To further clarify the embryonic phenotype after TC004374 RNAi, live imag-ing experiments were performed usimag-ing a transgenic line that expresses nuclear localized GFP in all embryonic cells (Sarrazin et al., 2012). Experimental setup,
controls and scoring of embryos were identical to the experiments performed for Tc-smurf RNAi 4.3.5.
TC004374 RNAi embryos exhibited several defects during development. The first defect observable for TC004374 RNAi embryos was a remarkable delay in forming the amniotic fold. Formation of the amniotic fold and gastrulation is a pro-cess that happens smoothly and very quickly in wild type embryos. Figure 4-33 shows selected frames of a control embryo undergoing gastrulation. Time points indicated in the given frames are referred to the time after flattening of the posteri-or pole became firstly visible (Tc-smurf live imaging chapter 4.3.5). Pictures were taken every 10 minutes. During each time interval a morphological advancement in development could clearly be identified for the control embryo. The amniotic fold formed about 30 minutes after formation of the differentiated blastoderm stage (Figure 4-33, 150 minutes frame) and within the next 30 minutes, it reached the ventral side of the embryo. 240–280 minutes after posterior flattening the embryo was completely covered with serosa and amnion. In control embryos the amniotic fold reached between 30 and 50 % of the anterior to posterior distance on the ven-tral side (Figure 4-33 frame 180 min) within 190 minutes after posterior flattening.
This was the case in 32 out of 35 embryos (mean time 177 minutes).
The TC004374 RNAi embryo appeared to be stuck at the differentiated blastoderm stage (Figure 4-34). After formation of the differentiated blastoderm only very low developmental progress was observed for almost 60 minutes. The embryonic cells moved slowly towards posterior and about 180 minutes after pos-terior flattening an indentation on the pospos-terior dorsal side occured. In the control the amniotic fold had already reached the ventral side of the amnion at this timepoint. Suddenly, between 210 and 220 minutes after posterior flattening the amniotic fold moved over the posterior pole and the embryo was covered by the extraembryonic membranes. A delay in the progress of the amniotic fold could be seen in a high number of TC004374 RNAi embryos: 10 out of 22 embryos needed over 210 minutes before the amniotic fold reached between 30 and 50 % of the anterior to posterior distance on the ventral side (mean time 215 minutes). Two other phenotypic aspects after TC004374 RNAi were observed during live imag-ing. 6 out of 20 TC004374 RNAi embryos scorable for the head showed clearly identifiable smaller heads (1 out of 49 in control embryos). 9 out of 26 embryos showed decaying tissue (control 1 out of 46), either in the anterior or laterally at
the border between embryonic and extraembryonic tissue. Cell death was identifi-able by loss of fluorescence in the embryo proper. In TC004374 RNAi embryos the embryonic rim often appeared to be less strictly defined than in the control. Alt-hough this observation would fit to the results for the TUNEL assay, this was diffi-cult to score with the given optical resolution. Figure 4-35 shows selected frames for the development of a TC004374 RNAi embryo displaying all phenotypic fea-tures mentioned. All movies are included on the DVD (folder Mov-ies/TC004374RNAi).
Figure 4-33 Time-lapse of control embryo undergoing gastrulation
0–280 minutes frames: lateral view. 900 minutes frame: ventral view. Given are 10 minutes time lapse frames of a control embryo with focus on the period of amniotic fold formation and embryo enveloping. Reference timepoint is posterior flattening (yellow arrowhead in 0 min frame) of the embryo before posterior pit formation.
120 minutes frame shows the differentiated blastoderm with clearly identifiable embryonic (e) and serosal (s) nuclei. Shortly later the dorsal edge of the embryonic tissue moved over the posterior of the embryo and formed the amniotic fold (arrowhead in 150–190 min frames). 190 min after posterior flattening over 30 % of the posterior embryo was covered with the extraembryonic membranes.
Figure 4-34 Amniotic fold delay in TC004374 RNAi embryo
Reference timepoint is posterior flattening (yellow arrowhead in 0 min frame) of the embryo before posterior pit formation. Lateral view, anterior to the left, dorsal side up. 120 minutes frame shows the differentiated blastoderm with distinguishable embryonic (e) and serosal (s) nuclei. Cells condensed at the anterior ventrally and in the posterior along the entire dorsal-ventral axis. The posterior cells moved slowly further posteriorly. A bend became apparent at the germ serosa border (arrowhead in 160 min frame). After 180 min an indentation was visible at the dorsal posterior end of the embryo. Only very little developmental progress could be seen until 210 min after posterior flattening. Then, suddenly, the amniotic fold (arrowhead in 220 min frame) quickly moved over the posterior pole and the embryo was covered with the extrembryonic membranes.
Figure 4-35 Selected frames of the development of a TC004374 RNAi embryo
Before turning of the embryo (about 360 min time frame) pictures show a slightly tilted ventral view. After-wards: lateral view. Reference timepoint is posterior flattening (yellow arrowhead in 0 min frame) of the em-bryo before posterior pit formation. 40 min after posterior flattening, emem-bryonic (e) and serosal (s) nuclei could
be distinguished and a clear differentiated blastoderm had formed after 120 min. The amniotic fold (arrowhead in 240 min frame) started covering the germ rudiment about 240 minutes after posterior flattening, and the serosal window (open arrowheads in 280–360 min time frames) started to close. Headlobes (H in 240–
360 min frames and in 600 min frame) were detectable but especially later (600 min) they were clearly re-duced in size (compare to control embryo in Figure 4-21 In vivo imaging of Tc-DsRed RNAi embryo (negative control)). The borders of the embryo appeared abnormally indistinct (arrowheads in 560 min frame) and the head never moved to the anterior as observed in the control. Later, fluorescence in the embryonic tissue vanished from the anterior germ band region, indicating potential cell death (arrowhead in 1070 min frame).
4.5 Isolation and characterization of Tribolium mothers against dpp