Cell dynamics during killifish embryos development and diapause

In this work I used time lapse brightfield microscopy to investigate cells division\u2019s kinetics during the first developmental stages of annual- and non-annual species belonging to the three different hylogenetic clades. Annual killifishes of all three clades had cleavage times significantly longer when compared to their non-annual sister taxa (average 35 min vs. average 75 min), showing, for the first time, that cell cycle rate during cleavage, a trait thought to be rather volutionary conserved can undergo convergent evolutionary change in response to variations in life-history

Studio della regolazione da parte di Tbx5 del miRNA 218 e del ruolo di quest'ultimo nello sviluppo cardiaco del Danio rerio.

Tbx5 è un fattore di trascrizione coinvolto nel differenziamento cellulare dei cardiomiociti e nella morfogenesi cardiaca. Nell’uomo la mutazione di questo gene causa diverse forme di malformazioni cardiache e degli arti superiori, che si riconoscono nella sindrome di Holt-Oram. Analisi bioinformatiche e studi di microarrays hanno posto in evidenza come a valle di Tbx5 vengano regolati tutta una serie di geni che sono a loro volta coinvolti nello sviluppo cardiaco. Inoltre, alcuni dei geni regolati da Tbx5 contengono al loro interno dei microRNA che vengono attivamente espressi, e partecipano a loro volta a regolare ed affinare tutto il processo di differenziamento cellulare e morfogenesi del tessuto cardiaco. Lo studio da me condotto prende in esame in particolare i geni slit 2 e slit 3, importanti per lo sviluppo neuronale, per i quali è stato recentemente dimostrato un ruolo nella morfogenesi cardiaca. Il mio studio evidenzia il loro rapporto di dipendenza da Tbx5, e valuta le possibili funzioni del miR-218, che si trova all’interno di un introne in entrambi i geni slit. Tutti gli esperimenti sono stanti condotti utilizzando Danio rerio come sistema modello, in quanto l’enorme prole prodotta giornalmente, la semplicità morfologica del cuore, ed il suo precoce sviluppo lo rendono ideale per studi di cardiogenesi. Inoltre, le prime fasi di sviluppo di Danio rerio avvengono anche in assenza di un apparato cardiocircolatorio funzionante, il che è ottimale per studiare totali o parziali disfunzioni di esso. I geni e i miRNA sopracitati sono stati studiati soprattutto tramite tecniche di microiniezione, valutando quindi i difetti morfologici e funzionali che una loro sovra- o sotto-regolazione provocavano nelle prime fasi di sviluppo. I mutanti ottenuti con le microiniezioni sono stati analizzati tramite ibridazioni in situ per caratterizzare i geni deregolati. L’espressione dei geni e dei microRNA in studio è stata, infine, alterata mediante microiniezione in linee transgeniche esprimenti GFP o RFP sotto il promotore di geni specifici per il miocardio (cmlc2) o per l’endotelio (flk1). Questo approccio ha permesso di valutare in maggiore dettaglio, tramite microscopia confocale, l’alterazione morfologica del cuore o dei vasi conseguente a tale deregolazione. Le analisi condotte hanno rivelato come la funzione di Tbx5 e del miRNA 218 siano strettamente connesse in processi riguardanti la morfogenesi cardiaca e come entrambi svolgano dei ruoli sia nella crescita che nella giusta strutturazione delle camere cardiache. Inoltre è stato definita una finestra temporale nella quale il miRNA 218 riesce a svolgere la sua azione, prima della quale non sembra avere effetto. Infine è stato dimostrato che la sovra-regolazione del miRNA 218 provoca un’alterazione del pattern di espressione del gene tie 2 un gene molto espresso a livello della valvola atrio-ventricolare

Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products

The chemo-mechanical cycle of the myosin II–actin reaction in situ has been investigated in Ca(2+)-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (P(i)) concentration. It was found that in control conditions (no added P(i)), shortening at V ≤ 350 nm s(–1) per half-sarcomere, corresponding to force (T) greater than half the isometric force (T(0)), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T(0) value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising P(i) concentration by 10 mm, which reduces T(0) and the number of motors by 40–50%, does not influence the dependence on V of number and strain. A model simulation of the myosin–actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that P(i) and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction. KEY POINTS: Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin–actin interactions increases with the increase in shortening velocity. . We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration. . A model simulation of the myosin–actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation. . These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction.

The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site.

Skeletal muscle shortens faster against a lower load. This force–velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (P(i)) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with P(i) release is still an unsolved question. Using nanometre–microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [P(i)]. We show that the rate constant of the working stroke is unaffected by [P(i)], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of P(i) from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere

Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon

Effect Of Priming On Seed Vigor Of Wheat (Triticum Aestivum L.)

Priming is a process that controls the process of hydration of seeds for the ongoing metabolic processes before germination. Research on priming was conducted at ICERI seed laboratory from May to September 2009 to evaluate the effect of different priming methods on wheat seed vigor. Physical properties and chemical composition of seed were evaluated before seeds were treated. The priming treatment were conducted by soaking 250 g of seed in 500 mL of solution for hydropriming and halopriming. Two seed lots of Nias and Dewata variety were subjected to heated and unheated distilled water for 12hours and subjected to KCl and CaCl2 at 10, 20, and 30 ppm and unprimed seed. The experiment were arranged in completely randomized design, replicated thrice. Vigor evaluation by observed seed germination, simultaneity growth, germination rate, seedling dry weight, electric conductivity of seed leakage and length of primary root. The results showed that highest germination, simultaneity growth, seedling dry weight, and length of primary root, were priming treatment with KCl 30 ppm and CaCl2 20 and 30 ppm. Priming with distilled water for 12 hours gave higher germination percentage and simultaneity growth

A computational framework for testing hypotheses of the minimal mechanical requirements for cell aggregation using early annual killifish embryogenesis as a model

Introduction: Deciphering the biological and physical requirements for the outset of multicellularity is limited to few experimental models. The early embryonic development of annual killifish represents an almost unique opportunity to investigate de novo cellular aggregation in a vertebrate model. As an adaptation to seasonal drought, annual killifish employs a unique developmental pattern in which embryogenesis occurs only after undifferentiated embryonic cells have completed epiboly and dispersed in low density on the egg surface. Therefore, the first stage of embryogenesis requires the congregation of embryonic cells at one pole of the egg to form a single aggregate that later gives rise to the embryo proper. This unique process presents an opportunity to dissect the self-organizing principles involved in early organization of embryonic stem cells. Indeed, the physical and biological processes required to form the aggregate of embryonic cells are currently unknown.Methods: Here, we developed an in silico, agent-based biophysical model that allows testing how cell-specific and environmental properties could determine the aggregation dynamics of early Killifish embryogenesis. In a forward engineering approach, we then proceeded to test two hypotheses for cell aggregation (cell-autonomous and a simple taxis model) as a proof of concept of modeling feasibility. In a first approach (cell autonomous system), we considered how intrinsic biophysical properties of the cells such as motility, polarity, density, and the interplay between cell adhesion and contact inhibition of locomotion drive cell aggregation into self-organized clusters. Second, we included guidance of cell migration through a simple taxis mechanism to resemble the activity of an organizing center found in several developmental models.Results: Our numerical simulations showed that random migration combined with low cell-cell adhesion is sufficient to maintain cells in dispersion and that aggregation can indeed arise spontaneously under a limited set of conditions, but, without environmental guidance, the dynamics and resulting structures do not recapitulate in vivo observations.Discussion: Thus, an environmental guidance cue seems to be required for correct execution of early aggregation in early killifish development. However, the nature of this cue (e.g., chemical or mechanical) can only be determined experimentally. Our model provides a predictive tool that could be used to better characterize the process and, importantly, to design informed experimental strategies