Heat shock induced VSR expression rescues abdominal segmentation

Next, I asked whether it was possible to suppress the RNAi effect by activation of VSR during GZ-elongation in order to regain previously silenced gene expression.

For that purpose, Tc-prd served again as control gene. Two transgenic hsVSR lines (hsCrPVi⁴⁷ and hsCrPVi⁴⁹) were generated, carrying CrPV1A under the control of a heat shock promoter (Schinko et al., 2012) (see materials and methods). Both insertion lines were tested in the rescue experiments. Heterozygous or homozygous hsVSR pupae were selected for their DsRed eye marker. Female pupae of these animals were injected with Tc-prd dsRNA (2.7µg/µl) and, subsequently, crossed to transgenic males for mating and egg deposition. Hence, at least 75% of the embryonic offspring should be heterozygous or homozygous for the transgene hsCrPVi. Parental RNAi against Tc-prd (pRNAi arrowhead in Fig. 4.12) should affect embryonic blastodermal patterning, leading to anterior structure defects in the gnathal and thoracic segments of the progeny. The subsequent heat shock mediated VSR activation (hsVSR in Fig. 4.12) during elongation should be able to recover the expression of the secondary pair-rule gene Tc-prd, resulting in a rescued abdominal segmentation (Fig. 4.12 increasing lines). The segmentation was considered to be rescued if the heat shocked Tc-prd RNAi cuticles revealed six to eight AS in contrast to the non-heat shocked control Tc-prd RNAis, which showed a strong abdominal phenotype with four to five AS (Fig. 4.13, Fig. 4.14 B). In order to establish the proper time point for the VSR pulse, RNAi embryos developed at 32°C until they reached germ rudiment to elongation stages (egg collections of 10-15h, 11-16h and 12-17h old embryos) and then heat shocked for 10 minutes at 48°C to activate the RNAi suppressor (see materials and methods) (Fig. 4.12, 4.13). In parallel, wt embryos of the same age were stained for Tc-wg expression to determine the embryonic stage at the heat shock treatment (Fig. 4.12). Furthermore, transgenic, non-heat shocked phenotype (Fig. 4.13 A-B right panels, Fig. 4.14 D). This phenotype was observed in each egg collection (Fig. 4.13). Especially, heat shocks of transgenic RNAi embryos

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61 at the age of 10-15h led to a high number of cuticles with restored abdomen (38% of line hsCrPVi⁴⁷ and 25% of hsCrPVi⁴⁹) (Fig. 4.13). The remaining treated embryos revealed either a shortened abdomen with 4-5 AS (Fig. 4.13, Fig. 4.14 C) or undefined segmentation number (called “no segmentation” in Fig. 4.13 right panels, Fig. 4.14 E).

Furthermore, the transgenic line hsCrPVi⁴⁹ tended to exhibit lower cuticle numbers with rescued AS than line hsCrPVi⁴⁷, indicating some position effect (Fig. 4.13, compare right panels of A and B).

Fig. 4.12: Model for the abdominal rescue approach by VSR activation.

Parental RNAi (arrowhead) of the secondary pair-rule gene Tc-prd silenced the expression (decreasing solid line represents the downregulated gene expression) in pupal, adult and embryonic stages. In order to rescue Tc-prd function during GZ-elongation, egg collections with embryos aged 10-15h, 11-16h and 12-17h (developed at 32°C) were heat shocked twice for 10 minutes at 48°C (see further details in materials and methods) to start VSR expression (bars indicate the age of the respective egg collections at the time when the first heat shock was performed). Suppressor activation in staged embryos resulted in rescued Tc-prd expression, associated with rescued abdominal segmentation (increasing lines represent rescued gene expression and rescued segmentation; solid line for 10-15h old embryos, dotted line for 11-16h old embryos and dashed line for 12-17h old embryos). Anterior segmentation was, however, impaired due to the knockdown of Tc-prd expression (decreased line during embryogenesis). Embryonic developmental stages corresponding to the timing of the first heat shock were determined by Tc-wg staining. As an example, a 10h old germ rudiment, a

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13h old germ band with five Tc-wg stripes, a 15h old elongating germ band with nine Tc-wg stripes and a fully elongated 17h old germ band stage with fifteen Tc-wg stripes are shown (anterior is up).

Fig. 4.13: Cuticle defects post parental Tc-prd RNAi followed by RNAi rescue in staged embryos

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63 (A, B)Two transgenic insertion lines carrying heat shock inducible CrPV1A were used (hsCrPVi⁴⁷ in A and hsCrPVi⁴⁹ in B) for the abdominal rescue approach. pRNAi against Tc-prd was followed by heat shock mediated CrPV1A activation in staged embryos (prd RNAi;+hs) (see respective age at the left).

Absolute numbers for each cuticle analysis are given at the right. The percentage of cuticle defects in head, thorax and urogomphi are shown in the left panels. The numbers of remaining abdominal segments are shown in the right panels. Transgenic, non-heat shocked Tc-prd RNAi embryos (prd RNAi; -hs) served as RNAi controls. Transgenic, non-injected but heat shocked embryos (hs control) served as heat shock controls. Abbreviations for the cuticle defects are given next to the corresponding bar once for each insertion line and category. An: antenna, Lr: labrum, Md: mandible, Mx: maxilla, Lb: labium, T1-T3: thoracic segments 1 to 3, Urog: urogomphi, 1AS-8AS: a total of one to eight abdominal segments, no s.: no visible abdominal segments, wt: wild type phenotype.

Fig. 4.14: L1 larval cuticle phenotype classes after Tc-prd pRNAi and RNAi rescue by VSR activation.

Anterior is to the left. (A-E) Lateral views. (B-E) Transgenic embryos carried hsCrPV1A (at least 75%).

(A) Wild type untreated L1 cuticle. (B) Non-heat shocked Tc-prd RNAi control cuticle represents a strong RNAi phenotype with missing gnathal segments (mandibles and labium) and thoracic segment T2 as well as with remaining 4 AS (white arrows). (C) Heat shocked Tc-prd RNAi cuticle with 4-5 AS (white arrows) and gnathal and thoracic defects like in B, indicating that the rescue was not successful either due to the age of the embryo or because the embryo did not inherit the construct (25%). (D) Heat shocked Tc-prd RNAi cuticle with 7 AS (white arrows) and gnathal and thorax defects like in B, indicating that the RNAi effect was successfully rescued. (E) Heat shocked Tc-prd RNAi cuticle represents the “no segmentation” phenotype. Cuticle shows gnathal defects like in B and missing thoracic (T2 and T3) and abdominal segments. Abbreviations: An: antenna, Lr: labrum, Md: mandible, Mx: maxilla, Lb: labium, T1-T3: thoracic segments 1 to 3, A1-A8: abdominal segments 1 to 8, wt: wild type.

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5 Discussion