An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants.

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

Impaired CaV1.2 inactivation reduces the efficacy of calcium channel blockers in the treatment of LQT8.

Mutations in the CaV1.2 L-type calcium channel can cause a profound form of long-QT syndrome known as long-QT type 8 (LQT8), which results in cardiac arrhythmias that are often fatal in early childhood. A growing number of such pathogenic mutations in CaV1.2 have been identified, increasing the need for targeted therapies. As many of these mutations reduce channel inactivation; resulting in excess Ca2+ entry during the action potential, calcium channel blockers (CCBs) would seem to represent a promising treatment option. Yet CCBs have been unsuccessful in the treatment of LQT8. Here, we demonstrate that this lack of efficacy likely stems from the impact of the mutations on CaV1.2 channel inactivation. As CCBs are known to preferentially bind to the inactivated state of the channel, mutation-dependent deficits in inactivation result in a decrease in use-dependent block of the mutant channel. Further, application of the CCB verapamil to induced pluripotent stem cell (iPSC) derived cardiomyocytes from an LQT8 patient demonstrates that this loss of use-dependent block translates to a lack of efficacy in correcting the LQT phenotype. As a growing number of channelopathic mutations demonstrate effects on channel inactivation, reliance on state-dependent blockers may leave a growing population of patients without a viable treatment option. This biophysical understanding of the interplay between inactivation deficits and state-dependent block may provide a new avenue to guide the development of improved therapies.

Histone variant HTZ1 shows extensive epistasis with, but does not increase robustness to, new mutations.

Biological systems produce phenotypes that appear to be robust to perturbation by mutations and environmental variation. Prior studies identified genes that, when impaired, reveal previously cryptic genetic variation. This result is typically interpreted as evidence that the disrupted gene normally increases robustness to mutations, as such robustness would allow cryptic variants to accumulate. However, revelation of cryptic genetic variation is not necessarily evidence that a mutationally robust state has been made less robust. Demonstrating a difference in robustness requires comparing the ability of each state (with the gene perturbed or intact) to suppress the effects of new mutations. Previous studies used strains in which the existing genetic variation had been filtered by selection. Here, we use mutation accumulation (MA) lines that have experienced minimal selection, to test the ability of histone H2A.Z (HTZ1) to increase robustness to mutations in the yeast Saccharomyces cerevisiae. HTZ1, a regulator of chromatin structure and gene expression, represents a class of genes implicated in mutational robustness. It had previously been shown to increase robustness of yeast cell morphology to fluctuations in the external or internal microenvironment. We measured morphological variation within and among 79 MA lines with and without HTZ1. Analysis of within-line variation confirms that HTZ1 increases microenvironmental robustness. Analysis of between-line variation shows the morphological effects of eliminating HTZ1 to be highly dependent on the line, which implies that HTZ1 interacts with mutations that have accumulated in the lines. However, lines without HTZ1 are, as a group, not more phenotypically diverse than lines with HTZ1 present. The presence of HTZ1, therefore, does not confer greater robustness to mutations than its absence. Our results provide experimental evidence that revelation of cryptic genetic variation cannot be assumed to be caused by loss of robustness, and therefore force reevaluation of prior claims based on that assumption.

CaV1.2 channelopathic mutations evoke diverse pathophysiological mechanisms.

The first pathogenic mutation in CaV1.2 was identified in 2004 and was shown to cause a severe multisystem disorder known as Timothy syndrome (TS). The mutation was localized to the distal S6 region of the channel, a region known to play a major role in channel activation. TS patients suffer from life-threatening cardiac symptoms as well as significant neurodevelopmental deficits, including autism spectrum disorder (ASD). Since this discovery, the number and variety of mutations identified in CaV1.2 have grown tremendously, and the distal S6 regions remain a frequent locus for many of these mutations. While the majority of patients harboring these mutations exhibit cardiac symptoms that can be well explained by known pathogenic mechanisms, the same cannot be said for the ASD or neurodevelopmental phenotypes seen in some patients, indicating a gap in our understanding of the pathogenesis of CaV1.2 channelopathies. Here, we use whole-cell patch clamp, quantitative Ca2+ imaging, and single channel recordings to expand the known mechanisms underlying the pathogenesis of CaV1.2 channelopathies. Specifically, we find that mutations within the S6 region can exert independent and separable effects on activation, voltage-dependent inactivation (VDI), and Ca2+-dependent inactivation (CDI). Moreover, the mechanisms underlying the CDI effects of these mutations are varied and include altered channel opening and possible disruption of CDI transduction. Overall, these results provide a structure-function framework to conceptualize the role of S6 mutations in pathophysiology and offer insight into the biophysical defects associated with distinct clinical manifestations.

Insulin signaling in the long-lived reproductive caste of ants.

In most organisms, reproduction is correlated with shorter life span. However, the reproductive queen in eusocial insects exhibits a much longer life span than that of workers. In Harpegnathos ants, when the queen dies, workers can undergo an adult caste switch to reproductive pseudo-queens (gamergates), exhibiting a five-times prolonged life span. To explore the relation between reproduction and longevity, we compared gene expression during caste switching. Insulin expression is increased in the gamergate brain that correlates with increased lipid synthesis and production of vitellogenin in the fat body, both transported to the egg. This results from activation of the mitogen-activated protein kinase (MAPK) branch of the insulin signaling pathway. By contrast, the production in the gamergate developing ovary of anti-insulin Imp-L2 leads to decreased signaling of the AKT/forkhead box O (FOXO) branch in the fat body, which is consistent with their extended longevity.