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JAK signaling is somatically required for follicle cell differentiation in Drosophila

University of Kentucky, T. H. Morgan School of Biological Sciences, 101 Morgan Building, Lexington, KY 40506, USA

View larger version (79K): [in a new window]   Fig. 1. The JAK ligand, Upd, is expressed in the follicular epithelium. (A) Expression of upd in the vitellarium is restricted to the two polar cells at the anterior and posterior end of each egg chamber. Within region 3 of the germarium (B), upd is expressed in the most posterior follicle cells (arrowhead). (C) A schematic representation of upd expression (shaded) illustrates the expression in the polar/stalk cell precursors at the posterior of the germarium and the polar cells in the vitellarium. (D) Stat92E is expressed strongly in the follicle cells of the germarium and terminal cells of chambers of the vitellarium up to stage 4, then weakly in later stages.

View larger version (84K): [in a new window]   Fig. 2. hop mutant ovaries contain fused egg chambers. Heteroallelic combinations of hop alleles show a range of severity of ovarian defects. (A-C) In comparison with wild type (A), loss of hop function results in chambers with additional germ cells, with penetrance and severity determined by allelic combination. Moderate allele combinations, such as hopmsv/hopGA32 (B), show frequent compound chambers. Severe allele combinations, such as hopmsv/hopM38 (C), result in extensive fusion of chambers, with no distinct, separated cysts. Orb antibody staining shows that the additional germ cells are the results of multiple germline cysts encapsulated within a single follicular epithelium. In wild-type ovarioles (D), Orb protein is dispersed throughout the germ cells of cysts within the germarium, but is concentrated in the oocyte of chambers in the vitellarium (Orb in red, DAPI in blue). In hopmsv/hopGA32 ovarioles (E), fused chambers contain multiple germline cysts (arrows), each with its own oocyte. Moreover, a chamber containing 4 fused cysts (F) has 60 ring canals (Kelch staining in red). The multiples of 15 ring canals (F', red) are consistent with chamber fusions rather than extra rounds of germline proliferation.

View larger version (83K): [in a new window]   Fig. 3. hop mutants produce polar cells at the expense of stalk cells. The identity of polar cell fates was assayed using the molecular markers Fas III (in red) and PZ80 (in green) with nuclear staining by DAPI (in blue). In wild-type ovaries (A), Fas III protein is found at high levels in the membranes of all follicle cells of the germarium, but is markedly reduced in all but the polar cells of egg chambers of the vitellarium. ß-galactosidase produced in the PZ80 enhancer trap is not detectable until approximately stage 4, after the egg chamber has exited the germarium. At that time, ß-galactosidase is visible specifically in the two polar cells at each end of the egg chamber. (B) In the intermediate mutant combination hopmsv/hopM75, there are extra polar cells, as indicated by the appearance of both Fas III and PZ80 (arrowheads). The number of polar cells is even greater in more severe mutant combinations, such as hopmsv/hopGA32 (C). The expression of the lacZ enhancer trap line, 93F, was used to mark the stalk cells in wild-type (D), and hop mutant (E and F) ovarioles. In wild type (D) 93F strongly marks the terminal filament (arrow) and the interfollicular stalk cells (arrowheads). In hopmsv/hopM75 (E), there are consistently fewer ß-galactosidase positive interfollicular cells (arrows). In strong mutant combinations, such as hopmsv/hopM38 (F), stalk cells are rare or absent in extensively fused ovarioles. Additional loss of one copy of the upd gene enhances the phenotype of hop mutants. The hopmsv/hopM4 heteroallelic combination shows nearly normal ovarioles (G), with only occasional extra polar cells, as indicated by Fas III (red) and PZ80 (green) and marked by arrows (see Table 1) and rare chamber fusions. However, these phenotypes are dramatically enhanced in hopmsv updYM55/hopM4 females (H).

View larger version (49K): [in a new window]   Fig. 4. Proliferation of follicle cells is not extended in hop mutants. In wild-type (A) ovarioles, follicle cells cease proliferation after stage 6, as indicated by the lack of any PH3 staining cells (in green, with Fas III in red, DAPI in blue) in post stage 6 chambers. As in wild type, in hopmsv/hopGA32 (B) and in hopmsv/hopM38 (C) ovarioles, no PH3 staining can be seen after stage 6. This restriction is visible even within fused chambers where cysts of different maturity are distinct (arrowhead). (D) In hopmsv/hopM75 ovarioles marked with PZ80, chambers with fusions of two consecutive cysts have islands of ectopic polar cells at the intersection of the fused cysts. The number of polar cells in those islands is represented in the graph.

View larger version (75K): [in a new window]   Fig. 5. Upd misexpression stimulates stalk cell production. In wild-type ovaries (A) enhancer trap A101 marks polar follicle cells in the vitellarium. When upd is misexpressed (B-F), polar and stalk cells are mis-specified. Chronic expression of hs-upd results from shifting adults to 30°C (B-F). (B) This treatment causes the frequent loss of polar cell clusters (arrowheads) and development of expanded and morphologically abnormal stalks (arrow). (C-F) The abnormal stalks are not monolayer and often traverse the outside of the chambers to form a continuous ‘rope’. (D, D') The cells in these ropes strongly express Fas III (green) which does not mark normal stalks. However, markers for mature stalk cells, 93F (blue stain in C) and -spectrin (yellow, E, E'), are also abundant in ropes. (F, F') Fusions of egg chambers and mislocalization of the oocyte (asterisk), similar to loss-of-function phenotypes, can occur in chronic upd misexpression, as revealed by Orb accumulation (red).

View larger version (83K): [in a new window]   Fig. 6. JAK pathway activity is required in the soma. Somatic mosaics of Stat92Ej6C8 show the same chamber fusion phenotype as the hop heteroallelic ovaries. Clones are marked by the loss of -Myc (green, mutant cells outlined) and Fas III staining is shown in red. (A) In an ovariole with nearly all mutant follicle cells in the vitellarium (brackets), multiple cysts are fused into a single egg (posterior). Approximately 8 stalk cells can be identified (arrow), all of which are wild type for Stat92E. (B) In a late stage fused chamber, the mutant clone is in the middle of the fusion, presumably corresponding to the anterior terminus of the older chamber and the posterior terminus of the younger chamber. (C) Consistent with the alteration of fate seen in hop mutant heteroallelic combinations, clones of hopc111 mutant cells (lack of green GFP) express the polar cell marker PZ80 (red).

View larger version (91K): [in a new window]   Fig. 7. JAK pathway function affects epithelial follicle cell differentiation. Loss of hop (A) or Stat92E (B-D) in mosaic animals alters epithelial cell fates. (A) In mutant clones (marked by loss of Ub-nGFP in green) generated 3 days before dissection, only cells of early stage chambers of the vitellarium maintain strong Fas III (red) expression (arrowhead). A mutant clone in a late stage chamber of the same ovariole retains little Fas III (arrow). Interestingly, some mutant cells in stage 7 or later chambers maintain Fas III in part of a clone (B-D). Typically the Fas III-positive cells are at the clonal boundary, adjacent to wild-type cells. Moreover, the Fas III-positive cells are almost always close to the terminus of the egg (asterisks).

View larger version (27K): [in a new window]   Fig. 8. Model for the functions of JAK signaling in the ovaries. Anteroposterior patterning of the follicular epithelium is accomplished through a series of cell signaling events. Each event progressively defines somatic fates. The differentiation events are represented as a cascade with the signaling pathways involved in each step indicated in blue. A diagram of an ovariole is colored to indicate the somatic cell identities and is keyed to the fates indicated in the cascade. See text for details.