expresses proteins characteristic of lens tissue (Kondoh et aL, 2000).

The nature of the mutation in you-too may result in a protein exhibiting dominant- negative function (Karlstrom et al., 1999). This indicates that target genes o f gli-2 are continually activated, even if HH signalling no longer prevails. It is speculated that anterior pituitary development requires a cessation of HH activity, as opposed to lens tissue, which requires continuous activation of the pathway (Kondoh et a l, 2000), thus leading to the observed fate transition. The implication on the floor plate phenotype and loss of wnt4b expression in you-too may be due to a repressive function of Gli-2, blocking transcription activation by other Gli-proteins.

The HH pathway in relation to anterior pituitary development is discussed further in chapter 3, and manipulated experimentally in chapter 6.

1.5.2 The Nodal pathway.

1.5.2.1 Zebrafish nodal genes.

The nodal gene was initially identified in mouse and is expressed in the primitive streak and then in the node tissue during gastrulation (Zhou et aL, 1993; Conlon et aL, 1994). These tissues give rise to the mesoderm and endoderm germ layers. Two genes related to the mouse nodal gene have been described in zebrafish, {cyclops/cyc and squint!sqt) which are believed to share between them homologous function with the mouse gene (Rebagliati et aL, 1998; Feldman et aL, 1998; Sampath et aL, 1998).

nodal genes are involved in a variety o f pattering events during development (Whitman, 2001). Mouse nodal is expressed in the epiblast prior to gastrulation, signalling to both the extra-embryonic ectoderm and visceral endoderm, tissues that subsequently function to establish the AP axis o f the embryo (Lu et aL, 2001; Beddington & Robertson, 1999). During gastrulation, nodal is expressed in the presumptive mesendoderm (primitive streak) and following gastrulation, it is expressed asymmetrically, exclusively in the left side of the lateral plate mesoderm, where it regulates the expression of genes implicated in LR patterning (Concha et aL, 2000; Wright, 2001).

Expression of zebrafish nodal genes {eye and sqt, previously nodal related! (ndr2) and nodal relatedl {ndrl) respectively) is similar to mouse Nodal expression, sqt is maternally expressed, and both eye and sqt are expressed prior to gastrulation in the marginal cells, then in the germ ring and later subdivide the organiser tissue into two zones, sqt is expressed in a small population of cells called the forerunner cells which will not involute, while eye is expressed in the hypoblast layer of the shield and is later also expressed later in the prechordal plate (Rebagliati et aL, 1998; Feldman et aL, 1998; Sampath et aL, 1998). Loss of eye and sqt expression (double mutants) causes a change o f fate specification for dorsal marginal cells, from endoderm and axial mesoderm to a neurectoderm fate. The involution o f these cells during gastrulation does not occur in mutants (Feldman et aL, 2000) and embryos show a lack o f all dorsal mesoderm and endoderm derived structures (reviewed in Schier & Talbot, 2001).

1.5.2.2 The Nodal signalling pathway

Nodal ligands, in common with other TGFB family proteins, signal by binding to serine-threonine kinase receptors bound to the cell membrane. The molecular

progression of the nodal signalling pathway in both mouse and zebrafish is shown in figure 1,5. There are two types of receptors: type I receptors, which activate intracellular proteins for signal transduction, and type II receptors, which activate type I receptors to transduce the signal (Massagué, 1998). Type I receptors include the structurally similar Alk4 and TARAM-A proteins (Reissmann et aL, 2001); type II receptors include ActRIIA and ActRIIB (Song et aL, 1999).

Additionally, co-receptors are involved in reception of the intercellular Nodal signal, which are members of the EGF-CFC family of proteins. One-eyed pinhead (Oep), a zebrafish co-receptor of Cyc and Sqt proteins, is required for effective Nodal signalling and its loss results in defective mesoderm and endoderm formation, disrupted organiser development and mis-positioning of the AP axis (Gritsman et aL, 1999). Unsurprisingly, this phenotype is identical to that observed in cyc!sqt double mutants, which lack functional nodal ligands (Feldman et aL, 1998), and oep'^' mutants are unresponsive to the over expression o f nodal genes (Gritsman et aL,

1999).

The Nodal signal is transduced from the membrane bound receptors to the nucleus by Smad proteins (Heldin et aL, 1997). In response to the activation of type I receptors by TGF13 signalling, Smad proteins are phosphorylated. Smad2 and SmadS are specific to TGFB (except BMP) and activin response, and Smadl, Smad5 and Smad8

are associated with BMP signalling. Smad4 appears to be an essential component of the transduction o f all TGFBs, including BMP, by forming heterodimers with other Smads. Smad6, Smad? and Smad9 have inhibitory functions (Baker & Harland,

1997; Kretzschmar & Massagué, 1998).

Mouse Smad2 mRNA injections into the early embryo are able to rescue the zebrafish phenotype (Gritsman et aL, 1999), providing evidence to directly link Smad2 function with Nodal signalling. In a Smad2 knockout mouse the node tissue and axial