NF-kappaB/Rel-mediated regulation of the neural fate in Drosophila

Abstract

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-kappaB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. We propose that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis.

Figure 1. Loss-of-function mutants of NFκB/Rel genes display both loss and gain of macrochaetes on the notum.

(A), a schematic representation of a wild-type heminotum, with the medial domain shaded yellow and the lateral domain orange. Large grey circles represent the positions of the eleven macrochaetes found on each heminotum. (B–D), heminota of flies homozygous mutant for dorsal (dl⁴/dl¹), Dif (Dif¹/Dif¹) and Relish (Rel^E20/Rel^E20); white arrows indicate ectopic bristles. Below, pie charts represent the percentage of heminota displaying ectopic bristles from a total of 200. Red sectors indicate the percentage with one ectopic macrochaete, yellow sectors two or more ectopic macrochaetes (p>0.001 when compared with wild type). (E, F), heminota of Ore-R and dl⁴/+ animals respectively, showing the lateral notum. Red arrows mark the pSA bristle, which is missing in a large fraction of dl⁴/+ (and also dl⁴/Df(2L)TW119, dl⁻ dif⁻ animals) (p>0.001). (G), the percentage of flies trans-heterozygous for various mutant combinations that display ectopic bristles (p>0.001).

Figure 2. Over-expression of NF-κB/Rel proteins can result in both loss and gain of macrochaetes.

scabrous[537.4]Gal4 was used to over-express Relish (A, B) Dorsal (C–F) and Dif (G) in proneural clusters. This resulted in a loss of bristles in lateral regions (red arrows in B, D, F and G). Ectopic bristles were present in medial regions of animals after over-expression of Relish and Dorsal (white arrows in A, E).

Figure 3. Toll-8 is expressed at varying levels in the disc epithelium and expression is extinguished in the neural precursors.

(A–B'), GFP expression driven by the Toll-8[MD806]Gal4 driver in discs from third instar larvae (A, A') and white prepupae (B, B') respectively. Expression is strong over the lateral notum but is excluded from the sensory organ precursors, which are stained for neuralized activity (A101, anti-β galactosidase; red). At the third larval instar, expression is clearly missing from the aPA precursor, and by prepupal stages, additional “holes” corresponding to the posterior supraalar (pSA) and sensilla trichoidea 1 (tr1) precursors are obvious in the lateral notum. (A') and (B') are higher magnifications of boxed areas in (A) and (B). (C), the expression of Toll-8 as revealed by in situ hybridisation with a Toll-8 RNA probe. Expression is strong in the lateral notum. (D), thorax of a fly of the genotype y f^36a abx>f⁺>Gal4; UAS Flp; Toll-8-Gal4. All MD806-positive cells in these flies simultaneously express flipase which induces high levels of FRT-mediated recombination. Consequently, all bristles marked with f^36a arise from cells expressing Toll-8. As can be seen in (3D), f^36a bristles can be seen even in the medial region of the notum. (E), thorax of a neurGal4>UAS-E(spl)m8 fly showing a complete loss of bristles. (F), thorax of Toll-8Gal4>UAS-E(spl)m8 fly showing a full complement of bristles, indicating that Toll-8 is not expressed in the precursors.

Figure 4. A protein composed of a fusion of Relish and VP16 can bind to the scute SOP-enhancer and activate transcription in vivo.

(A–C), expression of sc-SOPE-lacZ in wild type (A), Bx-Gal4>UAS-RelVP16 (B) and Bx-Gal4>UAS-ase discs (C). Over-expression of asense results in activation of the reporter gene over a larger area than over-expression of RelVP16. (D–F), expression of sc-SOPEα3⁻-lacZ (a version of the enhancer construct in which one of the NF-κB/Rel binding sites, α3, has been mutated) in wild type (D), Bx-Gal4>UAS-RelVP16 (E) and Bx-Gal4>UAS-E(spl)m7VP16 (F) discs. Expression is decreased in wild-type flies and after over-expression of RelVP16, but strongly increased in the presence of E(spl)m7VP16 whose activity does not rely on the same binding sites.

Figure 5. Expression of neuronal genes in Toll-8 and Relish mutants.

(A–I), in situ hybridisation with probes for scute, asense and senseless in wild type and in Toll-8¹ and Rel^E20 mutant discs. Each set of three discs was processed in the same way and stained for the same length of time. scute transcripts are present at higher levels in the proneural clusters and the clusters themselves appear enlarged (A–C). Expression of asense (D–F) and senseless (G–I) is also much stronger in the mutant discs in bristle precursors, the cells to which they are confined in the wild type. Staining in the mutants was already strong before any staining in the wild type had become visible. Arrows in (E) and (F) point to the precursors arising from the dorsocentral cluster and insets in (D) and (G) show SOPs in wild-type discs stained for 40 minutes. In addition, asense and senseless transcripts can be seen to accumulate ectopically over most of the epithelium at very high levels. (J–L), staining with an antibody against Senseless. The protein is present only in sensory organ precursors.

Figure 6. Transcript stability in Relish mutants correlates with the presence of a heptamer sequence in the coding regions of target genes.

(A), levels of heterologous (UAS) achaete, scute, asense and GFP transcripts driven by sca-Gal4 in wild-type (left column) and Rel^E20 (right column) flies. The ribosomal RNA RP49 was used as a loading control. Levels of scute and asense but not ac transcripts are elevated in the mutant. In addition levels of GFP transcript are elevated. (B), a sequence motif similar to that described in Sox9 and MyoD (ACTAGA) is present in scute, asense, senseless and a number of other genes involved in patterning the notum. The central five core element nucleotides, CTACA, are conserved in all cases. The 3′-most nucleotide, G, is replaced in most cases by a T or an A. The 5′-most nucleotide, A, is replaced by a G in three cases (shown in grey). The final base, G, is mostly substituted by A or T. (C), the sequence of scHw^Ua is presented. Transcription stops within the copia element whose sequence is given in red. The two MyoD motifs are outlined in grey; one, M2, is predicted to be absent from the truncated transcript. (D), a photograph of a scHw^Ua mutant fly showing the presence of an ectopic dorsocentral bristle (white arrow). (E), pie charts representing the percentage of heminota displaying ectopic bristles from a total of 200. Red sectors indicate the percentage with one ectopic macrochaete, yellow sectors two or more ectopic macrochaetes (p>0.001 when compared with wild type).

Figure 7. A model for the role of NF-κB proteins in buffering levels of the neural genes scute and asense.

Epidermal cells express high levels of Toll-8, which promotes high nuclear levels of NF-κB/Rel. Relish activates the transcription of an unknown target gene, whose activity results in the degradation of scute and asense transcripts. This activity extends throughout the neuro-epithelium of the disc. Once mature precursors are chosen, they stop expressing Toll-8 and low levels of the NF-κB/Rel proteins might be recruited to the scute SOPE, where they would synergize with the bHLH protein Scute to activate transcription of high levels of scute.