TC004374 was chosen as a potential interesting candidate after the screen-ing steps due to a penetrant head phenotype detected durscreen-ing cuticle analysis and a small or absent head phenotype detected in the Tc-wingless (Tc-wg) in situ hy-bridization screen in RNAi embryos (Figure 4-7 G-G’’).
To analyze the expression pattern of TC004374, RNA antisense probes were synthesized based either on the entire coding sequence (JS_M133) of the TC004374 gene or on a 3’-part, spanning 490 bp of exons 3+4 (JS_M255, se-quences on DVD, folder Genes_Sequences/iB_03735_TC004374/
M133_M264_M255). These probes were used for in situ hybridization on embryos of age 0-48 hours, development at 31°C. Sense probes served as negative con-trols. No localized expression for TC004374 could be detected in any stages ana-lyzed, suggesting uniform expression of this gene (not shown).
To ensure that the RNAi phenotype observed after injection of TC004374 dsRNA was not due to degradation of Tc-Cyclin J transcripts, Tc-Cyclin J cDNA was isolated from an embryonic cDNA collection using gene specific primers (JSP198 and JSP199). After parental RNAi against Tc-Cyclin J all progeny showed an empty egg (EE) phenotype in cuticle analysis and brief light micro-scope analysis of these embryos supported the assumption that the embryos did not develop at all. This result confirmed that the anterior patterning phenotype ob-served for TC004374 RNAi was not due to loss of Cyclin J function.
The cuticle phenotype resulting from TC004374 RNAi was characterized by anterior deletions of body cuticle, similar to the cuticle phenotypes seen after Tc-smurf RNAi. TC004374 RNAi phenotypes displayed head defects or broad dele-tions of anterior body cuticle (Figure 4-27, B+C). Two independent iBeetle dsRNA fragments (iB_03735_2, iB_03735_3), both partially overlapping with the original
iBeetle fragment (iB_03735_1) were injected for TC004374 (green annotations in Figure 4-25 B). Injection of the iB_03735_1 fragment resulted in the anterior pat-terning phenotype observed during the iBeetle screen. During injection of the two new non-overlapping fragments, only injection of the iB_03735_3 dsRNA fragment resulted in the anterior patterning phenotype. After injection of dsRNA against iB_03735_2 no cuticle defects were observed and almost all progeny of dsRNA injected females hatched. This gave rise to the assumption, that the anterior pat-terning phenotype depended on the 3’ region of the TC004374 gene. To further clarify this result dsRNAs based on two different sequence parts of the TC004374 cDNA were synthesized. One targeting exons 1 and 2 (Exon 1+2 fragment), and one targeting Exons 3 and 4 (Exon 3+4 fragment). These dsRNAs were used for a parental RNAi experiment in different Tribolium beetle strains to verify the frag-ment specific phenotype and to test for strain specific effects at the same time (Figure 4-25 B for fragments and Figure 4-28 for results of the injections). Injec-tions were done in the SB, Pig–19, and Black strains. Cuticle preparations on egg collections were done at three different time points to analyze a wearing off of the knock down effect (13, 20, and 27 days after injection, abbreviated dai). Buffer in-jections and RNAi against DsRed served as negative controls. Progeny of the in-jected females were counted and scored as described for the similar experiment for Tc-smurf (4.3.1).
Figure 4-27 Cuticle phenotype of TC004374
Anterior to the left for cuticles. A and B lateral views, C dorsal view. (A) Wild type L1 cuticle. ug: urogomphi, pg: pygopodia. (B) Mild TC004374 RNAi phenotype, broad regions of the anterior head are missing. (C) Stronger TC004374 RNAi cuticle phenotype. The entire head and thorax and part of the abdomen are miss-ing. This cuticle also shows a dorsal hole (arrowhead).
Similar to the observations seen for the RNAi experiments for Tc-smurf, the SB strain showed a relatively high number of unspecific background phenotypes observable in all different conditions tested (grey bars in in Figure 4-28). The frac-tion of these background phenotypes ranged between 15 % and 30 % in the con-trols. Background phenotype levels in the Black and in the Pig-19 strains never exceeded 10 %. Anterior defects were observed in 0–3 % of all analyzed progeny in the controls. Injection of the TC004374 Exon 1+2 dsRNA fragment resulted in 0–1,7 % anterior defects, confirming that dsRNAs targeting the first two exons of TC004374 did not lead to anterior patterning defects in any of the strains ana-lyzed. Injection of the TC004374 Exon 3+4 dsRNA fragment resulted in anterior patterning defects. The fraction of animals that showed defects or deletions of an-terior body parts ranged, depending on the strain, from 26,6 % to 56,5 % 13 dai and wore off during the following two weeks of the experiment. 27 dai the fraction of anterior defects observed among all progeny ranged between 2,6 % and 4,7 % and was thereby comparable or almost comparable to the controls. If at all, the amount of other defects was only slightly increased in TC004374 Exon 3+4 RNAi cuticles. The Pig-19 strain appeared to react stronger to knock down of TC004374 and the RNAi effect in this strain was also more stable over the time of the exper-iment. The fraction of anterior defects after injection in Pig-19 was perceptibly higher than in the other strains 13 and 20 dai. Additionally, after injection in the Black and the SB strain, a strong decline in the fraction of anterior defects was al-ready observed between 13 and 20 dai. After injection in the Pig-19 strain this fraction was almost consistent between these two time points, although a small increase in wild type progeny could be observed. Almost no anterior defects were observed 27 dai. The different strains showed a different susceptibility to TC004374 RNAi resulting in a different penetrance of the knock down effect. Qual-itatively, anterior defects could be observed in all strains after TC004374 RNAi.
Strain specific effects could therefore be excluded. Due to the observed higher penetrance, Pig-19 was used in all following experiments.
Figure 4-28 Fragment specific knock down phenotype of TC004374
Different strains showed a different ratio of background defects (grey bars). IP: Buffer injections. Only dsRNAs covering the region of Exon3+4 of the TC004374 gene led to high pro-portions of anterior defects (red bars). The anterior defect phenotypes were detectable in all strains analyzed. A decline in these phenotypes was observed two to three weeks after injection. See text for more details.
4.4.2 Immunohistochemistry and expression of marker genes in TC004374