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.