<i>cul-5</i> mutant ovarioles form aberrant egg chambers.

<p>Ovarioles from control (A) and <i>cul-5^EY21463</i> mutant (B–L) females, labelled for Vas (white) and DNA (blue). (A, B) Control (A) and many <i>cul-5</i> mutant (B) ovarioles show normal morphology. In a substantial fraction of <i>cul-5</i> mutant ovarioles (C–J), one or several egg chambers contain more than 16 germ line cells (aberrant egg chambers). (C, D) Cysts in region III of <i>cul-5</i> mutant germaria are often irregularly shaped and fail to take up a oval or round shape as wild type cysts do. (E) Frequently, several follicles containing excess germ cells are observed in a single ovariole. (F) More rarely, <i>cul-5</i> mutant egg chambers contain less than 16 germ line cells. (G) More mature aberrant egg chambers can develop relatively normally. (H) Aberrant egg chambers are also observed in a different allelic combination. (I) In some <i>cul-5</i> mutant ovarioles, individual egg chambers are not separated by a stalk, but by two layers of follicular epithelium. (J) A fraction of <i>cul-5</i> ovarioles has germaria that do not contain germ cells. (K, L) In aged flies, a fraction of ovarioles undergoes morphogenetic catastrophe and lose the highly ordered structure of normally developing ovarioles. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009048#pone-0009048-t001" target="_blank">Table 1</a> for quantification of this phenotype.</p

Follicular morphogenesis in aberrant <i>cul-5</i> mutant egg chambers.

<p>(A–H) Wild type (A–B) and <i>cul-5^EY21463</i> mutant (C–H) egg chambers were labelled for Vas (red) and FasIII (A, C–F) or EyA (B, G–H) (green in A–C, E, G or white in D, F, H). (A–C, E, G) are single confocal planes, while (D, F, H) are projections of Z-stacks spanning the entire corresponding egg chamber. (A) In wild type, FasIII accumulates in two pairs of polar cells at the anterior and posterior pole of the follicle (arrows). (B) EyA accumulates in a complementary pattern, and is excluded from the anterior and posterior polar cells while it accumulates in the nuclei of all other follicle cells. (C–D) In most aberrant egg chambers in <i>cul-5</i> mutants, only the normal two groups of polar cells are specified. (E–H) Only in small fraction of aberrant <i>cul-5</i> follicles, more than two groups of cells have upregulated FasIII (E–F) or downregulated EyA (G–H).</p

<i>cul-5</i> mutant follicles do not contain 2ⁿ germ line cells.

<p>The graph plots the number of germ cells (Y-axis) in 20 randomly chosen aberrant follicles (X-axis). The green and red lines indicate the normal 16 (green) germ cells per follicles, or multiples of 16 (red). For each follicle, we counted the number of germ line cells three times (error bars denote ±SD). The number at the bottom of the column indicates the number of Orb-positive cells (oocytes) found in that particular follicle.</p

<i>cul-5</i> phenotypes as a function of age.

<p>All numbers are percentages except for n, the number of ovarioles examined.</p>1<p>Unless otherwise indicated these phenotypes were observed in ovarioles that also contained ≥1 tumorous follicle, so they were included in the counts for that category.</p>2<p>Observed adjacent to a follicle containing >16 germ cells.</p>3<p>Observed in ovarioles without follicles containing >16 germ cells, therefore included in the category ‘others’ in the upper section.</p>4<p>Phenotypes observed at very low penetrance (<1%) include binuclear germ cells, multilayered follicle cells, or unusually long stalks.</p>5<p>This phenotype was only scored in 54/173 ovaries.</p

Germ line clusters overproliferate in the germarium of <i>cul-5</i> mutant ovarioles.

<p>(A–E) Wild type (A) and <i>cul-5^EY21463</i> mutant ovarioles (B–E) were stained for Vas (blue) and phospho-histone H3 (white). (A) In wild type ovarioles, small numbers of germ cells divide synchronously in the germarium. (B–E) In some <i>cul-5</i> mutant ovarioles, large numbers of germ line cells undergo mitosis in a germarium, while we never observe germ cell divisions in the distal germarium or in the vitellarium (B). (D–E) are projections of a Z-series taken through (C). The asterisk indicates dividing cells of somatic origin in the projections. (F) In wild type egg chambers, each oocyte (arrow) contains exactly four ring canals enriched in F-actin (labelled with Phalloidin). (G) In aberrant egg chambers in <i>cul-5</i> mutants, more than four ring canals are observed in oocytes (arrow) and nurse cells. (H–K) <i>cul-5</i> mutant germaria (J–K) feature large fusomes (labelled with mAb 1b1) spanning more than 16 cyst cells that are never observed in wild type (H–I) germaria. (H, J) are single 0.5 µm planes, while (I, K) are 3D reconstructions of corresponding Z-stacks spanning the width of the germaria. The bracket in K indicates a single polyfusome that is traceable through the entire Z-stack.</p