DMKPs provide a generalizable strategy for studying genes required for reproduction or viability in nontraditional model organisms.

The advent of CRISPR/Cas9-mediated genome editing has expanded the range of animals amenable to targeted genetic analysis. This has accelerated research in animals not traditionally studied using molecular genetics. However, studying genes essential for reproduction or survival in such animals remains challenging, as they lack the tools that aid genetic analysis in traditional genetic model organisms. We recently introduced the use of distinguishably marked knock-in pairs (DMKPs) as a strategy for rapid and reliable genotyping in such species. Here we show that DMKPs also facilitate the maintenance and study of mutations that cannot be maintained in a homozygous state, a group which includes recessive lethal and sterile mutations. Using DMKPs, we disrupt the zero population growth locus in Drosophila melanogaster and in the dengue vector mosquito Aedes aegypti. In both species, DMKPs enable the maintenance of zero population growth mutant strains and the reliable recovery of zero population growth mutant animals. Male and female gonad development is disrupted in fly and mosquito zero population growth mutants, rendering both sexes sterile. In Ae. aegypti, zero population growth mutant males remain capable of inducing a mating refractory period in wild-type females and of competing with wild-type males for mates, properties compatible with zero population growth serving as a target in mosquito population suppression strategies. DMKP is readily generalizable to other species amenable to CRISPR/Cas9-mediated gene targeting, and should facilitate the study of sterile and lethal mutations in multiple organisms not traditionally studied using molecular genetics.

Salt sensing: A new receptor for an ancient taste.

Regulation of salt intake is important for animals from flies to humans. A new study clarifies the molecular receptors mediating attraction to low salt concentrations in Drosophila, suggesting a surprisingly fly-specific solution to the challenge of detecting this universal tastant.

Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain.

Animals exhibit innate and learned preferences for temperature and humidity-conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior.

A Cut/cohesin axis alters the chromatin landscape to facilitate neuroblast death.

Precise control of cell death in the nervous system is essential for development. Spatial and temporal factors activate the death of Drosophila neural stem cells (neuroblasts) by controlling the transcription of multiple cell death genes through a shared enhancer. The activity of this enhancer is controlled by abdominal A and Notch, but additional inputs are needed for proper specificity. Here, we show that the Cut DNA binding protein is required for neuroblast death, regulating reaper and grim downstream of the shared enhancer and of abdominal A expression. The loss of cut accelerates the temporal progression of neuroblasts from a state of low overall levels of H3K27me3 to a higher H3K27me3 state. This is reflected in an increase in H3K27me3 modifications in the cell death gene locus in the CNS on Cut knockdown. We also show that cut regulates the expression of the cohesin subunit Stromalin. Stromalin and the cohesin regulatory subunit Nipped-B are required for neuroblast death, and knockdown of Stromalin increases H3K27me3 levels in neuroblasts. Thus, Cut and cohesin regulate apoptosis in the developing nervous system by altering the chromatin landscape.

Cell death regulates muscle fiber number.

Cell death can have both cell autonomous and non-autonomous roles in normal development. Previous studies have shown that the central cell death regulators grim and reaper are required for the developmentally important elimination of stem cells and neurons in the developing central nervous system (CNS). Here we show that cell death in the nervous system is also required for normal muscle development. In the absence of grim and reaper, there is an increase in the number of fibers in the ventral abdominal muscles in the Drosophila adult. This phenotype can be partially recapitulated by inhibition of cell death specifically in the CNS, indicating a non-autonomous role for neuronal death in limiting muscle fiber number. We also show that FGFs produced in the cell death defective nervous system are required for the increase in muscle fiber number. Cell death in the muscle lineage during pupal stages also plays a role in specifying fiber number. Our work suggests that FGFs from the CNS act as a survival signal for muscle founder cells. Thus, proper muscle fiber specification requires cell death in both the nervous system and in the developing muscle itself.

Detecting apoptosis in Drosophila tissues and cells.

In this chapter we discuss methods that can be used to study apoptotic cell death in the Drosophila embryo, ovary, as well as in cultured cell lines. These methods assay various aspects of the cell death process, from mitochondrial changes to caspase activation and DNA cleavage. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses. These techniques include Acridine Orange staining, TUNEL, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization.