Cockroach colony and animal sampling

Adult females of the cockroach B. germanica (L.) were obtained from a colony fed ad libitum on Panlab dog chow and water, and reared in the dark at 29±1°C and 60–70% relative humidity. Freshly ecdysed adult females were selected and used at appropriate ages. Mated females were used in all experiments (the presence of spermatozoa in the spermatheca was assessed to confirm that mating had occurred). All dissections and tissue samplings were performed on carbon dioxide-anaesthetized specimens under Ringer's saline (1.8 mM CaCl2, 154 mM NaCl, 2.68 mM KCl and 2.38 mM NaHCO3).

Cloning of Hippo cDNA

An 1884 bp fragment of B. germanica Hippo (BgHpo) was isolated from mRNA libraries representatives of different organs and stages of cockroach development (previously obtained in our laboratory). The fragment contained the coding region from the amino acid at position 30 through to the poly (A) tail, but lacked the N-terminal end. To complete it, 5′ rapid amplification of cDNA ends (RACE) was performed on RNA extracted from adult ovaries using the FirstChoice RLM-RACE Kit (Ambion, Huntingdon, Cambridgeshire, UK) according to the manufacturer's instructions. The sequence was amplified, cloned into the pSTBlue-1 vector (Novagen, Madison, WI, USA) and sequenced. Table S1 shows the primer sequences used in this amplification.

RNA extraction and expression studies

Total RNA was isolated using the GenElute Mammalian Total RNA Kit (Sigma, Madrid, SPAIN). A total of 400 ng from each RNA extraction was treated with DNAse (Promega, Madison, WI, USA) and reverse transcribed with Superscript II reverse transcriptase (Invitrogen, Carlsbad CA, USA) and random hexamers (Promega). RNA quantity and quality were estimated by spectrophotometric absorption at 260/280 nm in a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE, USA). The expression patterns of Kibra (BgKibra), Merlin (BgMer), expanded (BgExp), Hippo (BgHpo), salvador (BgSav), warts (BgWts), MATS (BgMats), yorkie (BgYki) and scalloped (BgSd) were determined by quantitative real time PCR (qRT-PCR) in the sixth instar nymph stage and during the first gonadotrophic cycle in the adult stage. Pools of 2 to 6 ovary pairs for each chosen adult age were used. The primers used in qRT-PCR expression studies were designed using Primer3 v.0.4.0 software [20] (sequences in Table S1). The actin-5c gene of B. germanica (Accession number AJ862721) was used as a reference for expression studies, and the eukaryotic initiation factor 4A, BgEIF4a (Accession number HF969254), for functional studies. PCR reactions were performed using the IQTM SYBR Green Supermix Kit (BioRad, Barcelona, SPAIN) with 200 nM of each specific primer (performed in triplicate). Amplification reactions were performed as follows: 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 30 s, using the MyIQ Single Color RTPCR Detection System (BioRad). After the amplification phase, a dissociation curve was produced to ensure that only one product was formed [15].

RNAi experiments

To knockdown BgHpo and assess the specificity of the phenotype, two dsRNAs (dsBgHpo-1 and dsBgHpo-2) were designed. The first, dsBgHpo-1, was 406 bp in length and comprised the subdomains V-IX of the protein serine/threonine kinase catalytic domain (nucleotides 382-788). The second, dsBgHippo-2, with 420 bp (nucleotides 1294-1714), belongs to a region where no conserved domain has been detected. Both RNAis were independently injected (1 µg/µl) into newly-emerged sixth nymphal instar females (N6D0). Since the same ovary phenotype was found using both dsBgHpo-1 and dsBgHpo-2, all RNAi treatments are hereafter referred to as dsBgHpo. To deplete BgWts, a 255 bp dsRNA (dsBgWts) was designed overlapping part of the catalytic domain from the Serine/Threonine Kinases, Large Tumor Suppressor subfamily proteins. dsBgWts (1 µg/µl) was injected into 0-day-old sixth nymphal instar females. For experiments on BgN knockdowns, a 363 bp dsRNA (dsBgN) was designed that encompassed a fragment of the Ankyrin domain of the protein. dsBgN (1 µg/µl) was injected into 6-day-old sixth nymphal instar females. To reduce the levels of BgN in dsBgHpo-treated females, double sequential RNAi treatment was performed on sixth nymphal instar females. First, dsBgHpo was injected into 0-day-old nymphs, and dsBgN injected when they reached 6 days. A 92 bp non-coding sequence from the pSTBlue-1 vector (dsMock) was used as control dsRNA (same dose and conditions as in dsRNA treatments). All four cDNAs were amplified by PCR and cloned into the pSTBlue-1 vector. Single stranded sense and antisense RNAs were obtained by transcription in vitro using either SP6 or T7 RNA polymerases from the respective plasmids, and resuspended in water. To generate the dsRNAs, equimolar amounts of sense and antisense RNAs were mixed, heated at 95°C for 10 min, cooled slowly to room temperature, and stored at −20 °C until use. The formation of dsRNA was confirmed by running 1 µL of the reaction products on 1% agarose gels. dsRNAs were suspended in diethyl pyrocarbonate-treated water and diluted in Ringer's saline.

Immunohistochemistry

Removed ovaries were immediately fixed in paraformaldehyde (4% in PBS) for 2 h, washed in PBT (PBS; 0.3% Triton-X100), treated with 50 µg/ml proteinase K for 2 min, washed for 2 min in 2 mg/ml glycine in PBT, washed for 10 min in PBT, and fixed again for 20 min in the same solution. After three washes with PBT, the tissues were placed in PBTBN (PBS with 0.1% Triton X-100, 0.5% BSA and 5% normal goat serum) for 1 h at room temperature. They were then incubated overnight at 4°C with the primary antibodies rabbit anti-PH3 (Cell Signaling Technology, Danver, MA, USA) (1∶250), mouse anti-ß-tubulin (1∶50), and mouse anti-NICD (Developmental Studies Hybridoma Bank, University of Iowa, Department of Biology, Iowa City, IA, USA) (1∶100). The tissues were washed with PBTBN three times and incubated for 2 h with either Alexa-Fluor 647 conjugated donkey anti-rabbit IgG or Alexa-Fluor 488 conjugated goat anti-mouse IgG secondary antibody (Molecular Probes, Carlsbad, CA, USA), both diluted to 1∶400 in PBTBN. In addition, ovaries were incubated at room temperature for 20 min in 300 ng/ml phalloidin-TRITC (Sigma) and then for 5 min in 1 µg/ml DAPI (Sigma) PBT. After three washes with PBT, ovaries were mounted in Mowiol (Calbiochem, Madison, WI, USA) and observed using a Zeiss AxioImager Z1 microscope (Apotome) (Carl Zeiss MicroImaging).

The number of cells in the follicular epithelia was estimated applying the function described in Pascual et al. [21].

Statistics

The data are expressed as means ± standard error of the mean (s.e.m.). Comparisons between tissue samples were made using the non-parametric Mann-Whitney test, employing GraphPad Prism 6 software. Comparisons between treatment and control groups were made using the Pair-Wise Fixed Reallocation Randomization Test (which makes no assumptions about distributions) [22], employing REST 2008 v. 2.0.7 software (Corbett Research).

The complete set of Hippo pathway components is expressed in the B. germanica ovary

The mRNA expression pattern of the Hippo pathway components (Kibra [BgKibra], Merlin [BgMer] and expanded [BgExp] [part of the apical complex], the kinase cassette composed of hippo [BgHpo], salvador [BgSav], warts [BgWts], MATS [BgMats], and the transcriptional co-activator yorkie [BgYki] and its partner scalloped [BgSd]), was examined in the B. germanica ovary in sixth (last) nymphal instar and young adult females (Figure 2). In the last nymphal instar the basal ovarian follicles complete their maturation, and follicular cells show the highest proliferation rate, which coincides with the highest rate of expression of all the components of the Hippo pathway. Of note, BgKibra, BgExp, BgSav and BgYki show especially high expression during the days closest to the imaginal moult. The scaffold protein BgMats and the coactivator BgSd show well-defined peaks on days 3 and 5 of the sixth nymphal instar, respectively (Figure 2). After the moult to adult, when basal follicles enter in vitellogenesis and become ready to uptake proteins, the expression of all the components of the Hippo pathway steadily decrease as the basal ovarian follicles mature. The lowest expression levels are reached just before oviposition, which take place 7 days after the emergence of the adult. These patterns clearly indicate that the main changes in the expression of Hippo pathway components occur during the sixth instar rather than during the adult stage, when the expression of all these genes gradually decreases.

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Figure 2. Expression pattern of the Hippo pathway components in ovaries of B. germanica.

mRNA expression pattern of the components of the Hippo pathway in ovaries in sixth nymphal instar and adult females during the first gonadotrophic cycle. The dashed line indicates the moult to adult. Data represent copies of mRNA per 1000 copies of BgActin-5c (relative expression) and are expressed as the mean ± s.e.m. (n = 3).

https://doi.org/10.1371/journal.pone.0113850.g002

Hippo is required for basal ovarian follicle growth and the mitosis-endocycle switch in follicular cells

To investigate whether hpo regulates the transition from mitosis to the endocycle in the panoistic ovaries of B. germanica, BgHpo transcripts were depleted by RNAi via the injection of dsBgHpo into 0-day-old sixth instar nymphs. In 5-day-old adults, the basal ovarian follicles in dsBgHpo-treated females (0.44±0.04 mm length; n = 27, Figure 3A) remained as small as in 0-day-old adult dsMock-treated females, and never reached the length of those observed in 5-day-old adult dsMock-treated females (1.55±0.08 mm; n = 12; P<0.0001; Figure 3B). Despite the strong size reduction of the basal ovarian follicles, the mRNA levels of BgHpo were significantly downregulated a 50% (fold change 1.67; Figure 3C) in the ovaries of 5-day-old adult females treated with dsBgHpo. Moreover, the depletion of BgHpo mRNA had no effect on the expression of BgYki, BgWts (downstream components of the pathway) or BgExp (an upstream component) (Figure 3C).

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Figure 3. BgHpo controls follicular cell proliferation in developing follicles.

Ovarioles from 5-day-old adult females treated with dsBgHpo (A), or dsMock (B). The posterior end of the ovarian follicles is towards the left. Scale bar: 100 µm. (C) Expression of BgHpo, BgWts and BgYki in ovaries from 5-day-old adult dsMock and dsBgHpo-treated females. BgHpo is significantly depleted in treated ovaries [P(H1)  = 0.004]. (D) Expression of BgVg in fat body from 5-day-old adult dsMock and dsBgHpo-treated females (no significant difference [P(H1)  = 0.098]. Data represent normalized values against the control (reference value  = 1) (n = 3). (E, F, F′) Follicular epithelium from 0-day-old adult females in which cells are dividing actively in dsMock- (E and Figure S1) and dsBgHpo- (F, F′) treated females. Scale bar: 10 µm. (G, H, H′) Follicular cells from 5-day-old adult females. In the dsMock (G and Figure S1) females, mitosis is arrested and patency is already apparent. In the dsBgHpo (H, H′) females, the cells are still dividing and the cytoskeleton is disorganized Scale bar: 10 µm. (I, J, K) Follicular cells in adult ovarioles labelled with an anti-phospho-histone 3 antibody (PH3). (I) Follicular cells from 0-day-old and (J) 5-day-old dsMock-treated females. (K) Follicular cells from a 5-day-old dsBgHpo-treated female. Scale bar: 20 µm. (L) Optical section of (K), the arrow indicates the layer of endosymbiont bacteriocytes. Oo: ooplasm. Scale bar: 10 µm.

https://doi.org/10.1371/journal.pone.0113850.g003

The follicular cells of 0-day-old adult dsBgHpo-treated females resembled those of dsMock-treated females (cuboid and tightly packed Figure 3 F, and Figure 3E and S1, respectively) and showed a similar number of cells undergoing mitosis. However, the arrangement of the F-actin microfilaments of the cytoskeleton was irregular, with random accumulations in different areas of the epithelium (Figure 3, F and F′). In 5-day-old dsMock-treated adult females, the follicular cells were larger, binucleated and the actin microfilaments were associated with cell membranes displaying lateral extensions, and therefore allowing large intercellular spaces (patency) (Figure 3G, and Figure S1). In contrast, in BgHpo knockdown females the cells remained smaller, showed a single nucleus, and maintained close contact each other (Figure 3H). Of note, in the cytoplasm of follicular cells of BgHpo knockdowns, the actin filaments became disorganized and showed strong labelling (Figure 3H′).

Further, while mitosis was arrested and the follicular cells became binucleated and entered the endocycle in 5-day-old dsMock-treated adults (Figure 3J), in 5-day-old BgHpo knockdown females these cells kept dividing (Figure 3K), with 30% more cells undergoing mitosis than in 0-day-old dsMock-treated females (Figure 3I). As a consequence of this overproliferation, the follicular epithelium became bilayered (Figure 3L, 4I and S2), mainly in the lateral and posterior regions of the basal ovarian follicle. This impedes the oocyte to reach its normal size and provokes the membrane folding (Figure S2). We attempted to quantify the cell number in the follicular epithelium of dsBgHpo-treated females, and despite the small size of the ovarian follicle, we found that in the most external layer, the number of cells was similar to that present in a dsMock-treated female. However, considering that the follicular epithelium in these treated females was bilayered (Figure 3L, 4I and S2), the total number of cells in dsBgHpo basal follicles would be approximately the double than in dsMock females. This overproliferation in dsBgHpo-treated females impaired the growth of the basal ovarian follicle, as it prevented vitellogenin (BgVg) uptake. Interestingly, BgVg was synthesized in the fat body of dsBgHpo-treated females, although at a somewhat lower levels compared to dsMock-treated females (Figure 3D).

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Figure 4. Loss of BgHpo generates long-stalks activating BgN.

(A) Stalk from a 5-day-old dsMock adult female. F-actin labelling in the apical tips of the cells. Scale bar: 20 µm. (B, B′, B″) Long-stalk phenotype from a 5-day-old dsBgHpo adult female. (B) F-actin labelling is concentrated in the central axis of the stalk. (B′) The cells are smaller but greater in number. (B″) Merging of (B) and (B′). Scale bar: 20 µm. (C) Ovariole from 0-day-old adult female showing NICD localization. (C′) Merge image of NICD and F-Actin labelling. The arrow indicates the labelling in the germarium. Scale bar: 50 µm. (D) Follicular cells from 0-day-old dsMock adult female; the dividing cells show strong NICD labelling. Scale bar: 20 µm. (E) Stalk from 0-day-old dsMock adult female showing NICD labelling. Scale bar: 10 µm. (F) follicular cells from 5-day-old dsMock adult female. Scale bar: 20 µm (the optical section of 5-day-old dsMock adult female is showed in Figure 5D). (G, H, I) Optical section of a basal ovarian follicle showing the different localization of NICD between the apical (Ap) and basal pole (Ba) of the follicular cells. (G) 0-day-old dsMock adult female. (H) 0-day-old and (I) 5-day-old dsBgHpo adult female. Arrowheads in (I) indicate NICD accumulation in the ooplasm. Scale bar: 20 µm. (J) Stalk in a 5-day-old dsBgHpo adult female ovariole. Oo: ooplasm, FC: follicular cells. Scale bar: 10 µm. (K) Basal ovarian follicle from a 5-day-old dsBgHpo adult female. Ov: oviduct. Scale bar: 50 µm. (L) mRNA expression of different components of the Notch pathway in ovaries of 5-day-old dsBgHpo adult females. BgSer, BgHnt, BgCut and BgEya were upregulated (P(H1)  = 0.0001, 0.010, 0.005 and 0.001 respectively); BgDl and BgN were not significantly affected. Data represent normalized values against dsMock controls (reference value  = 1, dashed line) (n = 3). In all images the posterior pole of the basal follicle is towards the left.

https://doi.org/10.1371/journal.pone.0113850.g004

BgHpo depletion produces long stalks

A longitudinal optical section of B. germanica stalk (Figure 4A) shows a tubular structure formed by a monolayer of differentiated follicular cells, with the actin microfilaments distributed mainly in the apical tips of the stalk cells and extending towards the lateral surface. Strikingly, the stalks in all dsBgHpo-treated females were unusually long, between 181% and 205% longer than in dsMock, a defect observed primarily between the basal and sub-basal follicles (Figure 4B). Although the stalks in BgHpo knockdowns were longer and cell numbers greater than in the dsMock group, no mitosis was detected (Figure 4B′). The cells were smaller and not well arranged into a monolayer, and the actin filaments were randomly distributed (Figure 4, B and B″).

This unexpected phenotype led us to confirm it by depleting another component of the pathway. We depleted BgWts as a part of the kinase cassette, downstream of Hippo. In 5-day-old dsBgWts-treated females the basal ovarian follicles were small and showed long stalk (Figure S3 C-E). The follicular cells were small and appeared very close to each other, without intercellular spaces, and they continued dividing as mitosis were detected with anti-PH3 labeling (Figure S3 F).

A long-stalk phenotype has been described in D. melanogaster when active Notch and Delta are expressed constitutively in ovaries [23], [24]. This suggests that, in B. germanica, Notch would be affected by the depletion of BgHpo. The involvement of Notch in the long-stalk phenotype in dsBgHpo females was examined using an antibody against the intracellular domain of D. melanogaster Notch (NICD). In ovarioles of 0-day-old dsMock-treated specimens, Notch labelling was detected in the basal ovarian follicle, in the stalk between the basal and sub-basal ovarian follicles, and in the germarium (Figure 4, C and C′). In the basal ovarian follicle, NICD was located differently in the apical pole of the follicular cells (Figure 4, E and G). However, labelling was more conspicuous in cells that were dividing, with NICD distributed throughout the cytoplasm (Figure 4D). In the stalk, of 0-day-old dsMock-treated females Notch was also mainly located in the apical pole of the cells (Figure 4E). However, in follicular cells from 5-day-old dsMock ovarian follicles NICD labelling was very faint (Figure 4F and 5D). NICD was also strongly apparent in the apical pole of the follicular cells of basal ovarian follicles from 0-day-old dsBgHpo adult females (Figure 4H). Moreover, in 5-day-old treated females, Notch continued to be present, and the labelling was extended to the lateral and the basal pole of the follicular cells (Figure 4I). An accumulation of NICD as discrete spots throughout the ooplasm, probably localized in vesicles, was also observed (Figure 4I). Unfortunately, the thickness of the full vitellogenic basal follicle in 5-day-old dsMock-treated specimens made the observation of Notch protein within the oocyte impossible. In 5-day-old dsBgHpo-treated females, NICD was abundant through the entire stalk (Figure 4J). A gradient of NICD labelling was in fact seen along the longitudinal axis of the basal ovarian follicle with more at the posterior pole (including the pedicel and the lateral oviduct), decreasing towards the anterior pole (Figure 4K).

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Figure 5. Notch depletion suppresses the stalk and reduces the long stalk phenotype in dsBgHpo knockdowns.

Ovariole from a 5-day-old adult female treated with dsBgN (A), and a basal ovarian follicle from a dsMock 5-day-old adult female (B). Scale bar: 100 µm. (C, D) Localization of Notch in ovarioles of 5-day-old adult dsBgN (C) and dsMock (D) females showing the position of the stalk (white box). In (C) there is no stalk. Notch was detected with an anti-NICD antibody. Scale bar: 20 µm. (E) mRNA relative expression of BgN, BgHnt, BgCut and BgEya in ovaries from 5-day-old adult dsMock- and dsBgN-treated females. BgN, BgCut and BgEya were significantly down-regulated (P(H1)  = 0.0001) in dsBgN-treated ovaries. (F) mRNA relative expression of BgHpo, BgWts, BgYki and BgN in ovaries from 5-day-old adult dsMock and dsBgHpo + dsBgN-treated females. BgHpo and BgN were significantly reduced (P(H1)  = 0.047 and 0.028 respectively). Data represent normalized values against the control (reference value  = 1) (n = 3). (G) Ovariole from a 5-day-old, adult, double knockdown female (dsBgHpo + dsBgN). Scale bar: 100 µm. (H) Follicular epithelium where no mitoses were detected in opposite to dsBgHpo treatment (see Figure 3K). Scale bar: 50 µm. (I–J) Different range of stalk phenotypes resulting from the double knockdown. Scale bar in I and K: 50 µm, in J: 100 µm. In all images the posterior end of the basal ovarian follicle is towards the left, except in (C) in which it is towards the bottom.

https://doi.org/10.1371/journal.pone.0113850.g005

Since Notch protein was detected in ovaries of 5-day-old adult BgHpo-treated females, the mRNA levels for a selection of Notch pathway components and Notch-dependent differentiation markers were measured. This selection, chosen with reference to the information available in D. melanogaster, includes Hindsight (BgHnt), Cut (BgCut) and Eye Absent (BgEya). No change was seen in Notch (BgN) mRNA levels, and no significant changes were observed in the expression of the ligand Delta (BgDl). However, the expression of Serrate (BgSer), the other ligand of Notch, was significantly upregulated (1.79 fold-change; Figure 4L). The increase in the expression of BgSer suggests a continuous activation of Notch signaling in these 5-day-old dsBgHpo-treated females. In addition, in these 5-day-old adult BgHpo-treated females BgHnt, BgCut, and BgEya were upregulated, with a 1.7 fold-change, 2.2 fold-change and 1.8 fold-change, respectively (Figure 4L).

Notch determines the correct structure of the stalk

To assess the function of Notch in the development of the stalk in panoistic ovaries, BgN was depleted by RNAi. dsBgN was injected into 6-day-old sixth instar females and the basal ovarian follicle was observed in 5-day-old adults. In BgN knockdowns, the basal ovarian follicles were significantly smaller (0.28±0.01 mm, n = 16; Figure 5A) than in the dsMock group (1.52±0.07 mm, n = 10; P<0.0001; Figure 5B), and even smaller than in 0-day-old dsMock-treated adult females (0.47±0.04 mm, n = 12; P<0.0001). Moreover, while the basal ovarian follicles of dsMock females showed the typical elliptical shape (Figure 5, B and D), those of 5-day-old dsBgN-treated females exhibited a sub-spherical shape (Figure 5A) the ovarioles showed no stalks (Figure 5, A and C) and were no follicular cells in mitosis (not shown). In the ovaries of dsBgN-treated females, the mRNA levels of BgN, BgCut and BgEya were significantly reduced (42-, 2.1- and 1.72-fold respectively; Figure 5E), indicating that Notch activates Cut and Eya. Interestingly, BgHnt expression was not affected (Figure 5E), suggesting that Notch does not act directly on Hnt to regulate Cut. In dsBgN-treated females, Notch labelling was not observed in any ovariole (Figure 5C), while in the basal ovarian follicles of dsMock females, faint labelling was observed, mainly along the stalks (Figure 5D). These results show that Notch is required for normal stalk formation in the panoistic ovaries of B. germanica.

Notch depletion recovers the phenotypes resulting from a reduction in Hpo

In the ovaries of BgHpo knockdowns, the follicular cells were unable to switch to the endocycle (Figure 3, H and L) and the stalks were longer than in ovarioles from the dsMock group (Figure 4B′) due to the continuous expression of BgN. To demonstrate a relationship between BgHpo and BgN, double sequential knockdowns of BgHpo and BgN were prepared. The corresponding mRNA levels were measured in 5-day-old adult females, which showed the expression of BgHpo to be significantly reduced (2-fold), and BgN to be dramatically depleted (38.5-fold; Figure 5F). These results were similar to those obtained for individual treatments (Figure 3C and 5E). Five-day-old double knockdowns females have ovarioles (Figure 5G) that showed reduced, and sometimes absent, stalks (Figures 5, I–J). In addition, 30% of the double knockdown females (n = 15) showed only a few dividing follicular cells, while the remaining 70% showed no mitosis (Figure 5H), a remarkable phenotype when compared with the continuous cell division found in dsBgHpo basal ovarian follicles (Figure 3L). These results suggest that low levels of BgN are necessary for the correct stalk formation and to stop the mitosis program in follicular cells.