Evolution of the regulation of developmental gene expression in blind Mexican cavefish.

Developmental evolution and diversification of morphology can arise through changes in the regulation of gene expression or protein-coding sequence. To unravel mechanisms underlying early developmental evolution in cavefish of the species Astyanax mexicanus, we compared transcriptomes of surface-dwelling and blind cave-adapted morphs at the end of gastrulation. Twenty percent of the transcriptome was differentially expressed. Allelic expression ratios in cave X surface hybrids showed that cis-regulatory changes are the quasi-exclusive contributors to inter-morph variations in gene expression. Among a list of 108 genes with change at the cis-regulatory level, we explored the control of expression of rx3, which is a master eye gene. We discovered that cellular rx3 levels are cis-regulated in a cell-autonomous manner, whereas rx3 domain size depends on non-autonomous Wnt and Bmp signalling. These results highlight how uncoupled mechanisms and regulatory modules control developmental gene expression and shape morphological changes. Finally, a transcriptome-wide search for fixed coding mutations and differential exon use suggested that variations in coding sequence have a minor contribution. Thus, during early embryogenesis, changes in gene expression regulation are the main drivers of cavefish developmental evolution.

Awakening adult neural stem cells: NOX signalling as a positive regulator of the quiescence-to-proliferation transition in the Xenopus retina.

A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.

The enzymatic and neurochemical outcomes of a mutation in Mexican cavefish MAO reveal teleost-specific aspects of brain monoamine homeostasis.

Monoamine oxidases (MAO; MAO-A and MAO-B in mammals) are enzymes catalyzing the degradation of biogenic amines, including monoamine neurotransmitters. In humans, coding mutations in MAOs are extremely rare and deleterious. Here, we assessed the structural and biochemical consequences of a point mutation (P106L) in the single mao gene of the blind cavefish, Astyanax mexicanus. This mutation decreased mao enzymatic activity by ∼3-fold and affected the enzyme kinetics parameters, in line with potential structure-function alterations. HPLC measurements in brains of four A. mexicanus genetic lines (mutant and non-mutant cavefish, and mutant and non-mutant surface fish) showed major disturbances in serotonin, dopamine, noradrenaline and metabolite levels in mutants and demonstrated that the P106L mao mutation is responsible for monoaminergic disequilibrium in the P106L mao mutant cavefish brain. The outcomes of the mutation were different in the posterior brain (containing the raphe nucleus) and the anterior brain (containing fish-specific hypothalamic serotonergic clusters), revealing contrasting properties in neurotransmitter homeostasis in these different neuronal groups. We also discovered that the effects of the mutation were partially compensated by a decrease in activity of TPH, the serotonin biosynthesis rate-limiting enzyme. Finally, the neurochemical outcomes of the mao P106L mutation differed in many respects from a treatment with deprenyl, an irreversible MAO inhibitor, showing that genetic and pharmacological interference with MAO function are not the same. Our results shed light on our understanding of cavefish evolution, on the specificities of fish monoaminergic systems, and on MAO-dependent homeostasis of brain neurochemistry in general.

Genetic identification and reiterated captures suggest that the Astyanax mexicanus El Pachón cavefish population is closed and declining.

The sizes of Astyanax mexicanus blind cavefish populations of North-East Mexico are demographic parameters of great importance for investigating a variety of ecological, evolutionary, and conservation issues. However, few estimates have been obtained. For these mobile animals living in an environment difficult to explore as a whole, methods based on capture-mark-recapture are appropriate, but their feasibility and interpretation of results depend on several assumptions that must be carefully examined. Here, we provide evidence that minimally invasive genetic identification from captures at different time intervals (three days and three years) can give insights into cavefish population size dynamics as well as other important demographic parameters of interest. We also provide tools to calibrate sampling and genotyping efforts necessary to reach a given level of precision. Our results suggest that the El Pachón cave population is currently very small, of an order of magnitude of a few hundreds of individuals, and is distributed in a relatively isolated area. The probable decline in population size in the El Pachón cave since the last census in 1971 raises serious conservation issues.

Influencing Elimination Location in the Domestic Cat: A Semiochemical Approach.

In the domestic cat, elimination at an inappropriate location is considered by cat owners and non-cat owners as an undesirable behaviour. The aim of the study was to assess the effect of a semiochemical formulation, reconstituted volatile fraction of cat anal gland secretions on the elimination behaviour of domestic cats. The study was conducted in four catteries, which housed 33 cats, using 37 litter trays and followed a randomised crossover design using the litter tray as the experimental unit. The parameters studied included daily elimination (urine plus stools) weight, urine weight, stool weight, elimination type and urine/stool quantity scoring. The parameters were analysed using GLMM with SAS 9.4 software. Four out of the six parameters studied showed a treatment effect, consistently in favour of cats defecating significantly less in the litter trays sprayed with the treatment versus litter trays sprayed with the control (elimination weight p = 0.0199; elimination type p = 0.0251; stool weight p = 0.0005 and stool quantity p = 0.003). These results demonstrate that an intraspecific semiochemical message originating from cat anal glands influences cats' defecation location.

Sex Differences in Mouse Exploratory Behaviour to Fel d 1, a Cat ABP-Like Protein.

Fel d 1 is a cat protein abundantly released and found in their habitat and is closely related to mouse androgen-binding proteins (ABPs). We hypothesized that mice have developed chemical communication mechanisms to detect and avoid this protein. We tested purified natural Fel d 1, a fox faeces molecule (TMT) as a positive control, and a negative control (purified water) in three different mouse groups (n = 14 each) to evaluate exploratory behaviour and stress responses. The mice did not show clear avoidance or stress responses to Fel d 1. Our results demonstrated a sex-treatment interaction for Fel d 1, with males spending more time in the areas treated with Fel d 1 than in the untreated areas (p = 0.018). This sex-treated area interaction was also not observed for either the blank or TMT. These results suggest that Fel d 1 from domestic cats could be recognized differently by male and female mice. These sex differences could be linked to the sexual role of ABP proteins and the ABP-like characteristics of Fel d 1.

The provision of toys to pigs can improve the human-animal relationship.

It is now widely recognised that a positive human-animal relationship is beneficial not only for farm animals' welfare but also for productivity and the quality of products. A better understanding of animal emotions is an important goal in disciplines ranging from neuroscience to animal welfare science, but few reliable tools exist for measuring these emotions. In this study, whether the provision of toys to solicit play behaviour in pigs is associated with a change in the human-animal relationship and the emotional state of pigs was investigated. We involved a group of sixteen mini-pigs housed in an experimental setting and the use of a preliminary test called the 'strange person' test. After a Control and a Play session (with medium-sized dog toys, balls with ropes), the strange person test was performed. During the test, a person wearing a colourful overall, a hood, a mask, gloves and boots (unknown person with an odd appearance) entered the pen, where 2 mini-pigs were housed, for a 2-min video recording. The strange person test results after the Play and Control sessions were compared. The results showed that the latency to approach the person (duration in seconds) and the duration for which the pig was distant from the strange person (duration in seconds) were significantly lower after the Play session than after the Control session (Degrees of Freedom =30; Statistic of the F test =39.1; p < 0.0001 and Degrees of Freedom =15; Statistic of the F test =54.3; p < 0.0001, respectively). The duration of direct contact (duration in seconds) (Degrees of Freedom =15; Statistic of the F test =14.8; p = 0.002), the need to separate the pig from the strange person (frequency) (Degrees of Freedom =30; Statistic of the F test =9.3; p = 0.005) and the duration of tail movement (duration in seconds) (Degrees of Freedom =15; Statistic of the F test =12.6; p = 0.003) were all significantly higher after the Play sessions than after the Control sessions. Overall, the results suggest a change in the human-animal relationship after the Play sessions: the pigs seemed to be less fearful and more inclined to interact with the strange person, showing a more positive emotional state. This preliminary study suggests that the provision of toys, and more precisely, the opportunity to perform object play behaviour, and sometimes, spontaneously, social play behaviour, can improve the human-animal relationship. Additional research to explore this topic thoroughly may yield interesting results because a positive emotional state of the animals and a good human-animal relationship are essential to ensure good quality of life of farm animals.

Single fluff-spray application of mother hen uropygial secretion analogue positively influences bursa of Fabricius development and the heterophil-to-lymphocyte ratio in ROSS 308 chicks.

Stress is an important cause of illness and mortality in chick production. Stressors such as manipulation, absence of maternal care, transport, and housing can lead to welfare issues, immunodepression, and decreased productivity. The mother hen uropygial secretion analogue (MHUSA), a synthetic analog of a maternal semiochemical secretion, has been proven to protect chicks and broilers against stress, significantly reducing the heterophil-to-lymphocyte ratio. The aim of the present study was to test the effects of the MHUSA on chicks' stress when single-sprayed on their fluff at the age of 1 d. Two-hundred eighty ROSS 308 chicks were included in the study. At day 1, each chick received a spray of 200 µL of a 2% MHUSA aqueous solution (140 chicks) or the same amount of the excipient (control group, 140 chicks), and then chicks were housed in 2 separate rooms. To assess the persistence of the MHUSA after this single application, fluff was sampled from 10 chicks every day for 7 d and at day 13 and 19, weighed, placed in dichloromethane, and analyzed by gas chromatography. Blood smears and the bursa of Fabricius were collected every 3 d from 10 chicks of each group for 36 d to assess the heterophil-to-lymphocyte ratio and the bursa weight-to-BW ratio, respectively. Gas chromatography analysis showed that the MHUSA was present on chick fluff until day 5. The statistical analysis revealed that the heterophil-to-lymphocyte ratio was lower in the MHUSA group at day 4, 7, and 9 (P < 0.0001 for day 4 and 7; P = 0.0377 for day 9). The bursa weight-to-BW ratio was significantly higher in the MHUSA group than in the control group from day 4 until day 29. These results confirm the beneficial effects of the MHUSA on chicks' adaptation to the new environment and on bursa of Fabricius development, suggesting its potential role in improving chicks' immune response.

Maternally regulated gastrulation as a source of variation contributing to cavefish forebrain evolution.

Sequential developmental events, starting from the moment of fertilization, are crucial for the acquisition of animal body plan. Subtle modifications in such early events are likely to have major impacts in later morphogenesis, bringing along morphological diversification. Here, comparing the blind cave and the surface morphotypes of Astyanax mexicanus fish, we found heterochronies during gastrulation that produce organizer and axial mesoderm tissues with different properties (including differences in the expression of dkk1b) that may have contributed to cavefish brain evolution. These variations observed during gastrulation depend fully on maternal factors. The developmental evolution of retinal morphogenesis and hypothalamic patterning are among those traits that retained significant maternal influence at larval stages. Transcriptomic analysis of fertilized eggs from both morphotypes and reciprocal F1 hybrids showed a strong and specific maternal signature. Our work strongly suggests that maternal effect genes and developmental heterochronies that occur during gastrulation have impacted morphological brain change during cavefish evolution.

Equine Activities Influence Horses' Responses to Different Stimuli: Could This Have an Impact on Equine Welfare?

The learning and cognitive challenges that horses may face differ according to the activities in which they are involved. The aim of this investigation was to study the influence of equine activities on the behavioral responses and autonomic nervous system (ANS) activity of adult horses. Forty-one horses were divided into four groups: dressage (9), jumping (10), eventing (13) and equine-assisted activity/therapy (9). A test was created to compare the horses' behavioral and physiological responses to different stimuli. The goal was always to obtain a treat. To study the ANS activity, heart rate variability was assessed using the standard deviation of the R-R intervals (SDNN), square root of the mean of the sum of the squares of differences between successive interbeat-intervals (RMSSD) and low frequency/high frequency (LF/HF). To assess behavioral responses, video analysis was performed considering the following behaviors: exploration, interactions with another horse, and latency to approach. Significant differences in SDNN (DF = 3; F = 3.36; p = 0.0202), RMSSD (DF = 3; F = 4.09; p = 0.0078), LF/HF (DF = 3; F = 4.79; p = 0.0031), exploration (DF = 3; F = 5.79; p = 0.0013) and latency to approach (DF = 3; F = 8.97; p < 0.0001) were found among horses from different equine activities. The activity that adult horses practice appears to influence behavioral and physiological responses to different stimuli, thus impacting equine welfare.

The Impact of Equine-Assisted Therapy on Equine Behavioral and Physiological Responses.

Equine-assisted therapies (EATs) have been widely used in the treatment of patients with mental or physical conditions. However, studies on the influence of equine-assisted therapy (EAT) on equine welfare are very recent, and the need for further research is often highlighted. The aim of this study was to investigate whether EAT creates negative or positive emotions in horses, and the influence of patients' expectations (one group of patients had physical and psychological expectations and one group of patients had only psychological expectations) on horses' emotional responses. Fifty-eight pairs (patient-horse) were involved in this study. Behaviors and heart rate variability (HRV) data were collected during a resting phase, a preparation phase in which the patients brushed and saddled the horse, and a working phase. Behaviors and HRV were compared between phases and among the groups of patients. Our results suggested that the EAT in this study was neither a negative nor a positive event. EATs with patients who had both physical and psychological expectations were more challenging for horses than those with patients who had only psychological expectations. Further research should focus on providing horses with positive stimulation and reinforcement to understand whether a positive association with EAT can be achieved.

Synthesis and Biological Evaluation of N-[2-(4-Hydroxyphenylamino)-pyridin-3-yl]-4-methoxy-benzenesulfonamide (ABT-751) Tricyclic Analogues as Antimitotic and Antivascular Agents with Potent in Vivo Antitumor Activity.

Benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) were modified to produce tricyclic quinazolinone 15-18 or benzothiadiazine 26-27 derivatives. These compounds were evaluated in cytotoxicity and tubulin inhibition assays and led to potent inhibitors of tubulin polymerization. N-[2(4-Methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16a) exhibited the best in vitro cytotoxic activity (GI50 10-66.9 nM) against the NCI 60 human tumor cell line and significant potency against tubulin assembly (IC50 0.812 µM). In mechanism studies, 16a was shown to block cell cycle in G2/M phase and to disrupt microtubule formation and displayed good antivascular properties as inhibition of cell migration, invasion, and endothelial tube formation. Compound 16a was evaluated in C57BL/6 mouse melanoma B16F10 xenograft model to validate its antitumor activity, in comparison with reference ABT-751 (1). Compound 16a displayed strong in vivo antitumor and antivascular activities at a dose of 5 mg/kg without obvious toxicity, whereas 1 needed a 10-fold higher concentration to reach similar effects.

Synthesis of triazoloquinazolinone based compounds as tubulin polymerization inhibitors and vascular disrupting agents.

A series of 1-phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5-ones designed as conformationally restricted CA-4 analogues, were tested for their tubulin polymerization and growth inhibitory activities. The 3-hydroxy-4-methoxy derivatives 11d and 12d are potent inhibitors of tubulin assembly but only the N-methylated amid counterpart 12d possesses potent anticancer activity in a large panel of cancer cell lines. Upon treatment with compound 12d, remarkable cell shape changes as cell migration and tube formation were elicited in HUVECs, consistent with vasculature damaging activity.