Summary Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X-chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human datasets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.

Estrogen-receptor alpha (ERα) neurons in the ventrolateral region of the ventromedial hypothalamus (VMHVL) control an array of sex-specific responses to maximize reproductive success. In females, these VMHVL neurons are believed to coordinate metabolism and reproduction. However, it remains unknown whether specific neuronal populations control distinct components of this physiological repertoire. Here, we identify a subset of ERα VMHVL neurons that promotes hormone-dependent female locomotion. Activating Nkx2-1-expressing VMHVL neurons via pharmacogenetics elicits a female-specific burst of spontaneous movement, which requires ERα and Tac1 signaling. Disrupting development of Nkx2-1+ VMHVL neurons results in female-specific obesity, inactivity, and loss of VMHVL neurons co-expressing ERα and Tac1. Unexpectedly, two responses controlled by ERα neurons, fertility and brown adipose tissue thermogenesis, are unaffected. We conclude that a dedicated subset of VMHVL neurons marked by ERα, NKX2-1, and Tac1 regulates estrogen-dependent fluctuations in physical activity and constitutes one of several neuroendocrine modules that drive sex-specific responses.

SUMMARY Axonal regrowth is crucial for recovery from CNS injury but is severely restricted in adult mammals. We used a genome-wide loss-of-function screen for factors limiting axonal regeneration from cerebral cortical neurons in vitro. Knockdown of 16,007 individual genes identified 580 significant phenotypes. These molecules share no significant overlap with those suggested by previous expression profiles. There is enrichment for genes in pathways related to transport, receptor binding, and cytokine signaling, including Socs4 and Ship2. Among transport-regulating proteins, Rab GTPases are prominent. In vivo assessment with C. elegans validates a cell-autonomous restriction of regeneration by Rab27. Mice lacking Rab27b show enhanced retinal ganglion cell axon regeneration after optic nerve crush and greater motor function and raphespinal sprouting after spinal cord trauma. Thus, a comprehensive functional screen reveals multiple pathways restricting axonal regeneration and neurological recovery after injury.

Activity of the RNA ligase RtcB has only two known functions: tRNA ligation after intron removal and XBP1 mRNA ligation during activation of the unfolded protein response. Here, we show that RtcB acts in neurons to inhibit axon regeneration after nerve injury. This function of RtcB is independent of its basal activities in tRNA ligation and the unfolded protein response. Furthermore, inhibition of axon regeneration is independent of the RtcB cofactor archease. Finally, RtcB is enriched at axon termini after nerve injury. Our data indicate that neurons have co-opted an ancient RNA modification mechanism to regulate specific and dynamic functions and identify neuronal RtcB activity as a critical regulator of neuronal growth potential.axon regeneration | RNA ligation | RtcB T he RNA ligase RtcB is the only known RNA ligase in metazoans. RNA ligation by RtcB is required for the maturation of intron-containing tRNAs (1-3), and also, it is required to process the transcription factor xbp-1 mRNA and activate the unfolded protein response (UPR) (4-6). Other than these two basic cellular processes, which are likely common to all metazoan cells, no functions for RNA ligation or RtcB are known. The nervous system is a site of expanded RNA processing after transcription. For example, neurons regulate alternative premRNA splicing in response to activity (7-10) and are highly enriched for mRNA editing (11-13). Here, we define a neuron-specific function for RtcB activity in regulating axon regeneration and show that this neuronal function is independent of RtcB's activities in tRNA and xbp-1 ligation.RtcB activity in neurons inhibits axon regeneration. We assayed axon regeneration in the GABA motor neurons of Caenorhabditis elegans using single-neuron laser axotomy (14). Mutants in the single C. elegans ortholog of RtcB, rtcb-1(gk451) (5), exhibited increased regeneration to the dorsal nerve cord (DNC) at 24 h after injury, consistent with previous data (Fig. 1 A and B) (15). Increased DNC regeneration was reduced to WT levels by introduction of a single-copy WT RtcB transgene (Fig. 1A). The increase in DNC regeneration is not caused by the trivial explanation that RtcB animals are narrower than the WT. At 6 h after injury, a time point at which neurons in WT animals are just initiating regeneration (15-17), a substantial fraction of axons in RtcB mutants had already regenerated to the DNC (Fig. 1C). Furthermore, WT animals did not regenerate as well as RtcB mutants, even when given additional time to regenerate (Fig. 1C). Finally, rescuing the overall growth defects of RtcB mutants did not alter the effect of loss of RtcB on axon regeneration (Fig. 2). Thus, loss of RtcB results in faster and more successful axon regeneration. Increased regeneration depends on loss of RtcB in neurons, because expressing WT RtcB under a GABA-specific promoter restored DNC regeneration to WT levels (Fig. 1A). DNC regeneration levels were not restored when the rescue construct contained a point mutation that eliminates ligase activity (H428A) ...

Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, KPC3/aex-5, and the neuropeptide NLP-40, and re-expression of these genes in the intestine of mutant animals is sufficient to restore normal regeneration success. Additionally, NPDC1/cab-1 and SNAP25/aex-4 genetically interact with rab-27 in the context of axon regeneration inhibition. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.