Tc-wingless expression in candidate RNAi embryos

The aim of this project was the identification of genes which are required for early anterior patterning processes. To this end RNAi embryos were analyzed to verify if knock down of the candidate genes resulted in specific anterior defects

during early embryonic stages or if the observed effects in larval cuticles were rather secondary due to general disruption of embryogenesis or later cuticle secre-tion. RNAi embryos were collected for all 26 injections from the non-overlapping fragment (NOF) injection in the SB strain experiment (4.2.3, Table 4-1). The em-bryo collections contained about 8–24 h old emem-bryos which developed at 31°C and were collected over a period of about one week. To facilitate phenotype identi-fication and interpretation the embryos were stained for expression of the segment polarity gene Tc-wingless (Tc-wg). Tc-wg is expressed in a segmental pattern in the Tribolium germ band (Nagy and Carroll, 1994). Germ band stage embryos were analyzed for phenotypic defects and Tc-wg misexpression. This experiment was done once for every candidate and some of the dsRNA injected females showed decreased egg deposition, resulting in limited RNAi embryos for pheno-type analysis. For this reason the results must be seen as preliminary. For 15 dsRNA injections the observed phenotype was wild type, or the amount of ob-tained germ band stage embryos was not sufficient to draw a conclusion (not shown). Figure 4-7 depicts embryos from those 11 embryo collections where mor-phological or Tc-wg-expression defects could be detected in at least three germ band stage embryos. For some of the candidate genes a head specific knock down effect could not be confirmed by NOF injection into the SB strain (marked by a red box in the right column of Figure 4-7). For many of the not confirmed candi-dates a high number of strong phenotypes or empty eggs (no detectable cuticle in the egg after cuticle preparation) was found during cuticle analysis. This might be an indication for a rather broad and more general developmental defect. It might also be that the phenotype observed after injection into the SB strain was stronger than the phenotype obtained in the Pig-19 strain. For other candidates the cuticle phenotype could be confirmed (green boxes) but the embryonic phenotype looked rather unspecific for head or anterior patterning defects (e.g. iB_00992, Figure 4-7 B–B’’; iB_01556 Figure 4-7 C–C’’; iB_03525, Figure 4-7 F–F’’). For these three candidates also high numbers of strong cuticle defects, not head specific defects, or empty eggs were documented for the cuticle analysis. Because the embryonic phenotypes indicated general developmental defects these candidates were no longer considered.

One interesting observation was the shift of the ocular Tc-wg-expression do-main towards a more anterior central position seen for the candidate iB_05754

(Figure 4-7 K–K’’). This effect was observed in several embryos. Although the cu-ticle preparation for this candidate showed a high number of empty eggs and strong defects and the head defects which were seen did not appear to be spe-cific, the embryonic phenotype suggests specific RNAi-mediated defects in the head tissue. It might well be that this gene is involved in head patterning as well as in other developmental processes during embryogenesis. This might result in plei-otropic effects on level of the L1 epidermis which hampers the interpretation of the cuticle phenotype. Finally, for two of the tested candidates specific anterior defects were observed in the cuticle preparations as well as in Tc-wg-stained RNAi em-bryos. For this reason they were selected for a detailed analysis.

iB_02881 showed a high penetrance of headless or head-defective cuticles in the cuticle collection. These phenotypes will be explained in more detail in 4.3.1, pictures are given in Figure 4-10. Headless or head size reduced embryos were also found in the RNAi embryos stained for expression of Tc-wg by in situ hybridi-zation. Additionally, anterior stripes for Tc-wg expression were often missing (Figure 4-7 E–E’’).

Two NOF dsRNAs, covering the original iBeetle fragment, were injected for iB_03735. Injection of the first NOF dsRNA against iB_03735 did not result in de-fects on the L1 cuticle, and also no developmental dede-fects were found in the RNAi embryos stained for Tc-wg expression. However, injection of a second NOF re-sulted with high penetrance in the cuticle phenotype observed in the screen and the analysis of RNAi embryos also confirmed specific anterior defects (Figure 4-7 G–G’’). Cuticle phenotypes for iB_03735 RNAi displayed head defects and miss-ing anterior body regions (shown in Figure 4-27). The different phenotypes ob-tained after injection of different dsRNAs for this candidate suggested a transcript- or splice variant-specific knock down effect for this gene. The gene model and the dsRNA fragments which resulted in head and anterior patterning phenotypes for iB_03735 will be shown later (4.4).

Figure 4-7 Tc-wg in situ hybridization screen in candidate RNAi embryos

Germ band stage embryos of different age were stained for the expression of the segment polarity gene Tc-wg. The column to the right gives a brief summary for the results of the corresponding cuticle analysis for NOF

injections in SB females. Green boxes: iBeetle phenotype confirmed, red boxes: iBeetle phenotype not con-firmed. Anterior to the left for all embryos. Embryos in one row are successively older from left to right. Not all embryos in one column are the same age. (A–A’’) In wild type embryos Tc-wg is expressed in every segment and in the posterior segment addition zone or growth zone (gz). Abbreviations for the segments are given next to the corresponding Tc-wg-stripe. oc: ocular, ic: intercalary. Abbreviations for the other segments are the same as in Figure 4-1 and can also be found in the abbreviation list. (B–B’’) Embryos after RNAi against iB_00992 showed missing or irregular Tc-wg stripes and sometimes abnormal morphology. (C–C’’) The RNAi phenotype of iB_01556 looked similar but stronger. (D+D’) iB_01574 RNAi could cause loss of almost all Tc-wg stripes, however morphological defects were not as severe as in B’’–C’’. (E–E’’) Knock down of iB_02881 resulted in smaller headlobes or deletions of entire anterior body regions. (F, F’’) iB_03525 RNAi could result in complete absence of Tc-wg-stripes associated with smaller headlobes. However, also relatively intact em-bryos with missing posterior Tc-wg stripes were observed in the embryo collection (F’). (G–G’’) Knock down of iB_03735 led to headless embryos (G’’) or reduced heads. Note the point-like ocular Tc-wg expression do-mains in G and G’ (arrowheads). (H) Early stage embryo of RNAi against iB_04050 which showed severe morphological defects and impaired Tc-wg expression. In later stage embryos, a thinner germ band with miss-ing posterior Tc-wg stripes was observed (H’). In some embryos the germ band was shortened (H’’). (I) Young germ band stage embryo after iB_06031 RNAi, morphologically similar to the phenotype seen in G. Also the ocular Tc-wg expression stripe was changed to a point like expression domain (arrowhead). (I’ and I’’) Tc-wg pattern of this embryo was only mildly affected, however, the embryo in I’’ showed strong defecs in Tc-wg pattern and general morphology. (J–J’’) Tc-wg expression was lost in some segments of iB_03951 RNAi em-bryos. (K–K’’) Random loss of segmental Tc-wg-expression was detected in iB_05754 RNAi emem-bryos. Note the anterior shift of the ocular (oc) Tc-wg expression in K’ and K’’ (compare to A’ and A’’). Lack of anterior tissue could potentially also be seen in the young embryo in K (black arrowhead).

4.3 Identification, isolation and characterization of iB_02881 as