Engrailed (En) is a homeodomain-containing transcription factor found in all bilaterian animals 
, but first identified in Drosophila melanogaster
, where it plays a crucial part in the patterning of body segments and limbs 
. However, the most highly conserved role of En is in neuronal development.
In vertebrates, En is required for cerebellar patterning 
, and formation of the retino-tectal projection 
. En regulates the development of spinal cord interneurons 
, and affects the survival of dopaminergic midbrain neurons 
. In Drosophila
and grasshopper CNS En controls neuron/glia fate decisions, neuronal identity and axon pathfinding 
, while in cockroach mechanosensory neurons, we showed that it also controls axon guidance, synaptic target recognition and, as a result, escape behavior 
Despite its well-known role in patterning the Drosophila
embryo, until recently there were few indications that En played any role in the adult nervous system. Now it is known that subsets of neurons in the peripheral and central nervous system express the en
gene through adulthood 
, and it has recently been shown that En expression, in combination with that of other transcription factors, is necessary for specifying olfactory sensillum identity and odorant receptor
) gene expression in the third antennal segment 
Engrailed is also expressed in the second antennal segment, or pedicel ( and ), in a spatially restricted subset of neurons that make up the mechanosensory Johnston’s organ (JO) 
. The JO is a chordotonal organ containing approximately 200 sensory units called scolopidia, each consisting of 2 or 3 neurons, the eponymous scolopale cell, and ligament and cap cells 
. The approximately 480 sensory neurons form a bowl-shaped agglomeration 
, divided anatomically into medial and posterior groups (). Electrophysiological and calcium-imaging studies have shown that some of the JO neurons, or JONs, detect sound (JO-AB neurons), while another subpopulation responds to gravity and wind (JO-CE neurons) 
. The JO-A subgroup shows calcium entry in response to sound in a wide range of frequencies ranging from about 100 Hz to 1000 Hz, while the JO-B neurons appear to respond better to lower frequencies 
. Although many other subsets of JONs can be distinguished with different GAL4
, the functional relevance of these is not clear. One possibility, therefore, is that En expression may distinguish a different, overlapping, subset of neurons that perhaps respond to high (or low) frequencies.
Structure of Drosophila Johnston’s Organ (JO).
En-expressing neurons in the Johnston’s Organ.
Extracellular recordings of sound-evoked potentials from the antennal nerve invariably show oscillations at twice the stimulus frequency 
. It is not absolutely clear how the mechanics of the insertion of the JO scolopidia at the pedicel-funicular joint gives rise to this frequency doubling. One model postulates that the posterior group of JONs is activated by air moving from the front towards the rear of the animal, and the medial group by air movements in the opposite direction 
(), although it was suggested that those JO scolopidia inserted above or below the pivot point (referred to as “v/d” in ) would perhaps be activated by both directions 
. In contrast, a more direct electrophysiological recording method suggests that some individual JO-AB neurons do in fact respond bidirectionally 
, although the anatomical reason for this is not clear.
En-expressing JONs appear to be predominantly located in one anatomical location, the posterior group, and are therefore suited to discriminating between these models – with the former, we would expect them to respond primarily to air moving from front to back. In this study, therefore, we investigate in more detail the anatomical and physiological properties of the En-expressing JONs, and in particular ask whether they respond to sound, and if so at which frequencies, and whether their responses are biased towards front-to-rear movements.