Previous to this work, and with the aim to study the role of the iab-8 ncRNA sequence in the repression of abd-A, R. Maeda generated a series of CRISPR-mediated deletions and inversions in the BX-C, which affected the transcriptional unit of the iab-8 ncRNA (Figure 7):
Figure 7 CRISPR-mediated recombination allowed for the production of four different recombinant lines, lacking different elements of the iab-8 ncRNA.
Representation of the mutant lines generated by CRISPR-mediated recombination used to study the expression of abd-A in the absence of certain elements of the iab-8 ncRNA (R. Maeda, unpublished).
- The mutant del(ex8+) carries a deletion of the area downstream from exon 2 of the iab-8 ncRNA to upstream from exon 8.
- The mutant del(ex8-) carries a deletion of the area downstream from exon 2 of the iab-8 ncRNA to downstream from exon 8.
- The mutant Inv carries an inversion of the same chromosomal area that was deleted in the previous mutant.
66
- The mutant DelEx8 carries a deletion that removes only exon 8 of the iab-8 ncRNA.
These internal deficiencies cause various degrees of ABD-A de-repression, illustrated in Figure 8 (R. Maeda, unpublished):
- Deletions del(ex8+) and del(ex8-) show a mild de-repression of ABD-A, reminiscent of the phenotype presented by the ΔmiR mutants.
- The inversion Inv show a complete de-repression of ABD-A in PS13 of the CNS, similar to the Fab-864 phenotype.
- The deletion DelEx8 does not have any effect on ABD-A expression.
Figure 8 The CRISPR-generated mutants for the iab-8 ncRNA present different degrees of ABD-A de-repression
Dissected CNS of WT, del(ex8+), del(ex8-), Inv and DelEx8 embryos immunostained against ABD-A (red) and EN (green). EN marks the anterior boundary of each parasegment. The scale bar corresponds to 20 µm. (R. Maeda, unpublished).
It is interesting to note that del(ex8+) and del(ex8-) show a similar level of abd-A de-repression as the one observed with a deletion of mir-iab-8 alone, indicating that no additional repressive elements lie within these large intervals. It also suggests that, whatever is the second repressive mechanism, it is functional despite the large regions that have been deleted.
67
In contrast, despite the fact that the inversion mutant contains the same regulatory elements as WT (simply inverted), we observe a complete de-repression of abd-A in PS13, indicating that the rearrangement interferes with the production of the iab-8 ncRNA (see next sections).
The iab-8 ncRNA terminates before reaching the abd-A locus in the CRISPR-mediated inversion of the BX-C
I reasoned that if transcriptional interference failure was the cause of abd-A de-repression in the Inv mutant, I would be able to detect a failure of the iab-8 ncRNA to reach the promoter of abd-A. I could not test this hypothesis using my probe against exon 8 of the iab-8 ncRNA, as this region is inverted in the mutant. However, using my intergenic probe, which lies just upstream from the abd-A promoter, I should be able to detect an absence of transcription of this genomic area in PS13 and PS14. This transcriptional readthrough, as explained in the first section, seems to be neuron-specific, so I looked at embryos of stage 14-15 (Figure 9). As I expected, the readthrough signal disappears in the Inv mutant, but it is detectable in the other lines.
Figure 9 The iab-8 ncRNA terminates prematurely in the CRISPR-mediated inversion of the iab-8 ncRNA transcriptional unit
In situ hybridization against the intergenic region between iab-8 and abd-A in WT embryos and the CRISPR-generated mutants del(ex8+), del(ex8-) and Inv.
68
The large CRISPR-mediated deletions affect the termination of the iab-8 ncRNA as well as the regulation of its promoter
To test if the level of ABD-A protein de-repression in the CRISPR-generated deletions and inversion correlates with the de-repression of the transcript, I have performed in situ hybridization in these mutants with the abdA 3'UTR probe (Figure 10).
While there is a good correlation between protein and RNA de-repression in the Inv mutant (10D), there is a surprising discrepancy in the del(ex8+) and del(ex8-) mutants, where the level of de-repression seems much higher at the transcriptional level (10 B,C).
In DelEx8 (10 E) there are high levels of transcription of the abd-A locus in PS13 and PS14, despite the total absence of protein in these parasegments in this mutant.
Figure 10 The CRISPR-mediated modifications of the BX-C affect the normal transcription in the abd-A region
Stage 14-15 CNS dissected out of WT, del(ex8+), del(ex8-), Inv and DelEx8 embryos after in situ hybridization with the abdA 3'UTR probe. The scheme to the right of each CNS illustrates the difference between the "domino" expression pattern of abd-A observed in WT in PS7-12 and the more uniform transcription characteristic of the iab-8 ncRNA detected in PS13-14.
69
Looking at embryos at earlier stages (8-11), I could make some observations that allowed me to interpret the previously described contradictions (Figure 11):
Figure 11 Deletions of part of the sequence of the iab-8 ncRNA affect its normal termination and increase the amount of readthrough transcription into the abd-A locus
Whole-mount embryos of stages 8-11 stained by in situ hybridization using the abdA 3'UTR probe. Comparison between WT (A), the two large CRISPR-mediated deletions del(ex8+) and del(ex8-) (B,C), the mediated inversion Inv (D) and the CRISPR-mediated deletion of exon 8 alone Del(Ex8) (E).
70
In the mutants del(ex8+) (11B) and del(ex8-) (11C), the abdA 3'UTR probe detects strong, uniform transcription in PS13 and PS14 of the ectoderm, being stronger in PS13. I have already stated that the abd-A promoter is never active in PS14 in WT or in any other characterized mutant contexts. In addition, it has been already shown that ABD-B is responsible for the repression of abd-A in PS13 of the ectoderm. The deletion of the microRNA mir-iab-8 has no effect over abd-A expression in PS13 at this embryonic stage, as I had observed by studying ΔmiR mutants (Appendix, Figure S2). Therefore, I deduce that the signal detected in PS13 and PS14 in these mutants correspond to the readthrough transcription of the iab-8 ncRNA, described in the first section.
Finally, the fact that exon 8 deletion alone DelEx8 presents a strong uniform signal in PS13 and PS14 (and given that in this mutant the expression of ABD-A protein is unaffected), confirmed me that this signal indeed emanates from the iab-8 ncRNA promoter. In this mutant, the iab-8 ncRNA does not have a terminator signal, and therefore all of the nascent transcripts that initiate in iab-8 are able to invade the abd-A locus.
Taking all of this into account, I am able to propose an explanation for the strong abdA-like signal that I can detect in the CNS of PS13 using the abdA 3'UTR probe in del(ex8+) and del(ex8-) mutants (Figure 12).
71
Figure 12 The cell-specific enhancers present in iab-2/iab-3 interact with the iab-8 ncRNA promoter in the del(ex8+) and del(ex8-) mutants
Graphical representation of the genetic interaction established between the abd-A and the iab-8 locus in del(ex8+) and del(ex8-) mutants, and dissected CNS of each genotype showing an in situ hybridization against abd-A.
Due to the large deletions in the del(ex8+) and del(ex8-) mutants, the enhancers present in iab-2/iab-3, responsible for the cell-specific expression pattern of abd-A, lie now closer to the promoter of the iab-8 ncRNA. Because of this, the iab-8 ncRNA changes its usually uniform expression pattern into the abd-A-like "domino" pattern in PS13 of the CNS (Figure 12, arrows). These deletions also cause an increase in the number of transcripts that are not able to terminate in exon 8 of the iab-8 ncRNA, either because this exon is absent or because of an increased number of transcripts that arrive to this area, due to the reduction of the size of the transcriptional unit of the iab-8 ncRNA. This is supported by the easy detection by in situ hybridization of readthrough transcripts in the ectoderm of mutant embryos of early stages 8-11, whereas in WT the readthrough transcription is CNS-specific (Figure 11 B,C). In addition, in these embryos the signal observed in PS13 is stronger than the one observed in PS14, which supports the idea that iab-2,3 enhancers (active in PS13, but not in PS14) are acting on the promoter of the iab-8 ncRNA. In the DelEx8 embryos (Figure 11E), on the other hand, there is not such difference, because in these embryos the enhancers that drive abd-A expression in PS13 are still far away from the iab-8 promoter.
72