SYCP1 head-to-head assembly is required for chromosome synapsis in mouse meiosis.

In almost all sexually reproducing organisms, meiotic recombination and cell division require the synapsis of homologous chromosomes by a large proteinaceous structure, the synaptonemal complex (SC). While the SC's overall structure is highly conserved across eukaryotes, its constituent proteins diverge between phyla. Transverse filament protein, SYCP1, spans the width of the SC and undergoes amino-terminal head-to-head self-assembly in vitro through a motif that is unusually highly conserved across kingdoms of life. Here, we report creation of mouse mutants, Sycp1L102E and Sycp1L106E, that target SYCP1's head-to-head interface. L106E resulted in a complete loss of synapsis, while L102E had no apparent effect on synapsis, in agreement with their differential effects on the SYCP1 head-to-head interface in molecular dynamics simulations. In Sycp1L106E mice, homologs aligned and recruited low levels of mutant SYCP1 and other SC proteins, but the absence of synapsis led to failure of crossover formation and meiotic arrest. We conclude that SYCP1's conserved head-to-head interface is essential for meiotic chromosome synapsis in vivo.

rRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

NOTCH Signaling Controls Ciliary Body Morphogenesis and Secretion by Directly Regulating Nectin Protein Expression.

Anterior segment dysgenesis is often associated with cornea diseases, cataracts, and glaucoma. In the anterior segment, the ciliary body (CB) containing inner and outer ciliary epithelia (ICE and OCE) secretes aqueous humor that maintains intraocular pressure (IOP). However, CB development and function remain poorly understood. Here, this study shows that NOTCH signaling in the CB maintains the vitreous, IOP, and eye structures by regulating CB morphogenesis, aqueous humor secretion, and vitreous protein expression. Notch2 and Notch3 function via RBPJ in the CB to control ICE-OCE adhesion, CB morphogenesis, aqueous humor secretion, and protein expression, thus maintaining IOP and eye structures. Mechanistically, NOTCH signaling transcriptionally controls Nectin1 expression in the OCE to promote cell adhesion for driving CB morphogenesis and to directly stabilize Cx43 for controlling aqueous humor secretion. Finally, NOTCH signaling directly controls vitreous protein secretion in the ICE. Therefore, this study provides important insight into CB functions and involvement in eye diseases.

CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.

Ethylphenidate as a selective dopaminergic agonist and methylphenidate-ethanol transesterification biomarker.

We review the pharmaceutical science of ethylphenidate (EPH) in the contexts of drug discovery, drug interactions, biomarker for dl-methylphenidate (MPH)-ethanol exposure, potentiation of dl-MPH abuse liability, contemporary "designer drug," pertinence to the newer transdermal and chiral switch MPH formulations, as well as problematic internal standard. d-EPH selectively targets the dopamine transporter, whereas d-MPH exhibits equipotent actions at dopamine and norepinephrine transporters. This selectivity carries implications for the advancement of tailored attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy in the era of genome-based diagnostics. Abuse of dl-MPH often involves ethanol coabuse. Carboxylesterase 1 enantioselectively transesterifies l-MPH with ethanol to yield l-EPH accompanied by significantly increased early exposure to d-MPH and rapid potentiation of euphoria. The pharmacokinetic component of this drug interaction can largely be avoided using dexmethylphenidate (dexMPH). This notwithstanding, maximal potentiated euphoria occurs following dexMPH-ethanol. C57BL/6 mice model dl-MPH-ethanol interactions: an otherwise depressive dose of ethanol synergistically increases dl-MPH stimulation; a substimulatory dose of dl-MPH potentiates a low, stimulatory dose of ethanol; ethanol elevates blood, brain, and urinary d-MPH concentrations while forming l-EPH. Integration of EPH preclinical neuropharmacology with clinical studies of MPH-ethanol interactions provides a translational approach toward advancement of ADHD personalized medicine and management of comorbid alcohol use disorder.

One year of transgenic overexpression of osteoprotegerin in rats suppressed bone resorption and increased vertebral bone volume, density, and strength.

RANKL is an essential mediator of bone resorption, and its activity is inhibited by osteoprotegerin (OPG). Transgenic (Tg) rats were engineered to continuously overexpress OPG to study the effects of continuous long-term RANKL inhibition on bone volume, density, and strength. Lumbar vertebrae, femurs, and blood were obtained from 1-yr-old female OPG-Tg rats (n = 32) and from age-matched wildtype (WT) controls (n = 23). OPG-Tg rats had significantly greater serum OPG (up to 260-fold) and significantly lower serum TRACP5b and osteocalcin compared with WT controls. Vertebral histomorphometry showed significant reductions in osteoclasts and bone turnover parameters in OPG-Tg rats versus WT controls, and these reductions were associated with significantly greater peak load in vertebrae tested through compression. No apparent differences in bone material properties were observed in OPG-Tg rat vertebrae, based on their unchanged intrinsic strength parameters and their normal linear relationship between vertebral bone mass and strength. Femurs from OPG-Tg rats were of normal length but showed mild osteopetrotic changes, including reduced periosteal perimeter (-6%) and an associated reduction in bending strength. Serum OPG levels in WT rats showed no correlations with any measured parameter of bone turnover, mass, or strength, whereas the supraphysiological serum OPG levels in OPG-Tg rats correlated negatively with bone turnover parameters and positively with vertebral bone mass and strength parameters. In summary, low bone turnover after 1 yr of OPG overexpression in rats was associated with increased vertebral bone mass and proportional increases in bone strength, with no evidence for deleterious effects on vertebral material properties.

RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.

INTRODUCTION: Ovariectomy (OVX) results in bone loss caused by increased bone resorption. RANKL is an essential mediator of bone resorption. We examined whether the RANKL inhibitor osteoprotegerin (OPG) would preserve bone volume, density, and strength in OVX rats. MATERIALS AND METHODS: Rats were OVX or sham-operated at 3 mo of age. Sham controls were treated for 6 wk with vehicle (Veh, PBS). OVX rats were treated with Veh or human OPG-Fc (10 mg/kg, 2/wk). Serum RANKL and TRACP5b was measured by ELISA. BMD of lumbar vertebrae (L(1)-L(5)) and distal femur was measured by DXA. Right distal femurs were processed for bone histomorphometry. Left femurs and the fifth lumbar vertebra (L(5)) were analyzed by muCT and biomechanical testing, and L(6) was analyzed for ash weight. RESULTS: OVX was associated with significantly greater serum RANKL and osteoclast surface and with reduced areal and volumetric BMD. OPG markedly reduced osteoclast surface and serum TRACP5b while completely preventing OVX-associated bone loss in the lumbar vertebrae, distal femur, and femur neck. Vertebrae from OPG-treated rats had increased dry and ash weight, with no significant differences in tissue mineralization versus OVX controls. muCT showed that trabecular compartments in OVX-OPG rats had significantly greater bone volume fraction, vBMD, bone area, trabecular thickness, and number, whereas their cortical compartments had significantly greater bone area (p < 0.05 versus OVX-Veh). OPG improved cortical area in L(5) and the femur neck to levels that were significantly greater than OVX or sham controls (p < 0.05). Biomechanical testing of L(5) and femur necks showed significantly greater maximum load values in the OVX-OPG group (p < 0.05 versus OVX-Veh). Bone strength at both sites was linearly correlated with total bone area (r(2) = 0.54-0.74, p < 0.0001), which was also significantly increased by OPG (p < 0.05 versus OVX). CONCLUSIONS: OPG treatment prevented bone loss, preserved trabecular architecture, and increased cortical area and bone strength in OVX rats.

Continuous RANKL inhibition in osteoprotegerin transgenic mice and rats suppresses bone resorption without impairing lymphorganogenesis or functional immune responses.

Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. The effects of RANKL are inhibited by a soluble decoy receptor called osteoprotegerin (OPG). Total ablation of RANKL in knockout mice leads to high bone mass, lymph node agenesis, and altered lymphocyte differentiation. In contrast, RANKL inhibition via OPG suppresses bone resorption but not inflammation in animal models of inflammatory bone loss. This suggests that the immune phenotype of RANKL knockout mice is related to total RANKL ablation. We hypothesized that prenatal RANKL inhibition via OPG overexpression would suppress bone resorption without influencing lymph node formation or subsequent immune responses. Transgenic rats were created, wherein soluble OPG was overexpressed by 100-fold vs wild type (WT) controls, by gestational day 11 (i.e., before lymph node formation). The structure of lymph nodes, spleen, and thymus of OPG-transgenic (OPG-Tg) animals were comparable to those of age-matched WT rats at gestational day 19 and in adulthood. The OPG-Tg neonates had elevated bone mass, confirming the prenatal inhibition of RANKL. Adult OPG-Tg rats and OPG-Tg mice exhibited no significant functional alterations relative to WT controls when subjected to immune challenges to test for altered innate and humoral responses (e.g., contact hypersensitivity to oxazolone, IgM response to Pneumovax, IgG response to keyhole limpet hemocyanin, or cytokine response to LPS). In summary, prenatal RANKL inhibition did not impair lymph node development, nor did continuous life-long RANKL inhibition cause obvious changes in innate or humoral immune responses in mice or rats.

Isoflurane with morphine is a suitable anaesthetic regimen for embryo transfer in the production of transgenic rats.

During our initial attempts to produce transgenic rats, we found that an anaesthetic combination typically used for embryo transfer (intramuscular injection of ketamine [90 mg/kg] with xylazine [10 mg/kg]) yielded extensive variation in both the depth and length of anaesthesia. In the present prospective study, we compared the reproductive outcomes afforded by using either isoflurane (5% for induction, 2% for maintenance, carried in 2 l/min of oxygen) with morphine (5 mg/kg s.c., given immediately after isoflurane induction) or ketamine/xylazine in adult (250-300 g), pseudopregnant Sprague-Dawley rats. Each animal was anaesthetized with either isoflurane/morphine or ketamine/xylazine, after which 30 microinjected eggs were transferred into the left uterine horn. The mean pregnancy rate for isoflurane/morphine (15%) was 50% greater than that achieved with ketamine/xylazine (10%). The mean number of live pups (just over five per litter) was comparable for both regimens. All rats given isoflurane/morphine quickly achieved a surgical depth of anaesthesia and experienced a rapid postoperative recovery (3-5 min). In contrast, 25% of rats injected with ketamine/xylazine did not reach a depth of anaesthesia within 10 min that was sufficient for laparotomy, and all that were anaesthetized successfully required an extended postoperative recovery period (60-90 min). These data show that isoflurane/morphine is well tolerated by microinjected embryos and suggest that its use during embryo transfer may provide a means for both reducing the number of pseudopregnant females used and increasing the speed with which rat transgenic projects are completed.

Strain variation of immature female rats in response to various superovulatory hormone preparations and routes of administration.

The purpose of this study was to examine the response of rats of different genetic backgrounds to various superovulatory hormonal treatments. Immature Sprague Dawley (SD), FBNF1, and F344 female rats (30 to 35 days of age) were used for this study as representatives of outbred, hybrid, and inbred strains respectively. Animals from each strain were allocated into four groups of hormone treatments as follows: 1) 30 IU pregnant mare serum gonadotrophin (PMSG) intraperitoneally (i.p.) followed 52 h later with 25 IU human chorionic gonadotrophin (HCG) i.p.; 2) 15 IU PMSG i.p. followed 52 h later with 7.5 IU HCG i.p.; 3) 1.0 IU follicle stimulating hormone (FSH) daily via Alzet mini-pumps for 60 h; and 4) 1.0 IU FSH daily via Alzet mini-pumps for 54 h followed by 10 mg luteinizing hormone (LH). The efficacies of the hormone treatments were evaluated using the following criteria: % mated, % ovulated, total oocytes per female, and % fertilized. The % mated of SD rats treated with PMSG(30)+HCG(25) was significantly higher (P < 0.05) than that of animals treated with PMSG(15)+HCG(7.5); in addition, the total oocytes per female was significantly higher (P < 0.05) for SD animals receiving PMSG(30)+HCG(25) than all other treatments. The % ovulated of SD rats was significantly lower (P < 0.05) in response to FSH alone as compared to all other treatments. The % ovulated for FBNF1 rats was significantly greater (P < 0.05) in response to both PMSG+HCG treatments as compared to FSH and FSH+LH. The % ovulated and % fertilized were significantly lower (P < 0.05) in F344 rats treated with FSH alone as compared to all other treatments. F344 rats produced significantly (P < 0.05) more oocytes per female in response to both PMSG+HCG treatments as compared to FSH and FHS+LH. The % ovulated of SD and F344 rats were significantly higher (P < 0.05) than that of FBNF1 rats in response to FSH and FSH+LH. SD rats produced a significantly greater (P < 0.05) number of oocytes per female than did FBNF1 rats in response to PMSG(30)+HCG(25) and a significantly greater (P < 0.05) number of oocytes per female as compared to those of FBNF1 and F344 rats in response to FSH and FSH+LH. The % fertilized of SD and FBNF1 rats were significantly higher (P < 0.05) than that of F344 rats in response to PMSG(15)+HCG(7.5). Our study demonstrates that treatment with PMSG+HCG is an effective method of eliciting superovulatory responses according to most criteria examined. We have also shown that outbred (SD) rats generally produced more oocytes per female in response to hormonal stimulation than did inbred and hybrid rats. Our results indicate that different strains of rats have various degrees of hormone sensitivity and response to different superovulation protocols.