El Estatuto Electoral aprobado por Decreto Ley N°14250

3.4.3.1.BrdU labelling

3.4.3.1.1.Epidermis

The S-phase marker BrdU reveals that cell proliferation occurs throughout the duration of opercular regeneration, and is not restricted to a dedicated growth zone such as a blastema. However, there are marked differences in the amount of BrdU label in early and later regenerates. Swelling stage and earlier regenerates (labelled 0-2 dpo) show at most a scatter of labelled cells (about 50 or fewer cells visible in dorsal view), mainly in the swelling region (Fig. 3.5A). From rim formation onwards, we find specimens with a high density of labelled cells in the entire opercular filament epidermis with the exception of the rim, plate and spine (Fig. 3.5B-D). Even in the most extensively stained specimens, these distal structures appear to lack staining (Fig. 3.5B and inset in 3.5D). While staining extends from the distal cup wall to the base of the peduncle, the surrounding head region does not appear to experience a similar level of proliferation. In later regenerates (> 6 dpo), BrdU label in the peduncle appears more spatially restricted, with stained cells concentrated laterally, and particularly in the developing wings (Fig. 3.5E-F). Table 3.1 summarises the numbers of specimens showing particular staining patterns in various age groups of regenerates.

Table 3.1. Distribution of epidermal staining patterns in BrdU-labelled whole opercular regenerates. “Complete” means dense epidermal staining throughout the peduncle and cup wall. “Partial” refers to dense staining in only part of the regenerate, e.g. peduncle only, cup only, or patchy. “Lateral concentration” denotes the concentration of staining in the lateral ridges and wings of the peduncle. Cups are generally densely and uniformly labelled in these specimens. “Limited” refers to the lower number of cells observed in early specimens as shown in Fig. 3.5. Numbers in brackets are percentages of the total number of specimens for that stage, rounded to nearest integer.

0-2 dpo 1-3 dpo 2-4 dpo 3-5 dpo 4-6 dpo 5-7 dpo 6-8 dpo 8-10

dpo Complete 0 5 (26%) 7 (39%) 25 (61%) 11 (30%) 2 (20%) 3 (16%) 0 Partial 0 2 (11%) 5 (28%) 7 (17%) 13 (35%) 3 (30%) 0 1 (4%) Lateral concentration 0 0 0 1 (2%) 9 (24%) 5 (50%) 15 (79%) 21 (88%) Limited 9 (25%) 0 0 0 0 0 0 0 No staining 27 (75%) 12 (63%) 6 (33%) 8 (20%) 4 (11%) 0 1 (2%) 2 (8%) Total 36 19 18 41 37 10 19 24

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3.4.3.1.2.Mesodermal tissues

Inside its thick layer of epidermal cells, the opercular filament contains longitudinal muscle fibres (ventrally located in the peduncle), connective tissue (dorsally concentrated in the peduncle, filling the cup) and a major blood vessel [Hanson, 1949]. To observe cell proliferation in these tissues, a small number of specimens were cut into blocks as described in section 2.3 and stained in the same way as whole heads. Overall, fewer cells are labelled in mesodermal tissues compared to the epidermis. There is also an apparent delay in the onset of BrdU incorporation, and mesodermal BrdU label disappears before epidermal staining (Fig. 3.5), although sample sizes for this experiment were quite small. Altogether, 5/6 of the 0-2 dpo specimens with a cup had epidermal but not internal staining (Fig. 3.5G), while 7/8 specimens labelled at 1-3 dpo showed strong mesodermal staining (Fig. 3.5H). The connective tissue inside the cup remains unstained throughout the stages investigated. The blood vessel is densely labelled at 4-6 dpo (Fig. 3.5K, 5/5 cups) but not in later regenerates (Fig. 3.5L, 7/7 cups at 8-10 dpo).

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Figure 3.5. BrdU labelling of whole opercular filaments and portions. A-F. Whole heads. A.

Early swelling stage specimen labelled 0-2 dpo. B. Slightly end-on view of rim stage specimen (labelled 1-3 dpo) showing unstained distal region and dense staining in the rest of the regenerate. Dark distal edge of the spine is homogeneous, non-specific staining. C. Groove stage specimen (labelled 2-4 dpo) with dense staining throughout the opercular filament. Inset shows close-up of the boxed area. D. Specimen labelled 4-6 dpo. Inset is a different view of the same specimen with the unstained opercular plate visible. E. Typical staining pattern for regenerates labelled 8-10 dpo. Staining in the peduncle is concentrated in the lateral ridges and wings, but the cup wall is still more or less evenly labelled. F. Right lateral view of the specimen shown in E. Inset is a magnification of the boxed area, showing the strongly stained right wing. G-L. Blocks cut from opercular regenerates to display mesodermal staining. Small diagrams indicate the rough stage of the specimen (see Fig. 3.1) and the location of the cut with the part of the regenerate not shown in the image faded out. Each image shows a view of the cut surface marked by the dashed line. Asterisks indicate the lumen of the opercular blood vessel. G. Section of early cup stage specimen (0-2 dpo). Arrowheads indicate the thickness of the epidermis. H. Cup stage specimen (1-3 dpo). I. Mid-peduncle cut of a typical 4-6 dpo specimen. J. The same cut in a typical 8-10 dpo specimen. K. Oblique cut through a 4-6 dpo cup. Faint staining inside the cup is in the epidermis on the far wall of the cup. L. 8-10 dpo cup with blood vessel unstained. Three portions of blood vessel lumen are visible due to the coiling of the vessel. Scale bars are approximately 0.5 mm in B-F, K and L and approximately 0.2 mm in A and G-J. Figure from Szabó and Ferrier (2014a).

3.4.3.2.Phosphohistone H3

Phosphorylation of the nucleosomal histone H3 at specific residues (chiefly Ser10 and Ser28) is widely used as a marker for mitotic cells. In regenerating opercular filaments, significant phosphohistone H3 (Ser28) immunoreactivity can only be observed in the epidermis. Generally, few cells are labelled, but these can occur anywhere along the peduncle and cup wall (Fig. 3.6). Because phosphohistone H3 detection does not require long live exposures, very early regenerates could also be tested. At 8 hpo, no staining is observed (n = 12). By 1 dpo (early swelling), specimens may have a scatter of stained nuclei (on the order of 10 cells at most). In general, phosphohistone immunohistochemistry produces much higher levels of background than BrdU staining.

Figure 3.6. Mitotic cells are distributed throughout the opercular filament. Labelled cells in three different regions from three specimens stained for phosphohistone H3 (Ser28). A indicates the rough location of the regions shown in B-D.B. Distal cup wall of a 3 dpo (groove stage) specimen. C.

Mid-peduncle region of a different 3 dpo specimen. D. Base of the peduncle in a 2 dpo (cup stage) specimen. All images are dorsal views. Scale bars are 100 µm. Figure from Szabó and Ferrier (2014a)

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3.5.Discussion