CNS-specific transcription of the iab-8 ncRNA
A) Immunostaining on a dissected embryonic CNS against ABD-A (red) and EN (green)
EN marks the anterior border of each parasegment. The scale bar corresponds to 20 µm.
58
B) RNA in situ hybridization on a dissected embryonic nerve cord using the abdA 3'UTR probe. While the structure of the abd-A transcription unit is drawn with exons in black, the iab-8 ncRNA is indicated with exons drawn in open box. The position of the abdA 3'UTR probe used in this experiment is indicated in red.
ABD-A protein (1A) is observed in the embryonic CNS from PS7 to the anterior border of PS13, being completely absent in PS6 and more anterior parasegments, due to Polycomb-mediated repression. In PS13 and PS14 the repression of abd-A is mediated by the iab-8 ncRNA and its associated microRNA (mir-iab-8).
However, when looking at the transcription of the abd-A locus by in situ hybridization using a probe against its 3’UTR (1B), I encounter a different situation. While in PS6 the abd-A domain is not transcribed, as one would expect from a Polycomb-repressed genomic area, PS13 and PS14 harbor a strong signal, which does not reflect the total absence of protein previously observed by immunostaining. Interestingly, this phenomenon had been already described in (Macias et al. 1994), where they proposed that this RNA expression in PS13-14 of the CNS could correspond to a "second wave" of activation of abd-A in this tissue.
However, I noticed that PS13 and PS14 present a different cell-specific pattern than the parasegments that belong to the domain where ABD-A protein is present. While in PS7-PS12 abd-A is strongly expressed in certain clusters of cells, forming a very characteristic
"domino" pattern, in PS13 and PS14 the staining is uniform.
According to previous studies (Karch et al. 1990), including the description of abd-A expression in different mutants and chromosomal rearrangements made in (Gummalla et al. 2012), ABD-A protein is never present in PS14 of the CNS, even in the mutants that show total de-repression of abd-A in PS13. Taking also into consideration the different cell-specific expression of the signal observed in these last parasegments, I tentatively conclude that the transcript observed in PS14 (and by extension, the one present in PS13 with the same expression pattern) cannot be due to the activity of the abd-A promoter.
59
So, what is the origin of this transcript? The closest promoter located upstream from the abd-A promoter is the iab-8 ncRNA promoter. Therefore, it could be possible that this transcript that arrives until the 3’UTR of abd-A corresponds to an extension of the iab-8 ncRNA. The fact that I detect this signal in PS13 and PS14, and that it is uniformly distributed inside each parasegment, supports this hypothesis, as this expression pattern is characteristic of the iab-8 ncRNA.
If the transcript detected in PS13 and PS14 is indeed the result of transcription from the iab-8 promoter, it should not be detected in embryos carrying a deletion of it, such as Fab-864. Figure 2 shows an Fab-864 homozygous embryo hybridized with the abdA 3'UTR probe. In this genetic context, there is a clear expansion of abd-A transcript into PS13, but the signal previously observed in PS14 is lost. The signal detected in PS13 is clearly the result of the de-repression of abd-A that happens in absence of expression of the iab-8 ncRNA. This is supported by the fact that PS13 presents, in Fab-864 embryos, the "domino" pattern characteristic of abd-A, in contrast with the uniform signal that it shows in WT embryos. The lack of uniform signal in PS14 provides a strong argument to conclude that this signal emanates from the iab-8 promoter, and therefore I can tentatively conclude that this is also the case for the uniform signal detected in WT in PS13.
60
Figure 2 The transcription of the abd-A locus detected in PS13 and PS14 disappears in Fab-864 embryos
Dissected embryonic nerve cords of WT (A) and Fab-864 embryos (B) stained by in situ hybridization with the abdA 3'UTR probe.
61
It is worthwhile noticing that the abdA 3'UTR probe is not able to detect any signal in PS13-14 in the embryonic epidermis (Figure 3B, arrow). This signal cannot be detected neither in the ectoderm of embryos of earlier stages (3C,arrow).
Figure 3 The transcription of the abd-A locus detected in PS13 and PS14 is CNS-specific
RNA in situ hybridization against abd-A in WT embryos of stages 14-15 (A,B) and 8-11 (C). A shows a dissected embryonic central nervous system, while B and C show whole-mount embryos.
To gain further insight, I generated a probe to detect specifically the expression of this extension of the iab-8 ncRNA. I took as template the genomic area comprised between exon 8 of the iab-8 ncRNA and the transcriptional start site of abd-A (Figure 4 C,D). To get a quantitative estimation of the amount of such readthrough transcript, I hybridized in parallel embryos with a probe generated from exon 8 of the iab-8 ncRNA (Figure 4 A,B).
62
The in situ hybridization experiments done using these two probes (Figure 4) confirmed the previous results, and allowed me to further characterize the tissue specificity of this readthrough transcription. In the early stages 8-11 the iab-8 ncRNA is strongly transcribed in the ectoderm, as shown in (4A), but it is efficiently terminated, not being able to invade the neighboring intergenic area between the iab-8 nc RNA and abd-A (4C).
In later stages 14-15, however, both probes are able to detect strong transcription in the CNS (4 B,D). Therefore, I could conclude that the readthrough transcription of the iab-8 ncRNA is CNS-specific.
Figure 4 The iab-8 ncRNA presents a CNS-specific transcriptional readthrough RNA in situ hybridization in WT embryos of stages 8-11 (A,C) and 14-15 (B,D) to detect the expression of the iab-8 ncRNA using a probe against exon 8 (A,B), or the production of the transcriptional readthrough inside the abd-A domain using a probe against the intergenic region between exon 8 of the iab-8 ncRNA and abd-A (C,D).
63
Knowing that the iab-8 ncRNA fails to terminate after exon 8 in the CNS, and invades the abd-A locus, the structure of this readthrough was still in question. It had been previously described that the 3'UTR of certain genes present an extension in their neural-expressed isoforms (Ji et al. 2009, Hilgers et al. 2011, Smibert et al. 2012).
However, this does not seem to be the case for the iab-8 ncRNA, as the characterization of alternative isoforms of the iab-8 ncRNA has not detected the existence of transcripts in which exon 8 has a different size.
Interestingly, (Gummalla et al. 2012) described an isoform of the iab-8 ncRNA that extends until the 3'UTR of abd-A, but that lacks the 5' exons of this Hox gene (Figure 5).
Figure 5 The iab-8 ncRNA has alternative isoforms
Structure of the main iab-8 ncRNA transcript (dark blue line). The detected isoforms resulting from alternative splicing and alternative polyadenylation are indicated in light blue. Modified from (Gummalla et al. 2012).
64
With the help of F. Cléard, we identified by RT-PCR different isoforms of the iab-8 ncRNA that spliced into abd-A. They are summarized in Figure 6. One feature shared by all of them was the absence of exon 8. In all cases, there was intergenic splicing happening from exon 7 or exon 6 of the iab-8 ncRNA into an internal exon (2, 4 or 5) of abd-A.
Figure 6 Five isoforms of the iab-8 ncRNA result from intergenic splicing between iab-8 and abd-A
Graphical representation of the five isoforms of the iab-8 ncRNA that result from splicing into the abd-A locus, identified by RT-PCR (F. Cléard, personal communication).
Taking into account all these observations, I can conclude that, in the embryonic CNS, iab-8 and abd-A occasionally undergo intergenic splicing. This phenomenon is caused by a failure of its termination upstream from the abd-A promoter, which has as a consequence the invasion of the abd-A locus, and the generation of a chimeric iab8/abdA RNA, which is not translated, as it lacks the 5' exons of abd-A. This alternative isoform is predominantly expressed in the CNS.
65