Teaching Fin Regeneration Using a Dominant Negative Receptor.

Rising in popularity as a model organism in the classroom, zebrafish have numerous characteristics that make them ideal for teaching. In this study, we describe an experiment that helps students better understand the concept of tissue regeneration and the genes that control it. This experiment utilizes a dominant negative transgene for fgfr1 and allows students to observe the consequences of its activation. The first part of the laboratory is hands-on, and includes details of the amputation of caudal fins, heat shocking, general fish care, and visual observations. Over the course of a week, students observed the differences between the activated and unactivated transgene in the zebrafish. The second part was literature based, in which students tried to determine which gene is responsible for inhibiting regeneration. This encouraged students to sharpen their skills of deductive reasoning and critical thinking as they conduct research based on the information they receive about dominant negative receptors and transgenes. Having both a hands-on and critical thinking component in the laboratory helped synthesize the learning goals and allowed students to actively participate.

Asymmetric fin shape changes swimming dynamics of ancient marine reptiles' soft robophysical models.

Animals have evolved highly effective locomotion capabilities in terrestrial, aerial, and aquatic environments. Over life's history, mass extinctions have wiped out unique animal species with specialized adaptations, leaving paleontologists to reconstruct their locomotion through fossil analysis. Despite advancements, little is known about how extinct megafauna, such as the Ichthyosauria one of the most successful lineages of marine reptiles, utilized their varied morphologies for swimming. Traditional robotics struggle to mimic extinct locomotion effectively, but the emerging soft robotics field offers a promising alternative to overcome this challenge. This paper aims to bridge this gap by studyingMixosauruslocomotion with soft robotics, combining material modeling and biomechanics in physical experimental validation. Combining a soft body with soft pneumatic actuators, the soft robotic platform described in this study investigates the correlation between asymmetrical fins and buoyancy by recreating the pitch torque generated by extinct swimming animals. We performed a comparative analysis of thrust and torque generated byCarthorhyncus,Utatsusaurus,Mixosaurus,Guizhouichthyosaurus, andOphthalmosaurustail fins in a flow tank. Experimental results suggest that the pitch torque on the torso generated by hypocercal fin shapes such as found in model systems ofGuizhouichthyosaurus,MixosaurusandUtatsusaurusproduce distinct ventral body pitch effects able to mitigate the animal's non-neutral buoyancy. This body pitch control effect is particularly pronounced inGuizhouichthyosaurus, which results suggest would have been able to generate high ventral pitch torque on the torso to compensate for its positive buoyancy. By contrast, homocercal fin shapes may not have been conducive for such buoyancy compensation, leaving torso pitch control to pectoral fins, for example. Across the range of the actuation frequencies of the caudal fins tested, resulted in oscillatory modes arising, which in turn can affect the for-aft thrust generated.

Genomic screening of fish-specific genes in gnathostomes and their functions in fin development.

In this study, we comprehensively searched for fish-specific genes in gnathostomes that contribute to development of the fin, a fish-specific trait. Many previous reports suggested that animal group-specific genes are often important for group-specific traits. Clarifying the roles of fish-specific genes in fin development of gnathostomes, for example, can help elucidate the mechanisms underlying the formation of this trait. We first identified 91 fish-specific genes in gnathostomes by comparing the gene repertoire in 16 fish and 35 tetrapod species. RNA-seq analysis narrowed down the 91 candidates to 33 genes that were expressed in the developing pectoral fin. We analyzed the functions of approximately half of the candidate genes by loss-of-function analysis in zebrafish. We found that some of the fish-specific and fin development-related genes, including fgf24 and and1/and2, play roles in fin development. In particular, the newly identified fish-specific gene qkia is expressed in the developing fin muscle and contributes to muscle morphogenesis in the pectoral fin as well as body trunk. These results indicate that the strategy of identifying animal group-specific genes is functional and useful. The methods applied here could be used in future studies to identify trait-associated genes in other animal groups.

Mechanics and properties of fish fin rays in nonlinear regimes of large deformations.

Fins from ray-finned fishes do not contain muscles, yet fish can change the shape of their fins with high precision and speed, while producing large hydrodynamic forces without collapsing. This remarkable performance has been intriguing researchers for decades, but experiments have so far focused on homogenized properties, and models were developed only for small deformations and small rotations. Here we present fully instrumented micromechanical tests on individual rays from Rainbow trout in both morphing and flexural deflection mode and at large deflections. We then present a nonlinear mechanical model of the ray that captures the key structural elements controlling the mechanical behavior of rays under large deformations, which we successfully fit onto the experiments for property identification. We found that the flexural stiffness of the mineralized layers in the rays (hemitrichs) is 5-6 times lower than their axial stiffness, an advantageous combination to produce stiff morphing. In addition, the collagenous core region can be modeled with spring elements which are 3-4 orders of magnitude more compliant than the hemitrichs. This fibrillar structure provides negligible resistance to shearing from the initial position, but it prevents buckling and collapse of the structure at large deformations. These insights from the experiments and nonlinear models can serve as new guidelines for the design of efficient bioinspired stiff morphing materials and structures at large deformations. STATEMENT OF SIGNIFICANCE: Fins from ray-finned fishes do not contain muscles, yet fish can change the shape of their fins with high precision and speed, while producing large hydrodynamic forces without collapsing. Experiments have so far focused on homogenized properties, and models were developed only for small deformations and small rotations providing limited insight into the rich nonlinear mechanics of natural rays. We present micromechanical tests in both morphing and flexural deflection mode on individual rays, a nonlinear model of the ray that captures the mechanical behavior of rays under large deformations and combine microCT measurements to generate new insights into the nonlinear mechanics of rays. These insights can serve as new guidelines for the design of efficient bioinspired stiff morphing materials and structures at large deformations.

Thyroid hormone regulates proximodistal patterning in fin rays.

Processes that regulate size and patterning along an axis must be highly integrated to generate robust shapes; relative changes in these processes underlie both congenital disease and evolutionary change. Fin length mutants in zebrafish have provided considerable insight into the pathways regulating fin size, yet signals underlying patterning have remained less clear. The bony rays of the fins possess distinct patterning along the proximodistal axis, reflected in the location of ray bifurcations and the lengths of ray segments, which show progressive shortening along the axis. Here, we show that thyroid hormone (TH) regulates aspects of proximodistal patterning of the caudal fin rays, regardless of fin size. TH promotes distal gene expression patterns, coordinating ray bifurcations and segment shortening with skeletal outgrowth along the proximodistal axis. This distalizing role for TH is conserved between development and regeneration, in all fins (paired and medial), and between Danio species as well as distantly related medaka. During regenerative outgrowth, TH acutely induces Shh-mediated skeletal bifurcation. Zebrafish have multiple nuclear TH receptors, and we found that unliganded Thrab-but not Thraa or Thrb-inhibits the formation of distal features. Broadly, these results demonstrate that proximodistal morphology is regulated independently from size-instructive signals. Modulating proximodistal patterning relative to size-either through changes to TH metabolism or other hormone-independent pathways-can shift skeletal patterning in ways that recapitulate aspects of fin ray diversity found in nature.

Proprioceptive wake classification by a body with a passive tail.

The remarkable ability of some marine animals to identify flow structures and parameters using complex non-visual sensors, such as lateral lines of fish and the whiskers of seals, has been an area of investigation for researchers looking to apply this ability to artificial robotic swimmers, which could lead to improvements in autonomous navigation and efficiency. Several species of fish in particular have been known to school effectively, even when blind. Beyond specialized sensors like the lateral lines, it is now known that some fish use purely proprioceptive sensing, using the kinematics of their fins or tails to sense their surroundings. In this paper we show that the kinematics of a body with a passive tail encode information about the ambient flow, which can be deciphered through machine learning. We demonstrate this with experimental data of the angular velocity of a hydrofoil with a passive tail that lies in the wake generated by an upstream oscillating body. Using convolutional neural networks, we show that with the kinematic data from the downstream body with a tail, the wakes can be better classified than in the case of a body without a tail. This superior sensing ability exists for a body with a tail, even if only the kinematics of the main body are used as input for the machine learning. This shows that beyond generating 'additional inputs', passive tails modulate the response of the main body in manner that is useful for hydrodynamic sensing. These findings have clear application for improving the sensing abilities of bioinspired swimming robots.

Flexibility is a hidden axis of biomechanical diversity in fishes.

Nearly all fish have flexible bodies that bend as a result of internal muscular forces and external fluid forces that are dynamically coupled with the mechanical properties of the body. Swimming is therefore strongly influenced by the body's flexibility, yet we do not know how fish species vary in their flexibility and in their ability to modulate flexibility with muscle activity. A more fundamental problem is our lack of knowledge about how any of these differences in flexibility translate into swimming performance. Thus, flexibility represents a hidden axis of diversity among fishes that may have substantial impacts on swimming performance. Although engineers have made substantial progress in understanding these fluid-structure interactions using physical and computational models, the last biological review of these interactions and how they give rise to fish swimming was carried out more than 20 years ago. In this Review, we summarize work on passive and active body mechanics in fish, physical models of fish and bioinspired robots. We also revisit some of the first studies to explore flexural stiffness and discuss their relevance in the context of more recent work. Finally, we pose questions and suggest future directions that may help reveal important links between flexibility and swimming performance.

Innervation and structure of the adipose fin of a lanternfish.

Adipose fins of teleost fishes have been shown to function as mechanosensory organs that respond to minute bending forces created by turbulence in fast-flowing streams. Nonetheless, adipose fins also exist in some fishes that occupy still waters, including lanternfish (Myctophidae) in the deep sea. The authors examined adipose fin structure in northern lampfish, Stenobrachius leucopsarus, from coastal British Columbia. After fixation, embedding and sectioning of the adipose and supporting tissue, it was evident that lanternfish adipose fins are stiffened by compound actinotrichia, acting like fin rays, that would create a higher aspect ratio. The actinotrichia converge at the base of the fin in a hinge point complex that anteriorly interacts with a cartilaginous endoskeletal rod, controlled by skeletal muscles. Afferent nerves enter the fin at this point and form fine branches as they track deeper alongside actinotrichia. The authors propose that the vertical nightly migration to surface waters, as well as predator evasion within large schools, results in microturbulence. In these circumstances, the adipose fin acts as a mechanosensor providing feedback to the caudal fin, as it occurs in salmonids and catfish.

Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses.

Successful regeneration requires the coordinated execution of multiple cellular responses to injury. In amputated zebrafish fins, mature osteoblasts dedifferentiate, migrate towards the injury, and form proliferative osteogenic blastema cells. We show that osteoblast migration is preceded by cell elongation and alignment along the proximodistal axis, which require actomyosin, but not microtubule (MT) turnover. Surprisingly, osteoblast dedifferentiation and migration can be uncoupled. Using pharmacological and genetic interventions, we found that NF-ĸB and retinoic acid signalling regulate dedifferentiation without affecting migration, while the complement system and actomyosin dynamics affect migration but not dedifferentiation. Furthermore, by removing bone at two locations within a fin ray, we established an injury model containing two injury sites. We found that osteoblasts dedifferentiate at and migrate towards both sites, while accumulation of osteogenic progenitor cells and regenerative bone formation only occur at the distal-facing injury. Together, these data indicate that osteoblast dedifferentiation and migration represent generic injury responses that are differentially regulated and can occur independently of each other and of regenerative growth. We conclude that successful fin bone regeneration appears to involve the coordinated execution of generic and regeneration-specific responses of osteoblasts to injury.

Bioinspired morphing fins to provide optimal maneuverability, stability, and response to turbulence in rigid hull AUVs.

By adopting bioinspired morphing fins, we demonstrate how to achieve good directional stability, exceptional maneuverability, and minimal adverse response to turbulent flow, properties that are highly desirable for rigid hull AUVs, but are presently difficult to achieve because they impose contradictory requirements. We outline the theory and design for switching between operating with sufficient stability that ensures a steady course in the presence of disturbances, with low corrective control action; reverting to high maneuverability to execute very rapid course and depth changes, improving turning rate by 25% up to 50%; and ensuring at all times that angular responses to external turbulence are minimized. We then demonstrate the developments through tests on a 1 m long autonomous underwater vehicle, namedMorpheus. The vehicle is capable of dynamically changing its stability-maneuverability qualities by using tuna-inspired morphing fins, which can be deployed, deflected and retracted, as needed. A series of free-swimming experiments and maneuvering simulations, combined with mathematical analysis, led to the design of optimal retractable morphing fins.

Zebrafish caudal fin as a model to investigate the role of probiotics in bone regeneration.

Probiotics are live microorganisms that confer several beneficial effects to the host, including enhancement of bone mineralization. However, probiotic action on bone regeneration is not well studied and therefore we analysed various effects of probiotic treatment on the caudal fin regeneration of zebrafish. Morphological analysis revealed an increased regenerated area with shorter and thicker lepidotrichia segments after probiotic treatment. Fourier transform infrared spectroscopy imaging analysis highlighted the distribution of phosphate groups in the regenerated fins and probiotic group showed higher amounts of well-crystallized hydroxyapatite. At the midpoint (5 days post amputation) of regeneration, probiotics were able to modulate various stages of osteoblast differentiation as confirmed by the upregulation of some key marker genes such as runx2b, sp7, col10a1a, spp1 and bglap, besides suppressing osteoclast activity as evidenced from the downregulation of ctsk. Probiotics also caused an enhanced cell cycle by regulating the expression of genes involved in Retinoic acid (rarga, cyp26b1) and Wnt/ß-catenin (ctnnb1, ccnd1, axin2, sost) signaling pathways, and also modulated phosphate homeostasis by increasing the entpd5a levels. These findings provide new outlooks for the use of probiotics as a prophylactic treatment in accelerating bone regeneration and improving skeletal health in both aquaculture and biomedical fields.

Osteoblasts pattern endothelium and somatosensory axons during zebrafish caudal fin organogenesis.

Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones and endothelium. In adult zebrafish, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development we show that the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.

An autonomously swimming biohybrid fish designed with human cardiac biophysics.

Biohybrid systems have been developed to better understand the design principles and coordination mechanisms of biological systems. We consider whether two functional regulatory features of the heart-mechanoelectrical signaling and automaticity-could be transferred to a synthetic analog of another fluid transport system: a swimming fish. By leveraging cardiac mechanoelectrical signaling, we recreated reciprocal contraction and relaxation in a muscular bilayer construct where each contraction occurs automatically as a response to the stretching of an antagonistic muscle pair. Further, to entrain this closed-loop actuation cycle, we engineered an electrically autonomous pacing node, which enhanced spontaneous contraction. The biohybrid fish equipped with intrinsic control strategies demonstrated self-sustained body-caudal fin swimming, highlighting the role of feedback mechanisms in muscular pumps such as the heart and muscles.

Changes in rays' swimming stability due to the phase difference between left and right pectoral fin movements.

Swimming motions of rays that swim using undulation locomotion are not always symmetrical; there may be a phase difference between the left and right pectoral fins. However, few studies on the swimming of rays have mentioned left and right pectoral fin movements. Moreover, the effects of movements of the left and right pectoral fins on swimming have not been clarified. This paper describes a computational study of phase differences of pectoral fin movements in the swimming of rays with the validity of fluid analysis methods. The movement and shape of the ray were made based on previous biological research and pictures. An overset grid was used to reproduce the ray's complex motions. The analysis was performed under four phase difference conditions: 0 [Formula: see text] ([Formula: see text] is the period), 0.25 [Formula: see text], 0.5 [Formula: see text], and 0.75 [Formula: see text]. The results show that a phase difference between the left and right pectoral fin movements affects swimming stability and maneuverability but not propulsive efficiency. We suggest that the phase difference in pectoral fin movements is essential for the swimming of rays, and rays adjust the phase difference between the movement of the left and right pectoral fins to suit their purpose.

Nuclear S-nitrosylation impacts tissue regeneration in zebrafish.

Despite the importance of nitric oxide signaling in multiple biological processes, its role in tissue regeneration remains largely unexplored. Here, we provide evidence that inducible nitric oxide synthase (iNos) translocates to the nucleus during zebrafish tailfin regeneration and is associated with alterations in the nuclear S-nitrosylated proteome. iNos inhibitors or nitric oxide scavengers reduce protein S-nitrosylation and impair tailfin regeneration. Liquid chromatography/tandem mass spectrometry reveals an increase of up to 11-fold in the number of S-nitrosylated proteins during regeneration. Among these, Kdm1a, a well-known epigenetic modifier, is S-nitrosylated on Cys334. This alters Kdm1a binding to the CoRest complex, thus impairing its H3K4 demethylase activity, which is a response specific to the endothelial compartment. Rescue experiments show S-nitrosylation is essential for tailfin regeneration, and we identify downstream endothelial targets of Kdm1a S-nitrosylation. In this work, we define S-nitrosylation as an essential post-translational modification in tissue regeneration.

High-speed camera recordings uncover previously unidentified elements of zebrafish mating behaviors integral to successful fertilization.

The mating behavior of teleost fish consists of a sequence of stereotyped actions. By observing mating of zebrafish under high-speed video, we analyzed and characterized a behavioral cascade leading to successful fertilization. When paired, a male zebrafish engages the female by oscillating his body in high frequency (quivering). In response, the female pauses swimming and bends her body (freezing). Subsequently, the male contorts his trunk to enfold the female's trunk. This behavior is known as wrap around. Here, we found that wrap around behavior consists of two previously unidentified components. After both sexes contort their trunks, the male adjusts until his trunk compresses the female's dorsal fin (hooking). After hooking, the male trunk slides away from the female's dorsal fin, simultaneously sliding his pectoral fin across the female's gravid belly, stimulating egg release (squeezing/spawning). Orchestrated coordination of spawning presumably increases fertilization success. Surgical removal of the female dorsal fin inhibited hooking and the transition to squeezing. In a neuromuscular mutant where males lack quivering, female freezing and subsequent courtship behaviors were absent. We thus identified traits of zebrafish mating behavior and clarified their roles in successful mating.

Modulation of bioelectric cues in the evolution of flying fishes.

Changes to allometry, or the relative proportions of organs and tissues within organisms, is a common means for adaptive character change in evolution. However, little is understood about how relative size is specified during development and shaped during evolution. Here, through a phylogenomic analysis of genome-wide variation in 35 species of flying fishes and relatives, we identify genetic signatures in both coding and regulatory regions underlying the convergent evolution of increased paired fin size and aerial gliding behaviors. To refine our analysis, we intersected convergent phylogenomic signatures with mutants with altered fin size identified in distantly related zebrafish. Through these paired approaches, we identify a surprising role for an L-type amino acid transporter, lat4a, and the potassium channel, kcnh2a, in the regulation of fin proportion. We show that interaction between these genetic loci in zebrafish closely phenocopies the observed fin proportions of flying fishes. The congruence of experimental and phylogenomic findings point to conserved, non-canonical signaling integrating bioelectric cues and amino acid transport in the establishment of relative size in development and evolution.

Evolution of touch and proprioception of the limbs: Insights from fish and humans.

The function of the hands is inextricably linked to cutaneous mechanosensation, both in touch and in how hand movement and posture (proprioception) are controlled. The structure and behavior of hands and distal forelimbs of other vertebrates have been evolutionarily shaped by these mechanosensory functions. The distal forelimb of tetrapod vertebrates is homologous to the pectoral fin rays and membrane of fishes. Fish fins demonstrate similar mechanosensory abilities to hands and other distal tetrapod forelimbs in touch and proprioception. These results indicate that vertebrates were using the core mechanosensory inputs, such as fast adapting and slow adapting nerve responses, to inform fin and limb function and behavior before their diversification in fish and tetrapod lineages.

Propolis Can Improve Caudal Fin Regeneration in Zebrafish (Danio rerio) Induced by The Combined Administration of Alloxan and Glucose.

Hyperglycemia, a primary symptom in diabetes mellitus, is associated with difficulties in wound healing and regeneration. This condition is due to the length of the inflammatory phase and free radicals. Furthermore, there is evidence that molecular pathogenesis is involved in impaired wound healing in diabetics. As an animal model, zebrafish have many shared orthologous genes with human that are involved in protein regulation of wound healing and regeneration. Little is known about natural drugs that may be used to treat complications of wound healing in diabetes. Propolis, however, is known to consist of various organic compounds such as phenols and flavonoids with antioxidant and anti-inflammatory activities. This research aims to study propolis' effect on caudal fin regeneration and relative expression of several genes belonging to Hedgehog, bone morphogenetic protein (BMP), and Wnt signaling hyperglycemic (HG) zebrafish. GC-MS analysis and antioxidant activity testing were performed on ethanolic extract of propolis (EEP). Caudal fin regeneration was analyzed using ImageJ; blood glucose levels were measured; and relative gene expression analysis of shha, igf2a, bmp2b, and col1a2 was performed by the real-time polymerase chain reaction method with the ß-actin housekeeping gene. Impairment of caudal fin regeneration in zebrafish hyperglycemia was characterized by a low percentage of regeneration and decreased relative gene expression. EEP at 15 ppm could increase the percentage of caudal fin regeneration and the expression of shha, igf2a, bmp2b, and col1a2. Based on the results, it appears that phenols and flavonoids from the EEP can improve the caudal fin regeneration of HG zebrafish.