Castor specifies NB5-2 late-born interneuron molecular identity and premotor connectivity.

Sensory-motor integration requires proper connectivity of interneurons within the central nervous system (CNS). Unique interneuron identity, defined by a collection of features that includes expression of transcription factors (TFs; molecular identity) and connectivity, is essential for proper circuit assembly. In Drosophila and mammals, interneuron identity is generated by sequential expression of a temporal transcription factor (TTF) cascade in progenitors which is then transiently maintained in post-mitotic progeny. In Drosophila, neural progenitors (neuroblasts; NBs) sequentially express the TTFs Hunchback (Hb) > Krüppel (Kr) > Pdm > Castor > Grainy head (Grh). While many studies have investigated the role of Hb in specifying early-born neuron fate, our understanding of whether late TTFs specify aspects of late-born interneuron identity is severely lacking. Here we investigate the role of Castor in specifying late-born interneuron molecular identity and connectivity using the NB5-2 lineage. NB5-2 predominantly produces interneuron progeny and late-born interneurons, Saaghi1-3 (SA1-3) and Jaam1-3 (JA1-3), contribute to a well characterized proprioceptive circuit. We find that NB5-2 Castor is required to close the Pdm expression window and promote Grh expression, consistent with other VNC NB lineages. Lineage specific misexpression of Castor in NB5-2 results in an increase in progeny expressing the late-born TFs, Runt and Nab. We identify SA1, SA3, and JA3 as Castor expressing NB5-2 progeny and that SA1 presynapses localize to discrete neuropil subregions. Presynapse number significantly increases in two SA1 subregions following NB5-2 Castor misexpression and disrupts proprioceptive circuit behavior. We conclude that NB5-2 Castor functions to specify late-born interneuron molecular identity and connectivity.