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ZIC2, a member of the zinc finger of the cerebellum family of transcription factors (ZIC1-5), is arranged in a divergently transcribed bigene pair with Zic5 on chromosome 13 in humans (14 in mice) and consists of three exons (Figure 1.2). Orthologues of the Drosphila odd-paired (opa), the ZIC proteins are defined by the presence of a zinc finger domain (ZFD) consisting of five tandem Cys2His2 zinc fingers (Aruga et al., 2006)(Figure 1.2). The ZFD mediates DNA binding and protein-protein interactions in all family members, and is essential for the nuclear localisation of at least one ZIC protein (ZIC3) (Ware et al. 2004; Brown et al. 2005; Bedard et al. 2007; Hatayama et al. 2008; Pourebrahim et al. 2011, reviewed in Houtmeyers et al. 2013). A similar domain can be found in GLI, GLIS and NKL proteins. In addition to the ZFD, ZIC2 contains a zinc finger N-terminally conserved domain (ZF-NC), the ZIC/Odd paired conserved motif (ZOC) (Aruga et al., 2006), as well as multiple polymeric amino acid tracts such as histidine, alanine and serine/glycine repeats. The presence of the ZOC domain and the makeup of the first zinc finger in ZIC2 results in its classification as a Subclass A ZIC protein, along with ZIC1 and ZIC3 (Houtmeyers et al., 2013). The ZOC domain is required for protein-protein interactions, whilst expansion of the C terminal alanine tract results in reduced DNA binding activity despite the ZFD domain remaining functional, and has been linked to multiple HPE cases (Brown et al., 2001; Himeda et al., 2013; Mizugishi et al., 2004). Recently, two additional domains were identified in ZIC1, 2 and 3: Subclass A N-terminally conserved domain (SANC) and Subclass A C-terminally conserved domain (SACC). Both domains are required for ZIC3 transactivation and presumably have a similar function in ZIC2 (Ahmed et al, manuscript under review).

During gastrulation Zic2, Zic3 and Zic5 are expressed in overlapping domains. It is this overlapping expression that is thought to contribute to functional redundancies between family members. Unique expression domains do exist, however, and it is these unique domains that have been attributed to the variety of diseases associated with Zic family members. At 5.5 dpc, Zic2, Zic3 and Zic5 are expressed in the ectoderm of the extra-embryonic and embryonic portions of the egg cylinder (Elms et al., 2004; Furushima et al., 2000). Transcripts have also been identified in the mesoderm that ingresses through the primitive streak, however expression is absent from the mesoderm that migrates to the extraembryonic regions. The distinctive expression of Zic2 begins at 7.0 dpc when it can be detected in the embryonic node (Figure 1.3). Though Zic3 is also expressed in the node, this does not occur until late gastrulation- early somite stages (7.75 - 8.0 dpc) after Zic2 node expression has ended (Elms et al., 2004). As the mesoderm ingresses into the primitive streak, Zic2 transcripts recede from the posterior primitive streak until expression in the primitive streak is ceased at the early headfold stage of gastrulation (7.75 dpc). It is during the establishment of the neural plate that Zic2 recedes from

Figure 1.2: The location and structure of ZIC2. (a) ZIC2 is arranged in a divergently transcribed pair with ZIC5 on chromosome 13 in humans (14 in mice), with three exons and two introns. (b) Like the other ZIC family members, the ZIC2 protein contains a highly conserved ZF-NC domain followed by five tandem C2H2 zinc finger domains which provide DNA binding activity. A ZOC

domain can also be found in ZIC2 towards the N-terminal, along with several low complexity regions such as alanine and histidine tracts and serine/glycine tracts. Figure is modified from (Houtmeyers et al. 2013).

Figure 1.3: Zic2 expression during mouse gastrulation. (a) A 5.5 dpc, pre-streak stage embryo. (b) A transverse section through the extra embryonic region of a pre-streak stage embryo at the level shown in (a). (c) A transverse section through the embryonic region of a pre-streak stage embryo at the level shown in (a). (d) A transverse section through the embryonic region of a 7.0 dpc, late-streak stage embryo at the level shown in (g). The primitive streak and node are to the right. (e) A 6.5 dpc, early-streak stage embryo at the onset of gastrulation. (f) A 6.75 dpc mid- streak stage embryo with mesoderm moving into the posterior amniotic fold. (g) A 7.0 dpc late- streak stage embryo. The node is now visible and Zic2 transcripts are seen in the node. (h) A 7.25 dpc early allantoic bud stage embryo. (i) A 7.5 dpc late allantoic bud stage embryo. Expression in the node and emerging head process is still seen. (j) A longitudinal section through a 7.5 dpc embryo such as that shown in (i). Zic2 transcripts are not found in the mesoderm of the extra embryonic region or of the proximal embryonic region. (k) A 7.75 dpc early head-fold stage embryo. Zic2 transcripts are now mainly confined to the anterior half of the embryo and the expression in the node has ceased. ae: anterior definitive endoderm, am: anterior mesoderm, hp: head process, n: node, paf: posterior amniotic fold. Scale bar, 50 mm (a – d), 100 mm (e – g), 200 mm (h, i and k) and 170mm (j). Figure taken from (Elms et al., 2004) with permission.

the posterior embryo proper and becomes restricted to the anterior neuroectoderm. By this time, Zic3 and Zic5 are also restricted to this region. The expression of the Zics in the neuroectoderm becomes limited to the dorsal region, which will eventuate into neural crest cells and dorsal neuron production (Elms et al., 2004; Houtmeyers et al., 2013). After the neural tube closes, high levels of Zic2 expression can be detected in the dorsal telencephalon (roof- plate and hippocampal primordium) (Cheng et al., 2006; Okada et al., 2008) and in the anterior and posterior ventral telencephalon (Okada et al., 2008). It is postulated that loss of Zic2 expression in the mid-gastrula node and developing forebrain results HPE.