Incorporación de Tecnología Tridimensional mediante Tomografía Microcomputarizada en el Estudio de la Anatomía de Salmónidos: Una Evaluación de Utilidad / Integration of Three-Dimensional Technology through Microcomputed Tomography in the Study of Salmonid Anatomy: A Utility Evaluation

Los salmónidos, durante su desarrollo, pueden experimentar ocasionalmente deformaciones esqueléticas. Para su diagnóstico se emplean diversas metodologías, entre las que se incluyen radiografías, técnicas histológicas, diafanización con tinciones de alizarina y azul de alcián, así como el uso del microscopio electrónico de barrido (SEM), cada una con sus inherentes ventajas y desventajas. Este estudio tuvo como finalidad evaluar y comparar la eficacia de la tomografía microcomputarizada (Micro-CT) para el análisis anatómico, reconstruyendo tridimensionalmente las imágenes y contrastándolas con los resultados obtenidos mediante la técnica de diafanización. Se analizaron las aletas caudales de cinco ejemplares de salmón Oncorhynchus kisutch: dos sujetos a diafanización y tres procesados para análisis mediante Micro-CT utilizando el equipo BRUKER SkyScan 1272. La técnica de Micro-CT demostró superioridad en la resolución de las estructuras óseas, facilitando una exploración detallada de las variaciones morfológicas y la distribución de la densidad mineral. Este enfoque permitió identificar anomalías en la morfología y crecimiento de las últimas vértebras y lepidotriquias dorsales, así como una densidad incrementada en lepidotriquias dorsales malformadas. La mayor resolución proporcionada por la Micro-CT no solo potencia nuestra comprensión de la ontogenia piscícola y su adaptación a ambientes diversos, sino que además inaugura perspectivas innovadoras para el estudio de la evolución de las estrategias locomotoras y las respuestas adaptativas frente a cambios ambientales a través del tiempo.

SUMMARY: During their development, some species of salmonids may occasionally experience skeletal deformations. Several methodologies are currently being used for the diagnosis of such malformations, among which X-rays, histological techniques, diaphanization coupled either with Alizarin Red or Alcian Blue stains, as well as Scanning Electron Microscopy (SEM) can be mentioned. Each one of those methods presents inherent advantages and disadvantages. The purpose of this study was twofold: Firstly, to evaluate and compare the effectiveness of microcomputed tomography (Micro-CT) technology for anatomical analysis, three-dimensionally reconstructing the obtained images; and secondly, to contrast those images with the results obtained through the diaphanization technique. The caudal fins of five specimens of the Oncorhynchus kisutch salmon were analyzed: Two specimens were subjected to diaphanization and three were processed for Micro-CT analysis, using the BRUKER SkyScan 1272 equipment. The Micro-CT technology demonstrated superiority in the resolution of bone structures, facilitating a detailed exploration of morphological variations, as well as the distribution of mineral density. This experimental approach allowed us to identify anomalies in the morphology and growth of the last vertebrae and dorsal lepidotrichiae, as well as an increased mineral density in the malformed dorsal lepidotrichiae. The higher resolution provided by Micro-CT not only enhances our understanding of the fish ontogeny and its adaptation to diverse environments, but also opens innovative perspectives for the study of the evolution of locomotor strategies and adaptive responses to environmental changes.

Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives.

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

Development and growth of the pelvic fin in the extant coelacanth Latimeria chalumnae.

The ontogeny of the paired appendages has been extensively studied in lungfishes and tetrapods, but remains poorly known in coelacanths. Recent work has shed light on the anatomy and development of the pectoral fin in Latimeria chalumnae. Yet, information on the development of the pelvic fin and girdle is still lacking. Here, we described the development of the pelvic fin and girdle in Latimeria chalumnae based on 3D reconstructions generated from conventional and X-ray synchrotron microtomography, as well as MRI acquisitions. As in other jawed vertebrates, the development of the pelvic fin occurs later than that of the pectoral fin in Latimeria. Many elements of the endoskeleton are not yet formed at the earliest stage sampled. The four mesomeres are already formed in the fetus, but only the most proximal radial elements (preaxial radial 0-1) are formed and individualized at this stage. We suggest that all the preaxial radial elements in the pelvic and pectoral fin of Latimeria are formed through the fragmentation of the mesomeres. We document the progressive ossification of the pelvic girdle, and the presence of a trabecular system in the adult. This trabecular system likely reinforces the cartilaginous girdle to resist the muscle forces exerted during locomotion. Finally, the presence of a preaxial element in contact with the pelvic girdle from the earliest stage of development onward questions the mono-basal condition of the pelvic fin in Latimeria. However, the particular shape of the mesomeres may explain the presence of this element in contact with the girdle.

3D + Time Imaging and Image Reconstruction of Pectoral Fin During Zebrafish Embryogenesis.

Morphogenesis is the fundamental developmental process during which the embryo body is formed. Proper shaping of different body parts depends on cellular divisions and rearrangements in the growing embryo. Understanding three-dimensional shaping of organs is one of the basic questions in developmental biology. Here, we consider the early stages of pectoral fin development in zebrafish, which serves as a model for limb development in vertebrates, to study emerging shapes during embryogenesis. Most studies on pectoral fin are concerned with late stages of fin development when the structure is morphologically distinct. However, little is known about the early stages of pectoral fin formation because of the experimental difficulties in establishing proper imaging conditions during these stages to allow long-term live observation. In this protocol, we address the challenges of pectoral fin imaging during the early stages of zebrafish embryogenesis and provide a strategy for three-dimensional shape analysis of the fin. The procedure outlined here is aimed at studying pectoral fin during the first 24 h of its formation corresponding to the time period between 24 and 48 h of zebrafish development. The same principles could also be applied when studying three-dimensional shape establishment of other embryonic structures. We first discuss the imaging procedure and then propose strategies of extracting quantitative information regarding fin shape and dimensions.

The pectoral fin muscles of the coelacanth Latimeria chalumnae: Functional and evolutionary implications for the fin-to-limb transition and subsequent evolution of tetrapods.

To investigate the morphology and evolutionary origin of muscles in vertebrate limbs, we conducted anatomical dissections, computed tomography and kinematic analyses on the pectoral fin of the African coelacanth, Latimeria chalumnae. We discovered nine antagonistic pairs of pronators and supinators that are anatomically and functionally distinct from the abductor and adductor superficiales and profundi. In particular, the first pronator and supinator pair represents mono- and biarticular muscles; a portion of the muscle fibers is attached to ridges on the humerus and is separated into two monoarticular muscles, whereas, as a biarticular muscle, the main body is inserted into the radius by crossing two joints from the shoulder girdle. This pair, consisting of a pronator and supinator, constitutes a muscle arrangement equivalent to two human antagonistic pairs of monoarticular muscles and one antagonistic pair of biarticular muscles in the stylopod between the shoulder and elbow joints. Our recent kinesiological and biomechanical engineering studies on human limbs have demonstrated that two antagonistic pairs of monoarticular muscles and one antagonistic pair of biarticular muscles in the stylopod (1) coordinately control output force and force direction at the wrist and ankle and (2) achieve a contact task to carry out weight-bearing motion and maintain stable posture. Therefore, along with dissections of the pectoral fins in two lungfish species, Neoceratodus forsteri and Protopterus aethiopicus, we discuss the functional and evolutionary implications for the fin-to-limb transition and subsequent evolution of tetrapods. Anat Rec, 299:1203-1223, 2016. © 2016 Wiley Periodicals, Inc.

Anatomy and evolution of heterocercal tail in lamniform sharks.

Lamniformes is a small shark group consisting of 15 extant species with remarkably diverse lifestyles and a wide range in heterocercal tail morphology. The caudal fin morphology must be related to their lifestyle because the tail is a main locomotive structure in sharks, but such relationships have remained largely uninvestigated. Here, the morphology-lifestyle relationship in lamniforms is examined through phylogenetic mapping. This study suggests that, within Lamniformes, caudal fins with a more horizontally directed curvature of the vertebral column are plesiomorphic, whereas those with a large dorsally directed curvature of the vertebral column are apomorphic. It also shows that caudal fins with posteriorly directed hypochordal rays are plesiomorphic, and that those with ventrally directed hypochordal rays are apomorphic within Lamniformes. Four basic caudal fin types are recognized in lamniforms on the basis of these skeletal variables in which one corollary is that the evolution of external morphology of caudal fin does not necessarily correspond to the evolution of its skeletal anatomy. This study also demonstrates that specific lifestyles seen in different lamniforms are indeed correlative with different caudal fin types in which a less asymmetrical heterocercal tail is a derived feature in lamniforms that evolved for fast swimming to capture fast swimming prey.

Functional implications of variation in pectoral fin ray morphology between fishes with different patterns of pectoral fin use.

In this study, I compare the morphology from the pectoral fin rays from the benthic longhorn sculpin (Myoxocephalus octodecimspinosus) to those from a species that does not use its fins for substrate contact, the yellow perch (Perca flavescens). I use CT scanning technology to compare the shape and structure of the paired hemitrichia that make up the pectoral fin rays between these species. I found that the structure of hemitrichia of the fin rays in yellow perch is consistent with previous descriptions for pelagic fishes. They are almost completely segmented, have a crescent shape in cross section, and are branched distally. In contrast, longhorn sculpin hemitrichia exhibit morphological regionalization along the proximo-distal length of the ray. The most proximal 20-50% of the length of the hemitrichia is unsegmented and cylindrical in cross section. Distally, the fin rays of longhorn sculpin are segmented and crescent-shaped but do not branch. I measured the second moment of area of the hemitrichia at distances of 10%, 30%, 50%, and 70% distance along the length of the fin rays. The cylindrical regions of the sculpin hemitrichia had a higher second moment of area than the crescent-shaped regions in either species. I hypothesize that that this regionalization of individual fin rays provides resistance to bending proximally and flexibility distally, features that may be useful during substrate contact. This combination of an elongate, unsegmented proximal region and segmented distal region in fin rays has not yet been described among extant ray-finned fishes. However, this structure is reminiscent of that of the elongate cylindrical region found in the fossil sarcopterygian fish Eusthenopteron.