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Dev Genes Evol (2003) 213:587–600 DOI 10.1007/s00427-003-0367-z
ORIGINAL ARTICLE
Ying Dong · Laurence Dinan · Markus Friedrich
The effect of manipulating ecdysteroid signaling on embryonic eye development in the locust Schistocerca americana Received: 12 August 2003 / Accepted: 8 October 2003 / Published online: 14 November 2003 Springer-Verlag 2003
Abstract Adult body plan differentiation in holometabolous insects depends on global induction and control by ecdysteroid hormones during the final phase of postembryogenesis. Studies in Drosophila melanogaster and Manduca sexta have shown that this pertains also to the development of the compound eye retina. It is unclear whether the hormonal control of postembryonic eye development in holometabolous insects represents evolutionary novelty or heritage from hemimetabolous insects, which develop compound eyes during embryogenesis. We therefore investigated the effect of manipulating ecdysteroid signaling in cultured embryonic eye primordia of the American desert locust Schistocerca americana, in which ecdysteroid level changes are known to induce three rounds of embryonic molt. Although at a considerably reduced rate compared to in vivo development, early differentiation and terminal maturation of the embryonic retina was observed in culture even if challenged with the ecdysteroid antagonist cucurbitacin B. Supplementing cultures with 20-hydroxyecdysone (20E) accelerated differentiation and maturation, and enhanced cell proliferation. Considering these results, and the relation between retina differentiation and ecdysteroid level changes during locust embryogenesis, we conclude that ecdysteroids are not an essential but possibly a modulatory component of embryonic retina development in S. americana. We furthermore found evidence that 20E initiated precocious epithelial morphogenesis of the Edited by C. Desplan Y. Dong · M. Friedrich ()) Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA e-mail:
[email protected] Tel.: +1-313-5779612 Fax: +1-503-2101221 L. Dinan Department of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter, Devon, EX4 4PS, UK
posterior retinal margin indicating a more general role of ecdysteroids in insect embryogenesis. Keywords Eye development · Evolution of development · Ecdysone · Metamorphosis · Schistocerca americana Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00427-003-0367-z
Introduction Insects evolved complex hormonal control mechanisms of development to coordinate their life cycle with environmental changes and to regulate growth dependent developmental transitions (Nijhout 1994; Truman and Riddiford 1999). Two major hormonal classes were employed in the control of insect development: (1) ecdysteroids, polyhydroxylated steroids present in a wide range of invertebrates; and (2) juvenile hormones (JH), sesquiterpenoids that have so far only been found in insects (Truman and Riddiford 2002). The life cycle stage-dependent functions and interactions of these hormones have been predominantly studied in holometabolous insect species such as the fruit fly, Drosophila melanogaster, or the tobacco hornworm, Manduca sexta, which progress through larval and pupal stages before reaching adulthood (Riddiford et al. 2001; Thummel 1996; Truman and Riddiford 2002). These different steps of postembryonic development are controlled by fluctuating ecdysteroid levels depending on peak duration and intensity as well as on the presence of JH. Ecdysone (E) and 3-dehydroxyecdysone (3dE) are the predominant ecdysteroids secreted from the prothoracic ring glands throughout postembryonic development. The majority of E and 3dE, which have only low biological activities, is converted in fatbody, midgut, and peripheral tissues to the biologically active form 20-hydroxyecdysone (20E). 20E peaks in the presence of JH to stimulate epidermal cells to
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prepare for molting and induce the formation of new cuticle to accommodate the growth of the larval stages. A moderate 20E peak in the absence of JH during the last larval instar, the commitment peak, induces behavioral changes to prepare for pupation. A subsequent 20E surge, the prepupal peak, which is associated with a concerted rise in JH levels, induces formation of the pupal cuticle. The final remodeling of the larval body plan into that of the adult via histolysis of larval tissues and differentiation of adult organs is orchestrated by a strong pupal ecdysteroid peak, when JH is downregulated. The regular array of the compound eye retina belongs to the suite of adult structures that form de novo during the last larval instar and pupation in holometabolous insects. Recent studies demonstrated several levels of ecdysteroids requirement for the postembryonic differentiation of the adult retina of D. melanogaster as well as M. sexta (Brennan et al. 1998, 2001; Champlin and Truman 1998). In both species, differentiation of the retina proceeds in a very similar manner in eye imaginal disc epithelium (Wolff and Ready 1993). The front of the differentiating eye field is marked by a narrow tissue indentation. This so-called morphogenetic furrow moves from posterior to anterior through the eye disc turning unpatterned epithelium into differentiating retina. Ommatidial preclusters are initiated at the posterior margin of the morphogenetic furrow in the form of R8 founder photoreceptor cells. As the regular spacing of the R8 founder cells anticipates the highly organized array of ommatidial facets in the adult retina, the morphogenetic furrow aligns with columns of developmentally synchronized ommatidial precursor cell clusters. Subsequent to R8 cell specification, additional photoreceptor cells are recruited to the developing ommatidial preclusters followed by cone and pigment cells. Eventually developmentally advanced ommatidia are positioned at the posterior margin of the eye disc such that a maturation gradient extends from the furrow to the posterior margin of the early developing eye field. Retina differentiation is initiated shortly before the ecdysteroid commitment peak and continues into early pupation, still overlapping with the prepupal peak. The subsequent pupal ecdysteroid peak induces cellular morphogenesis and terminal differentiation steps in the developing ommatidia throughout the retina in a concerted manner. Culturing experiments with Drosophila eye imaginal disc preparations indicated that ecdysteroids are essential for aspects of terminal retina differentiation (Li and Meinertzhagen 1995, 1997). Lens secretion from cone cells and screening pigment synthesis in pigment and photoreceptor cells were only observed in culturing media supplemented with 20E. Molecular genetic analyses converged with these results by discovering strong ommatidial disorganization and tissue degradation in late eye disc tissue clones mutant for components of the ecdysteroid signal transduction machinery, including the ecdysteroid receptor (EcR), the RXR homologous EcR co-receptor ultraspiracle (usp) and members of the Broad-complex (BR-C) ecdysteroid immediate early
response zinc-finger transcription factor family (Brennan et al. 2001; Oro et al. 1992). Also, targeted expression of a dominant negative EcR perturbed lens development and induced excessive cell death (Cherbas et al. 2003). Conflicting results however have been obtained from genetic and in vitro culturing studies regarding the role of ecdysteroids during early retinal differentiation in D. melanogaster. Progression of the morphogenetic furrow, photoreceptor differentiation, and photoreceptor axon elongation were reported to occur in D. melanogaster eye disc cultures lacking ecdysteroid supplements (Li and Meinertzhagen 1997). Reduction of ecdysteroid levels by genetic manipulation in D. melanogaster on the other hand caused strong defects of early retinal patterning including irreversible stall of furrow progression, incorrect initiation of R8 founder cells, and loss of cell proliferation, neuronal differentiation and expression of the furrow progression promoting signaling factor hedgehog (Brennan et al. 1998). The identity of the receptors involved in ecdysteroid signaling at the morphogenetic furrow has remained elusive as none of the classical nuclear receptors participating in ecdysteroid receptor dimer formation, such as EcR or DHR78, was required for normal patterning (Brennan et al. 2001). Nonetheless, a role of ecdysteroid signaling during early retina differentiation was further supported by consistent defects in furrow progression and R8 founder cell specification in tissue mutant for the BR-C isoform protein Z2 (Brennan et al. 2001). Although discrepancies between genetic and tissue culture experiments remain to be resolved, the available evidence indicates a complex action of ecdysteroid signaling-based control mechanisms in the developing D. melanogaster retina. This is further corroborated by the observation that loss of the ecdysteroid co-receptor USP leads to acceleration of furrow progression, which is associated with ectopic expression of the BR-C isoform protein Z1 anterior to the morphogenetic furrow (Ghbeish and McKeown 2002; Zelhof et al. 1997). An equally complex network of complementing mechanisms of transcriptional derepression and activation may underlie the differential regulation of early and terminal differentiation by ecdysteroids in the developing retina of M. sexta (Champlin and Truman 1998). Morphogenetic furrow progression, cell proliferation and photoreceptor differentiation could be observed in culture when 20E was added to a minimal concentration of 0.12 mM. A decrease of 20E concentration to below this threshold resulted in reversible stall of early development. If 20E levels were raised above 2 mM, proliferation and early development was irreversibly replaced by terminal differentiation involving induction of rhabdome formation in photoreceptor cells and lens secretion in cone cells. The dosage-dependent states of ecdysteroid control in the developing M. sexta eye reflect the two basic functions of ecdysteroid mediated control of insect development. The reversible block of early retina development controlled by low ecdysteroid levels regulates the transient halt of development during pupal diapause. The induction of terminal differentiation is a component of the postem-
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bryonic adult differentiation cascade driven by the strong pupal ecdysteroid surge. It is unknown whether similar mechanisms of developmental control already existed in more primitive forms such as a- and hemimetabolous insects, in which much of the adult body plan develops during embryogenesis. A general characteristic of embryogenesis in non-holometabolous insects is the occurrence of fluctuations in free ecdysteroid levels which, as studies in orthopterans have shown, induce three rounds of embryonic molts (Lagueux et al. 1979; Sbrenna et al. 1989). A requirement of ecdysteroids for embryonic cuticle induction also exists in higher insects. D. melanogaster experiences a single embryonic ecdysteroid peak which is associated with high levels of JH (Bender et al. 1997; Chavez et al. 2000; Riddiford 1993). In non-holometabolous insects, only the final embryonic ecdysteroid peak is linked to a rise in JH levels. The preceding peaks, which occur in the absence of JH and thus have no obvious equivalent in the embryogenesis of holometabolous insects, fall into the time window of early embryonic patterning and differentiation of adult structures. During postembryonic development, titer changes and functions of 20E and JH in nonholometabolous insects are very similar to those in holometabolous insects. 20E peaks in the presence of JH induce molts without morphological change until a major 20E surge in the absence of JH triggers adult maturation in the last immature instar (Truman and Riddiford 2002). Phylogenetic interpretation of the evolution of endocrine signaling suggests that the final ecdysteroid peak in primitive insects corresponds to the pupal differentiation peak in holometabolous insects (Truman and Riddiford 1999). Only the completion of wing and genitalia morphogenesis falls into the period of the final postembryonic ecdysteroid surge in primitive insects. It has therefore been asked “how the ecdysteroid signaling system has been able to ‘capture’ these patterning networks” which are involved in adult differentiation (Truman and Riddiford 2002). Alternatively, the shift of adult body plan differentiation into postembryogenesis may have been linked to a shift of embryonic ecdysteroid-mediated control mechanisms. Based on the observation of in vitro development of locust embryonic appendages it has been hypothesized that ecdysteroids may have little effect on embryonic development in primitive insects (Mueller 1963; Truman and Riddiford 2002). However, successful development in these experiments required the presence of yolk, which has been found to store large amounts of maternally supplied ecdysteroids (Hoffmann and Lagueux 1985). Interestingly, hemimetabolous insects are capable of undergoing diapause, in some cases in the embryonic stage, and diapausing forms have been reported to contain lower ecdysteroid levels than non-diapausing forms (Tawfik et al. 2002). These data raise the possibility that ecdysteroids may play a wider role in developmental control during embryogenesis of primitive insects besides induction of molting. To address this question we investigated the effect of ecdysteroids on embryonic eye
development in the locust Schistocerca americana, motivated by the fact that insect embryonic ecdysteroid levels are relatively well studied in the Orthoptera (Dinan and Rees 1981; Gande and Morgan 1979; Hoffmann and Lagueux 1985; Lagueux et al. 1979; Morgan and Poole 1976; Slama 2000; Tawfik et al. 2002), and that orthopteran usp and EcR orthologs have been identified (Hayward et al. 1999; Saleh et al. 1998).
Materials and methods Animal culture American desert locusts (S. americana) were raised on fresh wheat supplemented with dried wheat-germ in small cages at 31C and 60€5% relative humidity under a long daylight cycle (18 h light/6 h dark). Egg pods were collected daily from 12-cm diameter cups containing fine Vermiculite, cleaned, wrapped in Kimwipe moistened with 0.1% fungizone (Biowhittaker) and incubated in Parafilm-sealed petri dishes at 31C. Under these conditions, embryonic development was completed after 20 days consistent with the 5% developmental increments per day described by Bentley et al. (1979). In vitro culturing of embryonic eye lobes The in vitro culturing regimen for grasshopper embryonic eye lobes was developed by adapting a standard protocol for grasshopper primary tissue cultures (Myers and Bastiani 1993). Eggs were surface-sterilized and dechorionated by soaking for 3 min in commercial bleach. After two rinses in distilled water, eggs were transferred into Schneider’s Drosophila medium (GIBCO) supplemented with penicillin (10 U/ml), streptomycin (10 mg/ml) and glutamine (292 mg/ml; GIBCO). Embryos were dissected from the egg into the medium, cleaned from yolk and transferred into a second dish with fresh medium. To isolate individual eye lobes, the head was removed from the body and cut into halves along the midline with a sterile scalpel blade. Cultures were either started with the separate embryonic head hemispheres or with eye lobes that were further dissected from the head by cutting along the border between eye lobe and cerebral lobe. Eye lobes were transferred into a droplet of 50 ml culture medium on a sterile slide, which was placed into a 10020-mm petri dish on 0.1% fungizone moistened Kimwipe. The petri dishes were stored in polystyrene boxes together with moist tissue and incubated in a digitally controlled incubator at 31C for 48 h. Pharmacological perturbation 20-Hydroxyecdysone was purchased from Sigma (cat. no. H-5142; >95 purity) and dissolved in 100% ethanol to 10 mM stock concentration. Adding 20E to a final concentration of 2 mM, which is above the threshold level necessary to induce terminal differentiation events in cultured retina primordia of M. sexta (Champlin and Truman 1998), to eye lobe cultures induced cuticle detachment in numerous regions of the embryonic head including the eye lobes (see Electronic Supplementary Material). This was not observed in the reference tissue cultured without 20E demonstrating that 20E activated ecdysteroid signaling in the cultured grasshopper tissue consistent with previous reports in S. gregaria (Sbrenna et al. 1989). In these and all other experiments involving reference eye lobes, ethanol was added to the reference eye lobe culture at the same concentration as it was introduced to the experimental eye lobe by adding the supplemented drug. Inhibition of ecdysteroid signaling was conferred by applying the triterpenoid cucurbitacin B (cucB), which is a strong compet-
590 itive ecdysteroid inhibitor at the Drosophila EcR (Dinan et al. 1997). CucB purified from Iberis umbellata seeds (Cruciferae) was dissolved in 100% ethanol to 10 mM stock concentration. To test for potential cytotoxicity grasshopper embryos were incubated in final concentrations from 10–9 to 10–5 M. All embryos developed normally as judged by examination with brightfield microscopy. No defects were observed regarding eye morphology and pigmentation. However, later analysis of mitotic activity by propidium iodide staining revealed occasional hyperproliferation in retinas cultured at 10 mM cucB. To confirm that cucB was effective in blocking ecdysteroid, eye lobes taken from the same individual were cultured in 2 mM 20E-supplemented medium with and without co-supplementing cucB at a concentration of 10 mM (see Electronic Supplementary Material). The induction of apolysis by 20E was completely suppressed by application of cucB. Analysis of retina development The progress of retina differentiation was visualized by staining filamentous actin, which is enriched in the distal tips of developing ommatidia, with AlexaFluor 633 or rhodamine-conjugated phalloidin (Molecular Probes). The stained tissue was cleared in 70% glycerol containing 2.5% DABCO (Sigma). Confocal image stacks were taken with a Leica TCS SP2 laser scanning confocal microscope and processed using Leica Confocal Software (Simulated Fluorescence Projection and Z-projection) or ImageJ (http:// rsbweb.nih.gov/ij/). Brightfield images of in vivo developed tissue were taken with a SPOT RT CCD digital camera (Diagnostic Instruments) coupled to a Leica MZ 12.5 stereomicroscope or a Zeiss Axioscope. To quantify the progression state of retina differentiation, the number of ommatidial columns formed was counted along the eye lobe midline. Mitotic cells were identified based on chromatin condensation visualized by staining with propidium iodide (Orsulic and Peifer 1994). Quantitative analysis of mitotic cell frequency was done by comparison of the average numbers of mitotic cells anterior and posterior to the morphogenetic furrow counted within a window size of 245.5163.6 mm.
Results Time course of S. americana embryonic retina development in relation to ecdysteroid titer changes Ecdysteroid-mediated control of development is indicated by correspondence between the timing of developmental events and ecdysteroid level changes. Embryonic ecdysteroid levels have been reported by several groups for three orthopteran species, the migratory locust Locusta migratoria, the desert locust Schistocerca gregaria, and the house cricket Acheta domesticus (Gande and Morgan 1979; Lagueux et al. 1979; Scalia and Morgan 1982; Scalia et al. 1987; Tawfik et al. 2002; Whiting et al. 1993). Despite some discrepancy with regards to absolute titers, these studies converge on finding an increase from initially low free ecdysteroid titres during the second half of embryogenesis to a peak of at least tenfold higher than base levels at around 80% of development followed by a sharp drop before hatching. According to a study on Locusta by Lagueux et al. (1979), this major peak represents the last of four discrete peaks of free ecdysteroid, which correlate tightly with the timing of embryonic cuticle formation events. In the absence of original data for S. americana, we related the highly resolved time course of ecdysteroid level changes in Locusta to the
course of embryonic eye development in the former assuming evolutionary conservation of embryonic ecdysteroid regulation between such closely related species. As embryogenesis takes almost twice as long in S. americana (20 days) compared with Locusta migratoria (11 days), we used morphological and physiological landmarks of embryonic development to infer the timing of 20E level changes during S. americana embryogenesis (Fig. 1; Bentley et al. 1979). Accordingly, starting from egg deposition initial levels of maternally supplied free ecdysteroids remain constant. A first approximately threefold transient increase occurs at about 20% of development coinciding with cuticle deposition in the non-embryonic serosal membrane. The level of 20E remains low during this peak, which is also true for the second transient ecdysteroid titer increase at 35% of development, which correlates with formation of the first embryonic cuticle. A first moderate rise of 20E parallels the third ecdysteroid peak when free ecdysteroid levels increase threefold. This peak should correlate with deposition of the second embryonic cuticle between 50% and 55% of development. During the final ecdysteroid surge, which coincides with formation of the first nymphal instar cuticle at about 80% of development, ecdysone and 20E levels are eight- and sixfold increased respectively, and 20E is the predominant metabolite (45% of the total free ecdysteroid). If related to this time line of ecdysteroid level changes, formation of the visual primordium and initiation of early retina patterning occur at low ecdysteroid titers in S. americana. At the time of the first ecdysteroid peak, the eye lobes, the embryonic head compartments that give rise to both the compound eye retina and optic ganglia, are just being formed (Fig. 1B). Eye lobe compartment formation is completed by 30% of development and the morphogenetic furrow initiates at about 32.5% of development, i.e. shortly before the second embryonic ecdysteroid peak. Until the third peak of free ecdysone, the morphogenetic furrow progresses about 4 columns per day such that about 14 columns can be counted along the midline by 50% of development (Fig. 1D). Subsequent to the third peak, furrow progression accelerates to 10 new columns per day. The advancement of the eye field was not followed beyond 65% of development owing to insufficient labeling of whole-mount preparations. Nonetheless, the first nymphal instar compound eye measures about 70 ommatidial columns along the midline suggesting an average furrow progression rate of about 7 columns per day between 65% and 100% of development. Terminal differentiation of the retina fills much of the second half of embryogenesis. The first sign of retina maturation can be noticed shortly before 50% of development when a rim of pigmentation emerges at the posterior margin of the eye field owing to the onset of screening pigment synthesis in secondary pigment cells at the bottom of the retina (Fig. 1D). The onset of pigment formation thus seems to occur between the second and third ecdysteroid peaks. Following the morphogenetic furrow, the area of terminally differentiating ommatidia
591 Fig. 1A–J Grasshopper embryonic eye development in relation to ecdysteroid (20E) level changes. Anterior is to the left and dorsal is up unless indicated otherwise. Scale bars correspond to 100 mm. A Graph showing inferred fluctuations of free ecdysone (light line) and 20E (solid line) and the progress of retinal differentiation (dots linked by solid lines) during embryonic development of Schistocerca americana. The time points of serosal cuticle (SC), primary embryonic cuticle (1’EC), secondary embryonic cuticle (2’EC) and first instar nymphal cuticle (1’NC) synthesis, and major hallmarks of retina differentiation are indicated by arrows. B–E Lateral view of grasshopper eye lobe at 30%, 35%, 50% and 65% of embryonic development. Eye lobe and cerebral lobe (*) have been elaborated by 30% of development. F–I Projections of confocal image stacks taken from phalloidin-labeled eye lobes, corresponding to the stages shown in B–E, showing the progression of the retina. J Lateral view of grasshopper embryonic head at 80% of development
as characterized by screening pigment synthesis progresses anteriorly. About 25% of the eye field is pigmented by 65% of development (Fig. 1E). At this stage, pigmentation is also visible apically, as the pigment cell bodies begin outlining the emerging facets formed by cone and primary pigment cells. This pattern emerges at 55% of development (not shown; Bentley et al. 1979). The onset of rhabdomere formation occurs at 65% of development, which can be seen in phalloidin-stained tissue preparations by the presence of elongated actin-enriched structures at the posterior margin of the retina (Fig. 1I). By the time of the last ecdysteroid peak, approximately 80% of the retina has developed deep black pigmentation owing to pigment accumulation in photoreceptor and pigment cells (Fig. 1J). In summary, the fairly continuous progression of retina differentiation shows little correlation with the assumed episodic changes of free ecdysteroid levels (Lagueux et
al. 1979), which await experimental confirmation in S. americana. Nonetheless, aspects of terminal retina differentiation may be induced in response to the rising ecdysteroid levels during the second half of embryogenesis as found in several independent studies (Gande and Morgan 1979; Scalia and Morgan 1982; Scalia et al. 1987; Tawfik et al. 2002). In vitro development of the S. americana embryonic retina In vitro culturing has been instrumental in studying ecdysteroid requirement during eye development in higher insects as the ecdysteroid titer available to the tissue can be most stringently controlled (Li and Meinertzhagen 1995). We therefore investigated which aspects of embryonic retina differentiation in the grasshopper
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Fig. 2A–J Retina differentiation in cultured eye lobes. Projection images of confocal image stacks taken from phalloidin-labeled tissue. Morphogenetic furrow is indicated by an arrow. Scale bars correspond to 100 mm. A 0-h reference eye lobe of culture experiment started at 30% of development. No signs of retinal differentiation can be detected. B Experimental eye lobe from the same embryo as A after 48 h culture. C 48-h in vivo reference eye lobe from the same pod of embryos used for the experiment in A and B cultured for the same amount of absolute time as B. D 0-h reference eye lobe of a culturing experiment started at 42.5% of development. E 48-h experimental eye lobe from the same embryo as in D. F 48-h in vivo reference eye lobe of eye lobe seen in E. G– I High magnification view of the differentiating retina of eye lobes shown in D–F, respectively. Morphogenetic furrow and developing ommatidial clusters exhibit normal morphology in the cultured
could be reproduced under culturing conditions that had previously been developed to study aspects of central and peripheral nervous system development (Myers and Bastiani 1993). Ovipositing S. americana females lay pods containing between 40 and 80 eggs. Embryos from the same pod are synchronized in development with a small margin of variance (Bentley et al. 1979). Nonetheless, for the most accurate analysis of retinal differentiation, we cultured individual eye lobes such that the progress of retina differentiation could be determined by comparing the state of differentiation between eye lobes taken from the same embryo. To find out if standard culturing conditions allowed for in vitro initiation and progression of retinal differentiation, one eye lobe per embryo was fixed at the beginning of the experiment (0 h reference) and the other eye lobe was cultured for 2 days (48 h experimental) before fixation. At the end of the experiment, eye lobes from embryos of the same pod were dissected to serve as 48 h in vivo reference. When eye lobe cultures were started at 30% of development before morphological signs of furrow formation are detectable, experimental eye lobes initiated an intact morphogenetic furrow followed by a regular array of first developing ommatidial clusters (100% of n =4; Fig. 2A, B). Six new columns of ommatidia had been formed in the in vivo reference eye lobes corresponding to 40% of development, whereas only three columns had formed along the midline of cultured eye lobes. Consistent with the apparent delay of furrow progression in vitro, the eye field was also reduced at the lateral margins compared to in vivo reference eye lobes (Fig. 2B, C). If eye lobe cultures were started at 42.5% of development, the eye field also expanded but lagged considerably behind the size of the retina field of in vivo reference embryos with regards to both anteroposterior and dorsoventral width (Fig. 2D–F). Despite the slower rate of differentiation, furrow morphology and ommatidial precluster arrangement of in vitro developing retina tissue showed no major differences to that of in vivo developing tissue (Fig. 2G–I). The slowdown of in vitro retina differentiation compared to in vivo development could either be due to a continuously slower differentiation rate in vitro or to precocious arrest of an initially normal continuation of differentiation. To distinguish between these possibilities, experimental eye lobes were dissected after 24, 48 and 72 h of culture and compared to 0 h reference eye lobes (Fig. 2J). The average number of new ommatidial columns increased from 5.3 to 7.3 and 10 after 24, 48 and 72 h, respectively, indicating that the final number of newly formed ommatidia was the result of continuous
retina in H compared to in vivo developing tissue in G and I. The border between old posterior and newly formed anterior ommatidial columns in H is indicated by a white line. J Number of newly formed ommatidial rows at different time points of in vitro culturing. Error bars represent standard deviation. n =3 for all time points
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Fig. 3 Effect of activating and blocking ecdysteroid signaling on retinal differentiation. Graph showing results from four independent culture experiments comparing the average number of newly formed ommatidial columns in tissue cultured in medium only (S2), medium supplemented with 2 mM 20E (S2 + 20E) and in normally developing embryos (in vivo), or medium supplemented with 2 mM 20E and 10 mM cucB (S2 + 20E + cucB), and medium supplemented with 10 mM cucB (S2 + cucB). Each experiment involved embryos from the same pod. Each experimental group is based on a minimum number of five samples. Error bars indicate standard error
differentiation and that the rate of differentiation decreased within the first 24 h of culturing. Effect of modulating ecdysteroid signaling on furrow progression In order to determine a potential effect of 20E on the rate of retina differentiation, single eye lobe cultures were initiated for several embryos from a single pod. Experimental eye lobes were cultured for 48 h in S2 medium with or without a supplement of 20E at a concentration of 2 mM. The number of newly formed ommatidial columns per experimental eye lobe was determined by comparison with the 0 h reference eye lobes and the average number of newly formed columns compared between the two groups of treatment (Fig. 3). For comparison with in vivo development, the average number of ommatidial rows in 0 h reference embryos was deducted from that in eye lobes from embryos of the same pod dissected at the end of the experiment. In three replicas of this experiment, eye lobes cultured without 20E supplement formed on average 3.8–6.3 new columns, which compared to an average in vivo rate of 10.5–12.3 columns per 48 h. In spite of the variance in absolute numbers of new columns formed in different experiments, supplementing with 20E resulted in a consistent and significant increase of the eye field with an average of 6.8, but ranging up to 10, newly formed columns (t-test, p
