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Dissected presumptive pronephric mesoderm isolated from intermediate mesoderm at gastrula stages of development, does not form pronephros in saline solution (Brennan et al., 1998). Furthermore, transplantation of pronephric mesoderm to a heterologous site, away from the normal position of pronephros development, does not form pronephros in the transplanted region (Fales, 1935). Thus a signal, originating from tissues surrounding the presumptive pronephric intermediate mesoderm, is required for pronephric induction (Fig 1.4B and 1.5).

Figure 1.5 Transverse section of embryos at stage 26 and 35 highlights the cross- section arrangement of the pronephros. (A) At stage 26 the pronephros anlagen is not morphologically distinct or compartmentalized. (B) By stage 35 the pronephros has formed and the glomus, coelom, and tubules can all be observed by section. It is important to note that the glomus is medial to the tubules, not dorsal as many

The most important inductive signal to the intermediate mesoderm comes from the anterior somites (Seufert et al., 1999; Mauch et al., 2000; Mitchell et al., 2007). The nature and timing of this signal have not been elucidated, but the anterior somites are both necessary and sufficient to impart pronephric cell fates in dissected presumptive pronephric mesoderm (Holtfreter, 1933; Tételin, Thesis 2008). Whilst the transcriptional targets of this signal are unknown, it is likely to induce, either directly or indirectly, expression of genes encoding early markers of the pronephros anlagen, namely the paired box gene Pax-8 and the homeobox transcription factor Lim-1, which act synergistically to establish the early pronephric primordium (Carroll and Vize, 1999).

By mid-neurula stages of development the pronephros anlagen has been induced, but remains a mass of cells requiring patterning to allocate the different regions of the mature pronephros (Fig 1.4). Developmental cues pattern the pronephros anlagen and establish two molecularly defined axes, the medio-lateral axis across the proximal pronephros anlagen, and the proximal-distal axis across the entire lateral pronephros anlagen. The first genes that begin to pattern the pronephros are Wilms’ tumour gene-1 orthologue, WT-1, and the LIM homeodomain protein, Lmx1b(Carroll and Vize, 1996; Wallingford et al., 1998; Haldin et al., 2008). Lmx1b is believed to act upstream of WT-1, which is expressed from stage 18, but only in the medial region of the pronephros anlagen. Lmx1b and WT-1 initiate medio-lateral patterning and separation of the glomus (medial pronephric mesoderm) from the tubules (lateral pronephric mesoderm) (Figure 1.4A). WT-1 expression restricts expression of Pax-8, Lim-1 and other markers of the pronephros anlagen, such as Pax-2, to the lateral pronephric mesoderm, by inhibiting their expression in the

medial pronephric mesoderm (Vize et al., 1997). Some researchers do not include the medial pronephric mesoderm a part of the pronephros as it will form the vasculature that makes up the glomus, and is physically removed from the tubules by the coelomic cavity later in development. However, the medial pronephric mesoderm gives rise to a number of pronephric tissues, as shown by a series of experiments performed by Ruth Howland (1916) in the spotted salamander, Amblystoma punctatum. She showed that removal of the entire embryonic pronephros minus the medial pronephric mesoderm (the aorta anlagen), permitted the glomus to form and the nephrostomes to regenerate, but not the tubules. In conclusion the medial pronephric mesoderm in A. punctatum is necessary for formation of the nephrostomes, despite their final location on the lateral side of the coelomic cavity at the tips of the proximal tubules. Therefore the medial pronephric mesoderm is required for formation of the glomus, coelom and nephrostomes. Additionally, some authors contest what stage the pronephric corpuscle is specified. The proximal pronephros has been shown to be specified around stage 12.5, with the distal pronephros anlagen specified later, around stage 14 (Brennan et al., 1998). The glomus and tubules develop separately as a consequence of the proximal pronephros anlagen splitting down its midline, producing the lateral and medial pronephric mesoderms. The pronephric corpuscle is most likely specified at the same time (stage 12.5) as the rest of the proximal pronephros anlagen (Brennan et al., 1999a). Furthermore, despite surrounding tissues contributing signals to induce pronephros formation in the intermediate mesoderm, none have been shown to contribute cells to the pronephros anlagen that could potentially form the pronephric corpuscle. This supports the view that the pronephric corpuscle is derived from pronephric intermediate mesoderm.

In the literature, patterning of the lateral pronephric mesoderm is perceived to occur in both dorso-ventral and proximo-distal directions to form the proximal and distal tubules. Previous publications have concluded that the gene expression patterns ofWnt-4,Notch-1andSerrate-1suggest they have a role in regulation of this dorso- ventral patterning of the lateral pronephros mesoderm (Vize et al., 1997). However, results in this thesis show Wnt-4 and Notch signalling are not involved in dorsal- ventral patterning of the lateral pronephric mesoderm. Furthermore, our results suggest that this region undergoes anterior-posterior patterning rather than a dorsal- ventral patterning event. The lateral pronephric mesoderm forms one continuous tubule that extends from the base of the three nephrostomes to the cloaca and is subdivided functionally along its length; this subdivision being exemplified by specific gene expression patterns which define different functional domains (Raciti et al., 2008). In theory, there is no requirement for a dorsal-ventral signal. Examples of dorsal-ventral patterning during development include establishment of the dorso- ventral body plan of Drosophila (Roth, 2003) and vertebrates (Holley et al., 1995), the dorsal-ventral axis of the limb bud (Capdevila and Izpisua Belmonte, 2001; Robert, 2007), and dorsal-ventral boundary formation in the Drosophila imaginal wing disc (Irvine, 1999). All these examples require dorsal-ventral patterning as the dorsal side of the axis is morphologically and/ or molecularly distinct from the ventral side. The pronephric tubules do not differ across their dorsal-ventral axis; hence there is seemingly no requirement for molecular control of this axis during pronephros development.

Whilst the lateral pronephros mesoderm is not molecularly divergent across its dorsal-ventral axis, it is across its proximal-distal axis. Evidence for this is

provided from the differing developmental stages for the specification of the proximal (stage 12.5) and distal (stage 14) pronephros (Brennan et al., 1998). Furthermore, the molecular expression patterns of genes expressed in the tubules display distinct proximal-distal patterns (Reggiani et al., 2007), and the Notch signalling pathway has been proposed to regulate proximal-distal patterning across the whole pronephros (McLaughlin et al., 2000).

In conclusion, this interpretation of the literature combined with our new experimental data, leads us to conclude that the two axes important in patterning the pronephros at the molecular level are the medio-lateral axis across the proximal pronephros anlagen, and the proximal-distal axis across the lateral pronephric mesoderm. These two axes are fundamental to patterning of the pronephros, with the medio-lateral signal allocating the glomus, coelom and nephrostomes and the proximo-distal signal patterning the tubules.