SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs.

The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.

Photoperiod alters testicular methyltransferase complex mRNA expression in Siberian hamsters.

Exposure to long photoperiods stimulates, whereas exposure to short photoperiods transiently inhibit testicular function in Siberian hamsters via well-described neuroendocrine mechanisms. However, less is known about the intra-testicular regulation of these photoperiod-mediated changes. N6-methyladenosine (m6A) is one of the most common mRNA modifications in eukaryotes, with alterations in m6A mRNA methylation affecting testis function and fertility. We hypothesized that genes controlling m6A methylation such as methyltransferase-like-3 (Mettl3) and -14 (Mettl14) and Wilms' tumor-1 associated protein (Wtap), part of an mRNA methylating methyl-transferase complex, or the fat-mass-and-obesity-associated (Fto) and the α-ketoglutarate-dependent dioxygenase alkB homolog-5 (Alkbh5) genes responsible for m6A demethylation, may be differentially regulated by photoperiod in the testis. Male hamsters were exposed to long (LD, control) photoperiod for 14-weeks, short (SD) photoperiod for 2, 5, 8, 11 and 14-weeks to induce regression, or SD for 14-weeks followed by transfer to LD for 1, 2, 4 or 8-weeks to induce recrudescence (post-transfer, PT). SD exposure significantly reduced body, testis, and epididymal masses compared to all other groups. Spermatogenic index, seminiferous tubule diameters and testosterone concentrations significantly decreased in SD as compared to LD, returning to levels no different than LD in post-transfer groups. SD exposure significantly decreased Wtap, Fto, Alkbh5, but increased Mettl14 mRNA expression as compared to LD, with values in PT groups restored to LD levels. Mettl3 mRNA expression did not change. These results suggest that testicular recovery induced by stimulatory photoperiod is relatively rapid, and that the methyltransferase complex may play a role during photostimulated testicular recrudescence.

Sequential emergence and contraction of epithelial subtypes in the prenatal human choroid plexus revealed by a stem cell model.

Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair, their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells, derived CPECs (dCPECs) displayed canonical properties and dynamic multiciliated phenotypes that interacted with Aß uptake. Single dCPEC transcriptomes over time correlated well with human organoid and fetal CPECs, while pseudotemporal and cell cycle analyses highlighted the direct CPEC origin from neuroepithelial cells. In addition, time series analyses defined metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints, followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contracted. These temporal patterns were then confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs, these findings suggest new dynamic models of ChP support for the developing human brain.

Mosquito cryptochromes expressed in Drosophila confer species-specific behavioral light responses.

Cryptochrome (CRY) is a short-wavelength light-sensitive photoreceptor expressed in a subset of circadian neurons and eyes in Drosophila that regulates light-evoked circadian clock resetting. Acutely, light evokes rapid electrical excitation of the ventral lateral subset of circadian neurons and confers circadian-modulated avoidance behavioral responses to short-wavelength light. Recent work shows dramatically different avoidance versus attraction behavioral responses to short-wavelength light in day-active versus night-active mosquitoes and that these behavioral responses are attenuated by CRY protein degradation by constant light exposure in mosquitoes. To determine whether CRY1s mediate species-specific coding for behavioral and electrophysiological light responses, we used an "empty neuron" approach and transgenically expressed diurnal Aedes aegypti (AeCRY1) versus nocturnal Anopheles gambiae (AgCRY1) in a cry-null Drosophila background. AeCRY1 is much less light sensitive than either AgCRY1 or DmCRY as shown by partial behavioral rhythmicity following constant light exposure. Remarkably, expression of nocturnal AgCRY1 confers low survival to constant white light as does expression of AeCRY1 to a lesser extent. AgCRY1 mediates significantly stronger electrophysiological cell-autonomous responses to 365 nm ultraviolet (UV) light relative to AeCRY1. AgCRY1 expression mediates electrophysiological sensitivity to 635 nm red light, whereas AeCRY1 does not, consistent with species-specific mosquito red light responses. AgCRY1 and DmCRY mediate intensity-dependent avoidance behavior to UV light at different light intensity thresholds, whereas AeCRY1 does not, thus mimicking mosquito and fly behaviors. These findings highlight CRY as a key non-image-forming visual photoreceptor that mediates physiological and behavioral light responses in a species-specific fashion.