Timeline of Thermobia domestica embryogenesis at 37°C.

(A) diagrammatic representations of important events in embryogenesis. In the segmenting germ band, the arrow indicates the invagination of the mid abdominal region into the yolk. By the onset of katatrepsis (Panfilio, 2008), dorsal closure along the abdomen (red arrow) has moved the abdomen ventrally (solid black arrow) and the contraction of the serosa pulls the sides of the embryo towards the anterior pole (dashed black arrows). After katatrepsis the expanding lateral edges of the embryo displace cells of the amnion, gradually zipping up the dorsal thoracic midline (red arrow) until definitive dorsal closure is accomplished. (B) Summary of the progression of embryonic development at various times after egg deposition. The interception of each stage with the 50% line (dashed) is the basis for the embryonic time-line in (C). EEF: extraembryonic fluid. (C) Timeline of embryonic development of Thermobia domestica based on (B). Photomicrographs show the appearance of embryos at the indicated times; first and last embryos are covered by the egg chorion. White triangle: progression of dorsal closure; red triangle: eye pigmentation; blue triangle: expanded cerci after shedding of the E1 cuticle. Times of cuticle deposition based on Konopová and Zrzavý, 2005.

Titers of hormones and hormone related gene transcripts during embryogenesis of Thermobia domestica.

(A) Titer of juvenile hormone III (JH-III) during the last 60% of embryogenesis and the first eight days of juvenile life. The timing of various milestones of embryonic development are noted for this panel and those below. ecd: ecdysis; J#: start of second, third and fourth juvenile instars. (B) The ecdysteroid titer in 20-hydroxyecdysone (20E) equivalents during embryogenesis and the first six days of juvenile life. Dark bars indicate the approximate time of deposition of the first embryonic (EC1) epicuticle and procuticle, and the second (EC2) embryonic cuticle (based on Konopová and Zrzavý, 2005). epi: epicuticle deposition, pro: procuticle deposition. (C) The relative levels of transcripts of the JH receptor, Methoprene-tolerant (Met), the JH response gene Kruppel homolog 1 (Kr-h1), and the TGF-b family member Myoglianin (Myo) based on RT-PCR of stages through embryogenesis. The expression is related to the 12 hr timepoint which is given the value of 1. Each bar shows the mean (+/- S.D.) for three independent samples.

The effects of suppression of JH production on the embryonic development of Thermobia.

(A) Compared with solvent alone (Control), treatment of stage 2 juveniles with 7-ethoxyprecocene (7EP) resulted in a >75% reduction in JH-III levels as measured 1 day later. (B) The effect treating embryos at 6 d AEL with the cyclohexane solvent (blue) or 1 μg 7EP (orange) on their subsequent expression of Kr-h1, Met, and myo over the next three days as revealed by RT-PCR. Sub-groups of 7EP treated embryos were treated with 1 ng JHm at 7 d and measured over the next two days (green). Significant differences determined by t-test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001. (C) A group of 20 embryos were treated with cyclohexane (black) or 1μg 7EP (red) and their subsequent development monitored until hatching. Both groups started eye pigmentation at the same time, but resorption of the extraembryonic fluid and hatching was suppressed in the 7EP group, The latter two events were restored by treating embryos with the 1 ng JHm at 8.5 d AEL (green). (D) Confocal sections of the dorsal view of embryos treated as in “C” and examined at the time of hatching of the controls. 7EP treatment prevented extension of the foregut (fg) and crop (c) and the posterior displacement of the midgut (mg). Normal gut development was restored with JHm treatment at 8.5d. br: brain; muscle (green); propidium iodide staining (red). (E) Pseudo-transmitted light and confocal sections showing cuticle and nuclei of the eye region of 10 d embryos that had been treated with cyclohexane (control) or 7EP at 5 d AEL. A subset of the latter were given JHm on day 7.5. Control and JHm treated embryos show local apolysis of the eye cuticle and expansion of the depth of the eye due to growth of the rhabdoms; embryos treated with 7EP alone failed to show this growth. PI: propidium Iodide stain, + trans: pseudotransmitted light. (F) The relationship of the final phenotypes of embryos to the time of their treatment with 1 μg of 7EP. Arrows indicate the normal timing of the event. EEF: extraembryonic fluid.

Time-course of the rescue of 7EP-imposed developmental arrest by treatment with JHm.

(A) Groups of about 20 embryos were treated with cyclohexane (controls) or 1 µg 7EP at d3.5 AEL and then monitored daily for the time of reabsorption of the extraembryonic fluid and hatching. The vertical dashed lines indicate the 50% time for these two developmental events in the control group. Replicate groups were also given JHm (1 ng pyriproxyfen) at the indicated times and their development followed to hatching. (B) Summary of the timing of resorption of the extraembryonic fluid (white bars), as indicated by the appearance of air between the embryo and the eggshell, and of hatching (black bars) of 7EP-treated embryos that were then rescued by JHm treatment at the indicated times.

The response of Thermobia embryos to various doses of the JH mimic pyriproxyfen.

(A) Cartoon showing the time of JHm treatment versus the time of dissection. (B) Lateral projections of confocal stacks showing the appearance of typical embryos three days after treatment with the indicated dosage of pyriproxyfen. Embryos were stained for DNA (magenta) and chitin (green). The embryos were dissected away from the yolk which caused disruption in the dorsal thoracic and head regions of some embryos. (C) Photomicrograph of an embryo arrested in mid-katatrepsis. During its envelopment, the contraction of the amnion segregated the yolk (y) from the embryo which hangs in the ventral half of the egg. t: thorax, a: abdomen.

The response of Thermobia embryos to 1 ng JHm given at different times through embryonic development.

(A) Examples of the terminal phenotypes of embryos resulting from JHm being applied at the indicated times [days after egg laying (AEL). Yolk was dissected away from the first three embryos. The remaining embryos had undergone provisional dorsal closure with the amnion enclosing the yolk mass. (B) Graph showing the times when the indicated event can no longer be inhibited by treatment with 1ng of pyriproxyfen (JHm). The 50% point defines the Inhibitory Time50 (IT50). (C) Comparison of the IT50 with the time when 50% of the embryos reach corresponding developmental milestones (Developmental Time50; DT50). (D, E). Lateral confocal and pseudotransmitted light images of the eye region of 7.5 d embryos that had been treated with JHm at (D) 3.5 d or (E) 4.5 d AEL. In (D) only the posterior quarter of the eye placode has been patterned as seen by the posterior crescent of screening pigment and formation of only 3-4 proto-ommatidial clusters. In (E) the entire eye placode has acquired its final form with its 12 ommatidial clusters. The insets below show various Z depths of the boxed cluster in the merged image, with “0 μ” being at the surface. Nuclei of identifiable ommatidial cell types can only be recognized in (E). c: quartet if crystalline cone cells, p: crescent-shaped nuclei of the paired cells that secrete the cuticular lens.

Effects of early treatment with JHm on the subsequent growth and patterning of the limbs.

(A). Z-stack projections showing the lateral view of embryos that were treated with either cyclohexane (Control) or 1 ng of pyriproxyfen in cyclohexane (JHm) on d1.5 AEL and then dissected and stained at the indicated times thereafter. Propidium iodide (PI) (magenta) shows nuclei and anti-phosphohistone H3 (pHH3; green) shows dividing cells. The JHm series follows a subset of embryos that did not undergo katatrepsis (bottom) and a subset that did (middle). (B). Summary of the number of pHH3-positive cells in the developing limb buds of JHm-treated and control embryos through time. Each dot records the average number of pHH3-positive cells/limb from a single embryo. (C). Single optical sections through the limb buds of JHm-treated embryos that did not undergo katatrepsis and illustrating moderate (upper) and high (lower) levels of necrosis. Dead cells were evident as highly condensed PI-positive bodies (yellow triangles). (D). The time course of necrosis in the limbs of control and JHm-treated embryos. Necrosis was pronounced in embryos that failed katatrepsis (F) but rather mild in those that completed katatrepsis (K). (E). Levels of hormone-related transcripts in embryos treated at 2.5 d AEL with solvent alone (blue bars) or JHm (1 ng pyriproxyfen; pink) and then examined over the following two days. The expression is related to day 2.5, no treatment (grey) which was set as 1. Kruppel homolog 1 (Kr-h1), Methoprene tolerant (Met), myoglianin (myo). Significant differences determined by t-test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001. (F). Confocal images showing the effect of treating embryos at 2 d AEL with solvent (C) or JHm and subsequently immunostaining for Distal-less protein (Dll) at the indicated times thereafter. Embryos are shown from ventral view at 22h post-treatment, and then from lateral view thereafter. t: first thoracic leg.

The effects of JHm in inducing early differentiation and terminal maturation in embryos of Thermobia.

(A). Schematic showing the time of treatment with solvent or 1 ng pyriproxyfen (JHm) and the subsequent dissection and staining of the embryos. (B) Quantitation of the effects of JHm treatment on the appearance of striated myofibrils in the developing muscles of Thermobia embryos. Myofibrils normally appear around the time of definitive dorsal closure, but their appearance is advanced by three days in the JHm-treated embryos. (C-E). Confocal optical sections showing F-actin staining in developing longitudinal muscles in control (B) and JHm-treated (D,E) embryos from 4.5 to 8.5 d AEL. Yellow triangles indicate striations of the myofibrils. Insets show low power views of embryos with the magnified region boxed. green: F-actin shown by phalloidin binding, red: propidium iodide. JHm-treated examples are embryos that underwent katatrepsis (D) and ones that failed to complete katatrepsis (E).

Effects of 1ng pyriproxyfen (JHm) on the production of E1 and E2 cuticles.

(A, B) Projected confocal Z-stacks of embryos treated with solvent (Control) or JHm at 1.5d AEL and showing chitin staining of the developing legs at various times thereafter. The insets are PI staining of the same stack to show the form of the limbs on each day. In both groups no chitin was evident at 3.5d AEL, when only epicuticle is present (dots are due to surface debris). In control embryos, the procuticle of the first embryonic cuticle (EC1) (starting at 4.5 d AEL) stains weakly for chitin. The second embryonic cuticle (EC2) shows strong chitin staining (8.5d). JHm-treated embryos show enhanced chitin staining in their EC1 and they produce their EC2 a day early as demonstrated by the presence of cuticular hairs (white triangles). All cuticle images were made with the same laser power and gain. (C) Lateral view of a 5.5 d embryo that was treated with JHm on d1.5 and failed to undergo katatrepsis. The E1 cuticle covering its ventral surface contains higher than normal amounts of chitin staining. The arrow shows the plane of the transverse image the ventromedial region of the embryo is still invaginated into the yolk. PI: propidium iodide staining. (D, E) Projected confocal Z-stacks of untreated (D) and treated (E) embryos showing the effect of JHm treatment at 4.5 d AEL, prior to the production of the EC2 and examined around day 10. The cuticle of the control embryo (D) has a pebbly surface sculpturing, an egg tooth (blue triangle) but lacks cuticular eye lenses (red triangle). The JHm-treated embryo deposited a smooth cuticle that lacks an egg tooth but has abnormally formed cuticular lenses (red triangle). md: mandible.

Comparison of the sensitivity of different embryos to JH.

(A) The developmental responses to JH exposure vary through the embryogenesis of Thermobia domestica. Exogenous JH has little of no effect up through germ band formation. Coincident with the production of the first embryonic cuticle exogenous JH can suppress morphogenesis and evoke precocious differentiation. JH is normally released at definitive dorsal closure and supports terminal differentiation. (B) Comparison of the effects of exogenous JH on the development of different insects highlighting the importance of type of development (short germ band versus long germ band) and the time during development when ecdysteroids are first produced by the embryo. In the short germ band insects, Thermobia and Locusta, JH sensitivity is correlated with the ecdysteroid secretion that induces the first embryonic cuticle. Limb and body patterning is more advanced in Locusta versus Thermobia at that this initial molt, so its development is less repressed. Both embryos, though, undergo substantial body growth between the first molt and dorsal closure and this growth is suppressed in both. Long germ band embryos, by contrast, have finished their organ patterning by the time of the extended germ band and undergo body retraction, rather than growth as they progress to dorsal closure. They lack the intermediate growth period which is sensitive to JH in the short germ band embryos. Locusta ecdysone titers from Lagueux et al., 1977, and JH titers from Temin et al., 1986. Cuticle production and ecdysteroid titers in higher Diptera based on Calliphora (Bordes-Alléaume and Sami, 1987); JH inferred by embryonic expression of Kr-h1 (Beck et al. 2004).

Proposed scheme for the evolving role of JH to support the invention of complex life histories.

In all three grades of development, various lines of evidence suggest that JH interacts with systemic morphogen(s) but a complete picture of the latter has yet to be finished. Overall, the morphogen systems are involved in morphogenesis (M) while JH promotes and supports differentiation (D). In the ametabolous condition the developmental functions of JH are confined to embryogenesis where morphogens and JH act at different times to support morphogenesis followed by terminal differentiation. For hemimetabolous development, morphogens reappear during the postembryonic period to support wing formation, but the extension of JH into the postembryonic period along with this morphogen signaling results in the formation of a nymphal wing bud rather than a wing. The disappearance of JH in the last nymphal stage then allows unantagonized morphogen signaling to form the wing structure. In the holometabolous pattern, embryonic changes bring about a modified larval body plan with much of morphogenesis deferred into postembryonic life. JH continues to be needed in postembryonic life to allow the controlled growth of imaginal primordia rather than their metamorphic morphogenesis. The latter can then occur when JH is removed. The differentiation of these imaginal primordia to their pupal state requires the return of JH during pupal differentiation, reminiscent of the embryonic requirement of JH for juvenile differentiation in embryos of more basal insect groups. See text for more detail.

The percent of embryos attaining a particular developmental “milestone” as a function of the time after egg laying at 37°C. Age of each sample calculated from the midpoint of its 12 hr egg collection.

Age of samples extracted for Juvenile Hormone III measurements. Age of samples from midpoint of 12 hr egg collections. E: embryonic age; J: age of juvenile; n.d.: below range of detectability.

Age of samples extracted for ecdysteroid measurements (given as 20 hydroxyecdysone equivalents). Age of samples from midpoint of 12 hr egg collections. Hatching occurred at 11.5 days after egg laying (AEL)

Primers used for RT-PCR.

The progression of embryonic development of embryos treated with solvent or 7-ethoxyprecocene (7EP) at 3.5 days of development. Developmental rescue was attempted by subsequent treatment with a juvenile hormone mimic (pyriproxyfen) at the indicated developmental time thereafter. air: air appears between embryo and eggshell because of resorption of extraembryonic fluid; eye: appearance of eye pigment. Percent values are based on surviving embryos. *: these embryos blocked before dorsal closure because of the early timing of treatment with pyriproxyfen.

The relationship of the time of treatment with a JH mimic (1 ng pyriproxyfen) to when embryonic development subsequently stalled. EEF: extraembryonic fluid; pre-dorsal closure was evident by dorsal closure to the neck region, but the posterior head capsule had not fully formed.

The effects of early treatment with a JH mimic (1 ng pyriproxyfen) on the subsequent proliferative activity in the embryonic limb buds indicated by the number of limb cells expressing phosphor histone H3 (pPH3). N is the number of embryos scored for each treatment.