ROSAY's thesis: The NKD receptor

NKD promoter

First we work on the promoter region of NKD to know his expression and regulation. We compare results obtained with transgenic flies to transient transfections data. We localise the transcription start site and found transcriptional regulatory sites including potential E boxes. This indicates a possible regulation by bHLH-type of transcription factor. We used this promoter to drive a reporter gene expression in transgenic flies

Embryonic expression

Anti-ßgalactosidase staining start at stage 10, when cells' divisions that give rise to PNS begin. The stage 10 pattern represents transiently labelled ectodermal cells located at the posterior segment compartment. This pattern becomes restricted to large subectodermal cells in the three thoracic and nine abdominal segments. These are the posterior P cells because of their position relative to the tracheal pits. This corresponds also to the Ato location. Later on, after complete disappearance of the early PNS precursor's expression, the late CNS pattern appears. Atonal is one the proneural protein that confer the potential for some ectodermal cells to become neuronal precursors, that will be later internal sensory organs. They are chordotonal organs acting as proprioreceptors or stretch receptors.

Atonal and NKD

The panel on the left represents stage 11 embryos with NKD in situ , Ato in situ, anti-bodies staining for NKD from transgenic or double labelling for NKD transgenic and Ato in situ. NKD is expressed in an ato positive cell, a sensory organ precursor of the PNS, which are precursors of the lateral penta-scolopidial chordotonal organs (lch5) in the abdominal segments. The Ato/NKD co-expression indicates that Ato could interact with NKD. But other proteins are also expressed within the P cells. This was challenged by testing the pattern of NKD expression of the Drosophila transgenic NKD-ß-Gal in different mutated backgrounds.

The P cells

The P cells are known to expressed the bHLH proteins Ato, Ase and L'Sc. Therefore, we tested this expression in ato asc and da backgrounds. NKD-ß-Gal expression is unchanged in the complete absence of the AS-C. On the other hand, the expression is completely abolished in the ato background as well as in the da background. This is not surprising because Da is ubiquitously expressed and forms heterodimers with atonal. These results demonstrate that it is under regulation of Ato and Da directly or indirectly. We further investigated the possibility for Ato to acts on NKD transcription regulation

S2 cell line transient tranfection

Using the same NKD-ß-Gal constructs, we tested the transcriptional efficiency of the NKD promoter in transient transfection into Drosophila Schneider cells by measurement of ß-Gal activity. The basal transcriptional activities (in black) for all the promoter lengths are about twice the background of the S2 cells. With the knowledge that the in vivo expression is dependent on the proneural bHLH Ato and Da transcription factors, we therefore looked for transactivation of this promoter region by these transcription factors.

This results in a 10 fold stimulation of the transcriptional activity of the NKD promoter in S2 cells. Ato or Da alone doesn't stimulate significantly the NKD promoter. In addition, these effects are seen with 5 KB as well as with the 500 bp of proximal promoter region which contains two consensus sequences for E boxes.

If we remove NKDE1, the transactivation by the factors Ato/Da persists. No transactivation is observed with the -180 bp promoter deletion that removes NKDE2. Moreover, reinsertion of the NKDE2, but not that of the NKDE1, into the -180 bp deletion restores the transactivation. This demonstrates that in cell culture the NKDE2 box can mediates transactivation of the NKD promoter by Ato and Da proteins.

Gel shift

These proteins bind directly in gel shift assay to NKD-E2 (cf Rosay et al., 1995).

Dual organisation of the NKD promoter

To conclude, there is an early expression of NKD within precursors of internal sensory organs. These chordotonal organs depend on Ato and Da. NKD expression is mediated by bHLH proteins, via NKD-E2. In vivo, re-insertion of NKD-E2 restores the expression and the Ato dependence. NKD is also independently regulated from ASC. What about our neuropeptide receptor function in sensory precursors ?

The Peripheral Nervous System (PNS)

To answer this question, we examine the PNS of embryos when NKD is ectopicly expressed. The embryonic PNS develops in a stereotypical manner. PNS includes at least two types of sensory organs. We can see five spatial different patterns of sensory organs.

Lineage of the sensory organs

The sensory organs (neurone and support cells) arise through divisions of the ectodermal precursor cells. External sensory organs sense mechanical and chemical stimuli. Chordotonal organs work as proprioceptors to sense stretch.

One abdominal hemi-segment

Chordotonal organs such as lch5, are ato dependant. That's why, after disturbing NKD function at the precursor level, we stain embryos at stage 14 to 16 with anti-bodies against HRP, to study the sensory neurones and their projections.

Over expression under heat-shock

When NKD is expressed ectopicly, before the first PNS axonal migration, we observe a reduced number of lch5 at the lateral level (SN is the segmental nerve, ISN the intersegmental one).

Then NKD is may be involved in cell division. To complete the view of NKD function, we also build a transdominant negative form of the receptor (NKD204).

The mutant form of NKD

A normal state for signal transduction involves a specific ligand which binds to its receptor. The receptor will now be on an active form, able to transduce intracellular signal via the trimeric G proteins. A consensus is emerging to consider the third intracellular loop as responsible for the interaction between receptor and G proteins. We construct a mutated receptor, R204, by inverting charges of the amino-acids of the alpha helix. R204 will trap NKD ligand without transducing intracellular signal, and prevent endogenous NKD function.

NKD204

When expressed under heat-shock promoter, NKD204 produces PNS alterations, distinct from the previous one. We observe defects in axonal pathways.

Nerves originate from the lateral cluster, cross segment boundaries and extends to the neighbouring segment at the dorsal cluster.

Inter Segmental Nerve (ISN)

Axons from sensory neurons arise just after the first divisions, and fasciculate to form the ISN as they migrate ventrally to the CNS. Some motoneurons send projections dorsally. Among different substrates involved in ISN migration, are myoblasts, trachea or cell bodies of other sensory neurons. Time was lacking to perform experiments with UAS/GAL4 system to distinguish between an NKD204 expression within the precursor or within the neighbouring cells.