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Spatial transcriptomics is a new technology for capturing and mapping whole transcriptome gene expression on histological sections close to single-cell resolution. This technique allows researchers to gain insight into organs’ molecular morphology, as well as regional changes in gene expression during disease progression, development, and evolution. We apply this technique to the light-absorbing retina within the developing mouse eye. The mouse retina lacks internal blood vessels at birth but develops these structures over the first four weeks, which changes the diffusion dynamics for O2 and nutrients and affects the metabolic phenotypes of the retinal cells. Using spatial transcriptomics, we have generated spatial molecular atlases of the retina and extracted combined information on nutrient diffusion distances and whole transcriptome gene expression. With this technique, we can now provide a new quantitative understanding of the cellular responses to changes in O2 and nutrient availabilities during organ development. 

SEB Annual Conference 2022

Tracing the development of the eye using spatial transcriptomics

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
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Please register for the conference by clicking on this link:

https://www.sebiology.org/events/seb-annual-conference-2022/registration.html

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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

technical paper

We have developed a super resolution cryo-fluorescence optical microscope in order to perform correlative imaging with the cryo Soft X-ray Tomography (SXT) beamline at the Diamond Light Source. Using Structured Illumination Microscopy (SIM) the microscope generates images at almost twice the achievable conventional optical resolution in 3 dimensions. By reducing the gap between the resolution in optical fluorescence images and the X-ray tomography significantly more information can be extracted from the correlated image sets. However, there are many barriers to reliably produce high quality fluorescence images of 3D samples in cryo while preserving the sample integrity. The sample must be kept cold, condensation must be avoided on both the sample and the optics, sample position must be highly stable and light must be collected at as high a numerical aperture as possible. We have open source control software providing a simple interface with high speed control of the wide range of hardware involved as well as a high degree of flexibility to provide a range of experimental options. Despite, these challenges we have been able to reliably produce images with sub 200 nm resolution and achieve correlative alignment at sub 100 nm. The CryoSIM and cryoSXT instruments are available for open access via peer revied applications.

CryoSIM: the trials and tribulations of correlative super resolution cryo fluorescence imaging.

Bisphenols enter the environment from the production and degradation of plastics, and are ubiquitous pollutants with endocrine disrupting effects. Thyroid hormones are key regulators of vertebrate metabolism and muscle function, and represent one of many endocrine targets disrupted by bisphenol S (BPS). Our aim was to test whether BPS disrupts locomotor performance in zebrafish in a thyroid-dependent manner. First, we established that BPS at an environmentally relevant concentration impaired swimming performance (Ucrit). We then conducted two subsequent but separate, fully factorial experiments using BPS exposure (present and absent) and induced hypothyroidism (hypo- and normo- thyroid) as factors to assess mitochondrial function and muscle protein composition as potential mechanisms by which BPS could disrupt locomotor performance. Hypothyroidism but not BPS exposure reduced maximal substrate oxidation and proton leak. Metrics of mitochondrial efficiency were not impact by BPS exposure but were altered by both temperature and hypothyroidism. BPS exposure decreased AMPK activity (pAMPK/AMPKtotal) in skeletal muscle but increased fast myosin heavy chain (MHC) proteins. BPS exposure did not strongly impact slow MHC or myosin enhancer factor 2 (MEF2). Hypothyroidism decreased AMPK activity, and increased fast and slow MHC and MEF2 so that BPS and hypothyroidism affected muscle in the same direction. Since AMPK activity is important to sustained exercise, the BPS-induced decrease in AMPK activity could explain the observed decrease in swimming performance.

Bisphenol S Exposure reduces locomotor function by affecting muscle function but not mitochondria

First identified as sexual hormones, estrogens were shown to have a key role in the development of gonads but also to be involved in the skeleton development and homeostasis. In mammals, cytosolic estrogen receptors (ERa and ERb) are expressed in osteoblasts, osteoclasts, osteocytes as well as chondrocytes and chondroblasts, regulating their activity to build up and maintain cartilage and bone. 
To expand our knowledge of estrogen signaling in skeletal development outside of mammals, we used the European sea bass Dicentrarchus labrax, to test the impact of two concentrations of the natural estrogen 17b estradiol (E2, 0.4 and 40 ng/L) on 6-to-23-day old post-hatching larvae. 
We evaluated the effects of E2 on the morphological characteristics of the larvae as well as on the mineralization of the cranium, vertebrae and fins using alizarin red staining. In addition, qPCR measurements were performed to analyze the expression of various genes known to be involved in the formation of cartilage and bone in vertebrates. We showed that the effect of estrogens varied depending on the developmental stage of the larvae and the state of mineralization of their skeleton at the time of treatment, and that these effects were dose-dependent.











Estradiol effects on early skeletogenesis in the European sea bass Dicentrarchus labrax

Anthropogenic changes in the environment are ubiquitos and affect organisms in many ways. Flexibility of the endocrine system may help organisms to response to those changes. However, disruption in the female endocrine function may lead to altered transfer of hormones in eggs or embryos, ie. transgenerational endocrine disruption, which can have long-lasting effects on offspring. Maternally derived thyroid hormones (THs) are essential for development, but poorly studied, especially in wild birds. Here, we provide three case examples of studies on transgenerational TH disruption. First, we studied how parental exposure to metal pollution, using repeated sampling of polluted and control areas across Europe, influenced egg TH levels in a passerine bird, the great tit. Second, we studied how long-term experimental parental herbicide exposure influenced egg THs and offspring traits in Japanese quails. Third, we studied experimetally how limitation of iodine, a key element of the TH molecule, influenced parental and egg THs in rock pigeons. We found no overall effect of parental metal exposure on egg THs, but the effect was dependent on calcium availability. Parental herbicide exposure did not influence egg THs, but affected hatchability and embryo traits. Parental iodine availability did not influence parental or egg TH levels, but decreased egg laying rate. Results of these case examples suggest that transgenerational endocrine disruption is a complex phenomena and should be further studied, as such legacy effects can delay adapting to environmental changes.

Transgenerational endocrine disruption - a potential mechanism for legacy effects of anthropogenic disturbance

Nocturnin (NOC) is a phosphatase involved in the transformation of NADP(H) to NAD(H), performing a key role in the regulation of metabolism. In mammals, it has been purposed that nocturnin expression is induced by E-box element activation. However, nothing is known about the possible elements that modulate its expression in fish. Our aim was to undertake the study of the possible endocrine regulation and the intracellular pathways involved in the gene expression of three noc paralogs (noc-a1, noc-a2 and noc-b1) expressed in goldfish (Carassius auratus). For this purpose, we studied the effect of glucagon, adrenaline and different activators and inhibitors of protein kinase A (PKA) and protein kinase C (PKC) pathways on noc paralogs expression in hepatopancreas organotypic cultures. We found a concentration-dependent induction of noc-a1 expression by both glucagon and adrenaline at 2 and 6-h incubation time. Forskolin, an adenylyl cyclase activator, induced a 5-fold increase of the relative abundance of this paralog, meanwhile a 2-fold increase was obtained in the presence of phorbol 12-myristate 13-acetate, a PKC activator. The effects of glucagon and forskolin were fully blocked by a PKA inhibitor (H89), and partially by a PKC inhibitor (chelerythrine) and by a PLC inhibitor (U73122), suggesting a possible interaction between PKA and PKC pathways in the intracellular signaling of glucagon on noc-a1 expression. Regarding noc-a2 mRNA expression, our results show an induction by adrenaline at 2 and 6-h incubation time but not by glucagon, forskolin or phorbol 12-myristate 13-acetate. Then, noc-b1 seems to be insensitive to both hormones, and to PKA and PKC activators. Altogether, these results suggest a possible functional specialization of noc paralogs in hepatopancreas and aim to investigate the possible role of other metabolic regulators in the control of noc expression in goldfish.
Supported by the Spanish MICINN (PID2019-103969RB-C32). D.M and A.A-G are predoctoral fellows, UCM (CT42/18-CT43/18) and FPI (BES-2017-081398), respectively.

Glucagon and adrenaline: two modulators of nocturnin expression in goldfish?

How limited resources are physiologically directed towards somatic growth is a simple question, but poorly understood. The hormone - insulin-like growth factor 1 (IGF-1) - is an evolutionarily conserved hepatic peptide that is one of the more important hormones of growth regulation. For example, administration of IGF-1 to the nestlings of the pied flycatcher (Ficedula hypoleuca) induced growth to bigger pre-fledging body size. Moreover, administrating an IGF-1 receptor inhibitor (OSI-906) resulted in reduced growth. The growth rate is age-dependent and we investigated how IGF-1 levels changed with age and growth conditions in nestlings of two passerine species – great tits (Parus major) and jackdaws (Corvus monedula) – with different life histories. We showed experimentally in both species that levels of IGF-1 change positively with the improving food availability, both when observed in the short- (within 1h) and long-term (during the nestling phase). However, the effects of nestling age on IGF-1 levels as well as IGF-1 growth associations contrasted strongly between these species. Thus, IGF-1 appears to respond to environmental cues uniformly across species, but the timing of IGF-1 mediated investments into growth is dependent on the particular growth strategy. These findings together show that IGF-1 variability provides a promising avenue for studies of animals’ growth, of which the exploration has only just begun.

Physiology of somatic growth: insulin-like growth factor 1 and its variability in passerine species with different life-history

Shark skin is a composite of mineralized dermal denticles embedded in an internal collagen fiber network. Shark skin is sexually dimorphic; females have skin that is thicker, denticle density and degree of overlap is greater, and denticle morphology differs from males. Shark skin behaves with mechanical anisotropy, showing greater extensibility when tested along the longitudinal axis and increased stiffness along the hoop axis. As a result, shark skin has been hypothesized to function as an exotendon. The goal of this study was to quantify mechanical properties (tensile strain at maximum load (%), strength (MPa), stiffness (MPa), and toughness (MPa)) of shark skin between sexes and testing directions (longitudinal and hoop). We hypothesized that female sharks would have greater skin mechanical properties due to observed skin morphological and dermal density differences between sexes. We also hypothesized that skin tested in the hoop direction would be stiffer than skin tested longitudinally. We tested skin from two juvenile male and two juvenile female sharks from three species (bonnethead, Sphyrna tiburo; bull, Carcharhinus leucas; silky, Carcharhinus falciformis) in tension with 250N load cell on an Instron E1000 at a 2 mm/s strain rate. Refuting our hypothesis, we found that male shark skin was stiffer and stronger than female skin. Supporting our hypothesis, we found skin was stiffer when tested in the hoop direction compared to the longitudinal direction (across species and sexes). Our study provides evidence of sexual variation in shark skin mechanical behavior and further supports the exotendon hypothesis of shark skin.

Sexual dimorphisms in the mechanical properties of shark skin

The vocal repertoire of birds is incredibly diverse. They vocalize using a specific organ, the syrinx, producing a primary vibration at the origin of a primary sound. This sound propagates through the suprasyringeal system, composed of the trachea, the larynx, and the oral cavity. The large volume of this suprasyringeal system, compared to mammals of similar size, suggests that it plays a role in the modulation of the sound initially emitted. To investigate this, we filmed 21 species of dead birds and manipulated them under biplanar X-ray. Markers were implanted in tissues (e.g., on the trachea contour, glottis, beak) to calculate the passive deformations and volume changes of the vocal tract during a range of movements of the neck/head system. Our results show that neck extension and beak closing are associated with an elongation of the trachea of up to 20% of its initial length. We tested the effect of such elongation on sound modulation using computational simulations. Our work shows that the complexity of bird vocalizations is not only related to the syrinx, but also to the entire upper vocal system. This work provides new elements to better understand complex communication, one of the amazing characteristics we share with birds.

Influence of the avian upper vocal tract on sound modulation

Muscle shortening underpins most skeletal motion, and ultimately animal performance. Shortening is usually assumed to be homogenous within a muscle. However, in swimming fish, the whole body deforms like a bending beam: as the vertebral column flexes laterally, muscle shortening increases along a medio-lateral gradient. Recently it has been shown that fish also bend their bodies dorsally during feeding, resulting in dorsoventral shortening gradients in some regions. This suggests these dorsal motions also follow beam-like bending, where the curvature of the vertebral column predicts the linear, dorsoventral shortening gradient in the muscle. We tested this hypothesis by measuring vertebral curvature and dorsal body (epaxial) muscle shortening during feeding motions in rainbow trout (Oncorhynchus mykiss). We used XROMM to measure dorsal and lateral curvature across the anterior vertebral column and used fluoromicrometry to measure shortening between markers throughout the epaxial muscles. Vertebral curvature and muscle marker position were used to predict muscle strain following beam theory. Predicted strains were compared to the measured strains from fluoromicrometry. Trout flexed the anterior vertebrae dorsally and laterally during feeding strikes. When curvature and marker position in both planes were included, the model’s predicted strain matched the measured strain well across most vertebral regions. Our model confirms dorsal flexion during feeding in trout can be modelled with beam theory, even when accompanied by lateral flexion. We find evidence of linear dorsoventral and mediolateral shortening gradients in the epaxial muscles, although it remains to be determined if these reflect shortening of the fibres within these muscles.

Testing the relationship between muscle deformation and vertebral curvature during feeding in rainbow trout (Oncorhynchus mykiss).

Nuclear Pore Complexes (NPCs) are large macromolecular assemblies embedded into the nuclear envelope (NE). They are involved in the selective nucleocytoplasmic transport of macromolecules, chromatin organization, and DNA repair. NPCs are organized into sub-complexes composed of repetitions of about 30 highly conserved nucleoporins (NUPs). The NUP107–160 complex (also known as Y complex) is located in the outer ring and is conserved between distantly related eukaryotes such as yeast and mammals. Here, we are going to focus on two Arabidopsis mutants deficient for this sub-complex: Atnup160 and Atnup96, also known as suppressor of auxin resistance1 (sar1) and sar3, respectively. NUP160 and NUP96 are critical for the nucleocytoplasmic transport of mRNAs and play important functions in auxin signalling, plant growth and flowering time regulation, as well as in immune defence and cold stress tolerance. In the context of our global study about the possible role of NPCs during meiosis (the specialised cell division that leads to gamete formation), we decided to explore the potential function of NUP160 and NUP96 during this cell division because the corresponding mutants show reduced fertility. The results of our study have demonstrated that meiosis is altered in a percentage of the meiocytes from these mutants, which would explain the reduction in the number of seeds. Our observations have revealed that the alteration of the Y complex causes problems in meiotic recombination, the formation of meiotic structures (such as the synaptonemal complex), and viability of the meiocytes. The possible origins of these problems will be discussed.

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Tracing the development of the eye using spatial transcriptomics

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