C. capitata oogenesis is a suitable system for comparison
The C. capitata eggshell carries no structures that can be identified as homologues of the operculum, outward micropyle and dorsal appendages (Figure1B). Still, it is not entirely symmetrical, both over the anteroposterior axis and the dorsoventral axis. The anterior end of the chorion shows markedly stronger imprints of (previously present) follicle cells when compared to the posterior end. While we cannot say with certainty which side is dorsal and which is ventral, it is clear that one is more convex than the other. As both late stage egg chambers (Figure1A,D) and early embryos (data not shown) are clearly more convex at the ventral side, it is a reasonable assumption that the convex side of the egg is ventral.
From an initial observation of C. capitata oogenesis we can conclude first and foremost that it is a suitable system for comparison with D. melanogaster. C. capitata ovaries, like those of Drosophilidae, are meroistic polytrophic ovaries. While the egg chamber of C. capitata is usually larger than the corresponding stage in D. melanogaster, there is no notable difference in the number of cells that make up the follicular epithelium. Instead, the size of C. capitata follicle cells is increased with respect to those of D. melanogaster, thus contributing to a larger egg chamber as a whole (Figure1A).
The structure of the egg chambers as well as the progression of stages is nearly identical to that of Drosophila, providing a good basis for comparison (Figure1A). Starting at mid-oogenesis, we can observe the asymmetric localization of the oocyte nucleus (stage 8), as well as follicle cell migration (stage 9), and centripetal migration (stage 10B). Also visible is the dumping of nurse-cell content into the oocyte, as evidenced by the increasing size of the oocyte relative to the nurse cells, which disappear eventually. All these are important and stage-defining steps in Drosophila oogenesis. We will therefore refer to the stages defined in D. melanogaster when describing C. capitata oogenesis.
In addition to the migration of the main body follicle cells, a cluster of anterior follicle cells can be seen to migrate between the nurse cells at stage 9. Their migration ends at the posterior edge of the nurse cells, adjacent to the oocyte, where they are shortly joined by the centripetally migrating follicle cells. In D. melanogaster these cells are known as border cells, and can be identified by the expression of slbo, as well as with the polar-cell-specific label Fasciclin II. Both markers confirmed the identity of the border cell cluster in C. capitata (Figure1C,D). Interestingly, as the border cells have been associated in D. melanogaster with the formation of the micropyle, no obvious external micropyle can be seen on the C. capitata egg (Figure1B). However, upon closer examination of the newly formed eggshell we found a pore-like structure on the anterior side of the eggshell, likely homologous to the micropyle pore (Figure1E). This is consistent with the observed border cell localization in C. capitata, as these cells are known to form the pore of the micropyle, but not the outwardly visible structure.
Both EGFr and Dpp pathways are active in C. capitata oogenesis
In C. capitata ovaries, the initial activation of the dorsoventral patterning cascade by the ligand Gurken occurs similarly to D. melanogaster. In the early stages, the Cc-grk transcript is visible in the oocyte at the anterior cortex (data not shown), and around stage 8 the pattern becomes restricted to the putative dorsoanterior side of the oocyte (Figure3A,B). The transcript disappears around stage 11.
While we were unable to obtain patterns of EGFr activation because of practical difficulties, the fact that TGFα-EGFr signaling is conserved in insects as distant as Tribolium and Gryllus, functioning upstream of embryonic dorsoventral patterning even in drastically different systems of oogenesis, makes it unlikely that this would be any different in C. capitata. Indeed, we observed the dorsal repression of a known target of EGFr signaling in D. melanogaster: the gene pip (Figure4D,D’).
In contrast with oogenesis in D. melanogaster, Cc-dpp is not expressed in the somatic follicle cells, but instead in the germ line. Expression of Cc-dpp is first visible as early as the germarium. Once the egg chamber is formed, the dpp transcript localizes to the oocyte. When the oocyte increases in size, the mRNA seems to accumulate at the putative anterior end of the oocyte, in a ring around the edge, adjacent to the follicle cells (Figure3D,D’,I). Interestingly, this ring is reminiscent of the D. melanogaster pattern, where dpp is expressed in the stretched follicle cells as well as a few anterior rows of columnar follicle cells, resulting in a similar ring of dpp expression around the anterior end of the oocyte (Figure3C’). The main difference, of course, is that the transcript is located in different cell types.
One exception to the exclusive germ line expression of Cc-dpp is the border cell cluster. This migrating group of anterior follicle cells is not known to express dpp in D. melanogaster, but is the only group of somatic cells during oogenesis to express Cc-dpp. Expression is visible around stage 8, when the cell cluster is defined (Figure3D, empty arrowhead), and persists through migration until the edge of the nurse cells is reached.
A possible second group of Cc-dpp expressing follicle cells was identified using fluorescent in situ hybridization (FISH). This group of cells is centrally located between the nurse cells and the oocyte in late stage 11 (Figure3I’). Due to the very small sample size we cannot say with certainty whether these cells are the border cells or part of the follicle cells that have centripetally migrated inwards. As the signal of Cc-dpp expression does not persist in the border cell cluster after migration is completed, the observation could either indicate a new round of Cc-dpp expression in this cluster should these cells indeed be border cells, or it could point to conservation of dpp expression in the leading edge of centripetally migrating follicle cells.
While expression of the ligand may differ somewhat between the two species, downstream signaling is remarkably similar. The expression of the homologue of the Dpp pathway type I receptor tkv is not visibly different in C. capitata from D. melanogaster: Cc-tkv is expressed in the follicular epithelium (Figure3E,F), and disappears around stage 11 or 12. More importantly, the activity of the pathway, shown through immunohistochemistry for the phosphorylated form of Mad (pMad), is initially not different between the two species, despite the altered localization of the dpp transcript (Figure3G,H).
Differences in Dpp pathway activation between C. capitata and D. melanogaster start around stage 10B, when expression of Dm-tkv becomes restricted to the Br-positive cells of the appendage primordia, naturally affecting pMad patterns[51, 52]. These dynamics were not observed in C. capitata, where no Br-positive domains are formed (Figure4F’).
Patterning of the follicular epithelium downstream of EGFr and Dpp
The dynamics of EGFr and Dpp signaling and subsequent epithelial patterning in D. melanogaster egg chambers are key in defining the appendage primordia. Identifying the point in the genetic network where C. capitata no longer resembles D. melanogaster is therefore an important step in understanding the evolution of the dorsal appendages, as it could indicate the point where the network was co-opted.
Our first candidate for co-option was found when we saw that no expression of mirr could be detected in C. capitata egg chambers (Figure4B). The probe against Cc-mirr did reveal clear expression in the C. capitata embryo, in a pattern familiar from expression in D. melanogaster (Figure4B’).
mirr regulates the transcription of br in those cells that will give rise to the dorsal appendages (Figure4E’). Unsurprisingly, the br-positive domains do not appear on the C. capitata stage 10B follicular epithelium (Figure4F’), nor during any other stage of oogenesis. Early expression of br could be seen uniformly in the follicular epithelium, as in D. melanogaster, but the late expression dynamics, both the dorsal-anterior repression and the appearance of the two domains, were not observed; instead, expression diminished around stage 11 and had disappeared entirely by stage 12.
Preliminary results indicate that two other genes relevant for D. melanogaster epithelial patterning do not play a role in the C. capitata dorsal-anterior epithelium: expression of pnt, encoding the transcription factor responsible for the midline repression of br, could not be detected in the dorsal-anterior follicular epithelium of C. capitata. A second known expression domain of pnt at the posterior pole of the egg chamber was clearly visible from an early stage (stage 8), providing a positive control for the in situ hybridization and the pnt probe (Additional file1). Transcription of the gene rho was also not detected in either the early broad dorsoanterior domain, or in the late hinge-shaped patterns adjacent to the br expressing domains (Additional file1). However, as both early rho expression and the dorsoanterior domain of pnt can be difficult to detect in D. melanogaster egg chambers as well, we cannot be completely certain of the absence of pnt and rho transcripts in the dorsoanterior follicular epithelium of C. capitata.
Conserved expression of pip
Interestingly, especially in the light of the absence of detectable Cc-mirr expression, Cc-pip is repressed dorsally: the transcript is expressed asymmetrically, and clearly localizes to the ventral follicular epithelium. In a similar dynamic—though not precisely identical—to D. melanogaster, Cc-pip expression starts at stage 8 in follicle cells at the posterior pole of the egg chamber (Figure4D). This posterior expression domain during stages 8 and 9 is well known in D. melanogaster[38, 40]. During early stage 10, ventral follicle cells start expressing Cc-pip, and by late stage 10 expression in ventral and posterior follicle cells is of equal strength (Figure4D’). The pattern at this stage is identical to the expression pattern of Dm-pip (Figure4C’), including the sharp on-off boundary between ventral and dorsal cells. These results were obtained using two separate probes: one against the common part of all pip isoforms, and one specific to the homologue of isoform A (or pipe-ST2), confirming that the same isoform is used in C. capitata oogenesis as is known to function in D. melanogaster.