Can mechanical forces be responsible for novel bone development and evolution in fishes?

Mechanical forces influence the induction, growth and maintenance of the vertebrate skeleton. Using the zebrafish, Danio rerio, we explore the hypothesis that mechanical forces can ultimately lead to the generation of skeletal evolutionary novelties by modifications of the mechano‐responsive molecular pathways. Locomotion and feeding in zebrafish larvae begin early in ontogeny and it is likely that forces incurred during these behaviours affect subsequent skeletal development. We provide two case studies in which our hypothesis is being tested: the kinethmoid and intermuscular bones. The kinethmoid is a synapomorphy for the order Cypriniformes and is intricately linked to the bones of the protrusible upper jaw. It undergoes chondrogenesis within a ligament well after muscular forces are present within the head. Subsequent ossification of the kinethmoid occurs at sites of ligamentous attachment, leading us to believe that mechanical forces are involved. Unlike the kinethmoid, which has evolved only once, intermuscular bones have evolved several times during teleostean evolution. Intermuscular bones are embedded within the myosepta, the collagenous sheets between axial muscles. The effect of mechanical forces on the development of these intermuscular bones is experimentally tested by increasing the viscosity of the water in which larval zebrafish are raised. Since locomotion in high viscosity requires greater muscular forces, we can directly test the influence of mechanical forces on the development of intermuscular bones. Using developmental techniques paired with outgroup comparison for the kinethmoid, and direct experimentation for intermuscular bones, our case studies provide complementary insights into the effects of mechanical forces on the evolution of skeletal novelties in fishes.     

Development of the escape response in teleost fishes: do ontogenetic changes enable improved performance?

Teleost fishes typically first encounter the environment as free-swimming embryos or larvae. Larvae are morphologically distinct from adults, and major anatomical structures are unformed. Thus, larvae undergo a series of dramatic morphological changes until they reach adult morphology (but are reproductively immature) and are considered juveniles. Free-swimming embryos and larvae are able to perform a C-start, an effective escape response that is used evade predators. However, escape response performance improves during early development: as young fish grow, they swim faster (length-specific maximum velocity increases) and perform the escape more rapidly (time to complete the behavior decreases). These improvements cease when fish become juveniles, although absolute swimming velocity (m s(-1)) continues to increase. We use studies of escape behavior and ontogeny in California halibut (Paralichthys californicus), rainbow trout (Oncorhynchus mykiss), and razorback suckers (Xyrauchen texanus) to test the hypothesis that specific morphological changes improve escape performance. We suggest that formation of the caudal fin improves energy transfer to the water and therefore increases thrust production and swimming velocity. In addition, changes to the axial skeleton during the larval period produce increased axial stiffness, which in turn allows the production of a more rapid and effective escape response. Because escape performance improves as adult morphology develops, fish that enter the environment in an advanced stage of development (i.e., those with direct development) should have a greater ability to evade predators than do fish that enter the environment at an early stage of development (i.e., those with indirect development).     

Ectodermal P2X receptor function plays a pivotal role in craniofacial development of the zebrafish

P2X receptors are non-selective cation channels operated by extracellular ATP. Currently, little is known concerning the functions of these receptors during development. Previous work from our lab has shown that zebrafish have two paralogs of the mammalian P2X3 receptor subunit. One paralog, p2rx3.1, is expressed in subpopulations of neural and ectodermal cells in the embryonic head. To investigate the role of this subunit in early cranial development, we utilized morpholino oligonucleotides to disrupt its translation. Loss of this subunit resulted in craniofacial defects that included malformation of the pharyngeal skeleton. During formation of these structures, there was a marked increase in cell death within the branchial arches. In addition, the epibranchial (facial, glossopharyngeal, and vagal) cranial sensory ganglia and their circuits were perturbed. These data suggest that p2rx3.1 function in ectodermal cells is involved in purinergic signaling essential for proper craniofacial development and sensory circuit formation in the embryonic and larval zebrafish.     

Endogenous rhythms and entrainment cues of larval activity in the horseshoe crab Limulus polyphemus

The American horseshoe crab, Limulus polyphemus (Linnaeus), typically inhabits estuaries and coastal areas with pronounced semi-diurnal and diurnal tides that are used to synchronize the timing of spawning, larval hatching, and emergence. Horseshoe crabs spawn in the intertidal zone of sandy beaches and larval emergence occurs when the larvae exit the sediments and enter the plankton. However, L. polyphemus populations also occur in areas that lack significant tidal changes and associated synchronization cues. Endogenous activity rhythms that match predictable environmental cycles may enable larval horseshoe crabs to time swimming activity to prevent stranding on the beach. To determine if L. polyphemus larvae possess a circatidal rhythm in vertical swimming, larvae collected from beach nests and the plankton were placed under constant conditions and their activity monitored for 72 h. Time-series analyses of the activity records revealed a circatidal rhythm with a free-running period of ≈ 12.5 h. Maximum swimming activity consistently occurred during the time of expected falling tides, which may serve to reduce the chance of larvae being stranded on the beach and aid in seaward transport by ebb currents (i.e., ebb-tide transport). To determine if agitation serves as the entrainment cue, larvae were shaken on a 12.4 h cycle to simulate conditions during high tide in areas with semi-diurnal tides. When placed under constant conditions, larval swimming increased near the expected times of agitation. Thus, endogenous rhythms of swimming activity of L. polyphemus larvae in both tidal and nontidal systems may help synchronize swimming activity with periods of high water and inundation.     

Development of zebrafish (Danio rerio) pectoral fin musculature

During posthatching development the fins of fishes undergo striking changes in both structure and function. In this article we examine the development of the pectoral fins from larval through adult life history stages in the zebrafish (Danio rerio), describing in detail their pectoral muscle morphology. We explore the development of muscle structure as a way to interpret the fins' role in locomotion. Genetic approaches in the zebrafish model are providing new tools for examining fin development and we take advantage of transgenic lines in which fluorescent protein is expressed in specific tissues to perform detailed three-dimensional, in vivo fin imaging. The fin musculature of larval zebrafish is organized into two thin sheets of fibers, an abductor and adductor, one on each side of an endoskeletal disk. Through the juvenile stage the number of muscle fibers increases and muscle sheets cleave into distinct muscle subdivisions as fibers orient to the developing fin skeleton. By the end of the juvenile period the pectoral girdle and fin muscles have reoriented to take on the adult organization. We find that this change in morphology is associated with a switch of fin function from activity during axial locomotion in larvae to use in swim initiation and maneuvering in adults. The examination of pectoral fins of the zebrafish highlights the yet to be explored diversity of fin structure and function in subadult developmental stages. J. Morphol. (c) 2005 Wiley-Liss, Inc.     

Skeletal development in the fossorial gymnophthalmids Calyptommatus sinebrachiatus and Nothobachia ablephara

The development of the cartilaginous and bony elements that form the skull and axial and appendicular skeleton is described in detail for the post-ovipositional embryonic development of the fossorial gymnophthalmid species Calyptommatus sinebrachiatus and Nothobachia ablephara. Both species have a snake-like morphology, showing an elongated body and reduced or absent limbs, as well as modifications in skull bones for burrowing, such as complex articulation surfaces and development of bony extensions that enclose and protect the brain. Similar morphological changes have originated independently in several squamate groups, including the one that led to the snake radiation. This study characterizes the patterns of chondrogenesis and osteogenesis, with special emphasis on the features associated with the burrowing habit, and may be used for future comparative analyses of the developmental patterns involved in the origin of the convergent serpentiform morphologies.     

Swimming performance in early development and the "other" consequences of egg size for ciliated planktonic larvae

The evolutionary significance of egg size in marine invertebrates is commonly perceived in energetic terms. Embryonic size should also have direct effects upon the forces that govern swimming, a behavior common to early larval development in the plankton. If swimming is ecologically important, early larvae may need to perform to a certain "standard", or threshold of speed and/or stability. The existence of performance standards in early development could therefore act to constrain the evolution of egg size and the evolution of development. Here we present the key parameters that characterize the upward swimming speed of ciliated spheroidal larvae moving at very low Reynolds numbers. The dependence of maximum supported mass upon larval size, and the independence of neutral-weight swimming speed from size, lead to hypotheses about scaling of swimming speed with size. Experimental studies with thirteen broadcast-spawning planktotrophs demonstrate that free-living embryonic swimmers in all of these species conform to a strong negative scaling of density with size that offsets increases in mass with increasing size. This trend suggests that swimming ability is broadly under selection in early development. In experimental studies and in a hydrodynamic model of larval swimming, the performance of trochophore larvae provides support for our hypothesized scaling relationships, and also for the concept of a standard in swimming speed. Echinoid blastulae, however, show relationships between speed and size that are not predicted by our scaling arguments. Results for echinoids suggest that differences in ciliary tip speed, or possibly in spatial density of cilia over the blastula's surface, result in significant differences in species' performance. Strong phyletic differences in the initial patterning and growth of structures used for swimming thus appear to cause significant differences in the relationship of swimming ability with embryo size.     

Swimming Speed of Larval Snail Does Not Correlate with Size and Ciliary Beat Frequency

Many marine invertebrates have planktonic larvae with cilia used for both propulsion and capturing of food particles. Hence, changes in ciliary activity have implications for larval nutrition and ability to navigate the water column, which in turn affect survival and dispersal. Using high-speed high-resolution microvideography, we examined the relationship between swimming speed, velar arrangements, and ciliary beat frequency of freely swimming veliger larvae of the gastropod Crepidula fornicata over the course of larval development. Average swimming speed was greatest 6 days post hatching, suggesting a reduction in swimming speed towards settlement. At a given age, veliger larvae have highly variable speeds (0.8–4 body lengths s⁻¹) that are independent of shell size. Contrary to the hypothesis that an increase in ciliary beat frequency increases work done, and therefore speed, there was no significant correlation between swimming speed and ciliary beat frequency. Instead, there are significant correlations between swimming speed and visible area of the velar lobe, and distance between centroids of velum and larval shell. These observations suggest an alternative hypothesis that, instead of modifying ciliary beat frequency, larval C. fornicata modify swimming through adjustment of velum extension or orientation. The ability to adjust velum position could influence particle capture efficiency and fluid disturbance and help promote survival in the plankton.     

Shh and Fgf8 act synergistically to drive cartilage outgrowth during cranial development

Much of the skeleton and connective tissue of the vertebrate head is derived from cranial neural crest. During development, cranial neural crest cells migrate from the dorsal neural tube to populate the forming face and pharyngeal arches. Fgf8 and Shh, signaling molecules known to be important for craniofacial development, are expressed in distinct domains in the developing face. Specifically, in chick embryos these molecules are expressed in adjacent but non-overlapping patterns in the epithelium covering crest-derived mesenchyme that will give rise to the skeletal projections of the upper and lower beaks. It has been suggested that these molecules play important roles in patterning the developing face. Here, we directly examine the ability of FGF8 and SHH signaling, singly and in combination, to regulate cranial skeletogenesis, both in vitro and in vivo. We find that SHH and FGF8 have strong synergistic effects on chondrogenesis in vitro and are sufficient to promote outgrowth and chondrogenesis in vivo, suggesting a very specific role for these molecules in producing the elongated beak structures during chick facial development.     

Early development of the postcranial skeleton of the pikeperch Sander lucioperca (Teleostei: Percidae) relating to developmental stages and growth

The early development of the postcranial skeleton (pectoral girdle, pelvic girdle, vertebral column and fins) in pikeperch (Sander lucioperca (L.)) was studied from hatching to days 47 and 43 post fertilization (dpf) at two different rearing temperatures, 15.5 and 18.0°C. Four embryonic and six larval stages were described, ranging from 3.4 ± 0.3 mm to 21.8 ± 2.1 mm in total length. The crucial point in larval development is swimbladder inflation, which enables larvae to swim energy efficiently. Until this time point, only the most essential skeletal elements to enable swimming movements have developed. As the larvae become neutrally buoyant, they grow and differentiate postcranial elements rapidly. Concurrently, swimming performance and foraging success seems to improve. A specific size is correlated with a distinct developmental stage defined by a set of traits that includes the skeletal elements. The developmental sequence of skeletal structures is temperature independent, although growth is slower and the individual developmental stages are reached later at 15.5°C than at 18.0°C. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Developmental plasticity in the cardiovascular system of fish,with special reference to the zebrafish

During development the circulatory system of vertebrates typically starts operating earlier than any other organ. In these early stages, however, blood flow is not yet linked to metabolic requirements of tissues, as is well established for adults. While the autonomic nervous system becomes functional only quite late during development, in the early stages control of blood flow appears to be possible by blood-borne and/or local hormones. This study presents methods based on video-imaging techniques and fluorescence microscopy to visualize cardiac activity, as well as the vascular bed of developing lower vertebrates, and tests the idea that environmental factors, such as hypoxia, may modify cardiac activity, or even the early formation of blood vessels in embryos and larvae. In zebrafish larvae, adaptations of cardiovascular activity to chronic hypoxia become visible shortly after hatching, and the formation of some blood vessels is enhanced under chronic hypoxia. Exposure of early larval stages of zebrafish to a constant water current induces physiological adaptations, resulting in enhanced swimming efficiency and increased tolerance towards hypoxia. Furthermore, application of hormones such as NO can modify cardiac activity as well as peripheral resistance, and they can stimulate blood vessel formation. In consequence, even during early development of fish or amphibian larvae, the performance of cardiac muscle and of skeletal muscle can be modified by environmental influences and peripheral resistance can be adjusted. Even blood vessel formation can be stimulated by hypoxia, for example, or by the presence of specific hormones. Thus, at approximately the time of hatching the physiological performance of vertebrate larvae is already determined by the combined action of environmental influences and of genetic information.     

Energetics of larval swimming and metamorphosis in four species of Bugula (Bryozoa)

The amount of energy available to larvae during swimming, location of a suitable recruitment site, and metamorphosis influences the length of time they can spend in the plankton. Energetic parameters such as swimming speed, oxygen consumption during swimming and metamorphosis, and elemental carbon and nitrogen content were measured for larvae of four species of bryozoans, Bugula neritina, B. simplex, B. stolonifera, and B. turrita. The larvae of these species are aplanktotrophic with a short free-swimming phase ranging from less than one hour to a maximum of about 36 hours. There is about a fivefold difference in larval volume among the four species, which scales linearly with elemental carbon content and, presumably, with the amount of endogenous reserves available for swimming and metamorphosis. Mean larval swimming speeds (in centimeters per second) were similar among species. Specific metabolic rate and larval size were inversely related. For larvae of a given species, respiration rates remained similar for swimming and metamorphosis; however, because metamorphosis lasts about twice as long as a maximal larval swimming phase, it was more energetically demanding. Larger larvae expended more energy to complete metamorphosis than did smaller larvae, but in terms of the percentage of larval energy reserves consumed, swimming and metamorphosis were more "expensive" for smaller larvae. A comparison of the energy expended during larval swimming calculated on the basis of oxygen consumption and on the basis of elemental carbon decrease suggests that larvae of Bugula spp. may not use significant amounts of dissolved organic material (DOM) to supplement their endogenous energy reserves.     

Development of mandibular,hyoid and hypobranchial muscles in the zebrafish: homologies and evolution of these muscles within bony fishes and tetrapods

Background  

During vertebrate head evolution, muscle changes accompanied radical modification of the skeleton. Recent studies have suggested that muscles and their innervation evolve less rapidly than cartilage. The freshwater teleostean zebrafish (Danio rerio) is the most studied actinopterygian model organism, and is sometimes taken to represent osteichthyans as a whole, which include bony fishes and tetrapods. Most work concerning zebrafish cranial muscles has focused on larval stages. We set out to describe the later development of zebrafish head muscles and compare muscle homologies across the Osteichthyes.     

Loss and gain of the juvenile rudiment and metamorphic competence during starvation and feeding of bryozoan larvae

SUMMARY In many animals, larval structures and juvenile rudiments develop independently. One advantage of this independence is that juvenile rudiments can be expended as a nutrient reserve or for energy conservation. When bryozoan cyphonautes larvae were starved, structures required for settlement and metamorphosis shrank. When the larvae were again fed, these structures grew back. Starvation reduced the size of both the internal sac, a rudiment of postlarval juvenile structures, and the pyriform organ, which functions in sensing and crawling on the substratum at settlement. In contrast, starvation affected neither the size of the larval shell nor the lengths of the ciliary bands used in swimming and feeding. Starved larvae that had reduced the pyriform organ and internal sac did not metamorphose in response to stimuli from a laminarian alga. The laminarian alga did stimulate metamorphosis of the same larvae after renewed feeding, when the larvae had regrown these structures. Thus starved larvae expended body parts needed for settlement and metamorphosis when food was scarce while retaining structures for feeding, swimming, and defense. Starved larvae thereby retained the capacity to regrow structures needed for settlement and metamorphosis when they again encountered food. Advantages from expendable juvenile rudiments may enhance selection for their being developmentally distinct from structures for larval swimming and feeding.     

Altered timing of the extracellular-matrix-mediated epithelial-mesenchymal interaction that initiates mandibular skeletogenesis in three inbred strains of mice: development, heterochrony, and evolutionary change in morphology

Subtle changes in embryonic development are a source of significant morphological alterations during evolution. The mammalian mandibular skeleton, which originates from the cranial neural crest, is a complex structure comprising several components that interact late in embryogenesis to produce a single functional unit. It provides a model system in which individual developmental events at the basis of population-level evolutionary change can be investigated experimentally. Inbred mouse strains exhibit obvious morphological differences despite the relatively short time since their divergence from one another. Some of these differences can be traced to small changes in the timing of early developmental events such as the formation of the cellular condensations that initiate skeletogenesis. This paper examines an even earlier event for changes in timing, the epithelial-mesenchymal interaction(s) required to initiate chondrogenesis of Meckel's cartilage and osteogenesis of the dentary bone. Using three inbred strains of mice (CBA, C3H and C57) we found that, within each strain, cartilage and bone are induced at the same time and by the same (mandibular) epithelium, that chondrogenesis and osteogenesis are initiated by a matrix-mediated epithelial-mesenchymal interaction, and that timing of the interactions differs among the three inbred strains. These results are discussed with respect to the possible molecular basis of such temporal shifts in inductive interactions and how such studies can be used to shed light on heterochrony as a mechanism of evolutionary change in morphology.     

The development of an artificial hybrid and revealing elements of reproductive isolation between sympatric forms of Dryagin’s char and <Emphasis Type="Italic">Salvelinus alpinus</Emphasis> complex (Salmonidae) from Sobachye Mountain Lake (Taimyr)

The embryonic-larval development (at 3°C) of a hybrid between sympatric lake forms of Arctic char Salvelinus alpinus complex (large females of Dryagin’s char x male goggle-eyed char) from the polar mountain Lake Sobachye (the Norilo-Pyasina water system, Taimyr) was studied. No morphological disturbances were found in the embryonic membrane during development. After hatching, because of yolk sac soaking and blood circulation disturbances along its vessels, about 15% of specimens perished. The most mass disturbances in the development of free embryos and larvae that were manifested in the body curvature, loss of mobility, and subsequent death of a greater proportion of specimens were caused by modifications of differentiation of mesenchymal cells responsible for the axial skeleton formation. It is suggested that anomalies in osteogenesis and a high variation of age when dermal cranial bones (praeoperculum and frontale) appear that bear seismosensory system canal are related to the conflict of genetic programs of the development of parental forms. The rate of embryonic larval development of hybrid specimens exceeds that in the lake small form of Arctic char from Lake Davatchan (Transbaikalia) reared under the same conditions. The reproductive isolation of sympatric Dryagin’s char and goggle-eyed char in Lake Sobachye is provided by both pre- and postcopulation mechanisms. A possible mechanism of formation of reproductive isolation of sympatric forms related to colonization by them of a deep-water biotope is discussed.     

Vestigial ophiopluteal structures in the lecithotrophic larvae of Ophionereis schayeri (Ophiuroidea)

Evolution of echinoderm development from a feeding to a non-feeding mode can be examined by studying non-feeding larvae with structures that appear to be vestiges derived from a feeding ancestral state. The lecithotrophic larvae of the Australian brittle star Ophionereis schayeri possess such features, and the early development of this species was documented by light and scanning electron microscopy. The embryos undergo irregular cleavage, resulting in the formation of different sized blastomeres, with subsequent development through a wrinkled blastula stage. The lecithotrophic larva of O. schayeri possesses several vestigial ophiopluteal structures, including a continuous ciliated band, a larval gut, and a larval skeleton. The ciliated band is a reduced expression of the continuous ciliated band typical of ophioplutei. The larval gut is a transiently complete system, but an esophageal plug and rapid closure of the blastopore renders it nonfunctional. The larval skeleton, though reduced, consists of four rods corresponding to the body, posterolateral, anterolateral, and postoral rods characteristic of an ophiopluteus. Due to a heterochrony in larval skeletogenesis, the postoral rods develop early and simultaneously with the other rods. Compared with the larvae of other lecithotrophic ophiuroids, the larva of O. schayeri is one of the most reduced ophiopluteal forms reported to date.     

Vitellogenin 1 is essential for fish reproduction by transporting DHA-containing phosphatidylcholine from liver to ovary

Vitellogenins (Vtgs) are essential for female reproduction in oviparous animals, yet the exact roles and mechanisms remain unknown. In the present study, we knocked out vtg1, which is the most abundant Vtg in zebrafish, Danio rerio via the CRISPR/Cas 9 technology. We aimed to identify the roles of Vtg1 and related mechanisms in reproduction and development. We found that, the Vtg1-deficient female zebrafish reduced gonadosomatic index, egg production, yolk granules and mature follicles in ovary compared to the wide type (WT). Moreover, the Vtg1-deficient zebrafish diminished hatching rates, cumulative survival rate, swimming capacity and food intake, but increased malformation rate, and delayed swim bladder development during embryo and early-larval phases. The Vtg1-deficiency in female broodstock inhibited docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) transportation from liver to ovary, which lowered DHA-PC content in ovary and offspring during larval stage. However, the Vtg1-deficient zebrafish increased gradually the total DHA-PC content via exogeneous food intake, and the differences in swimming capacity and food intake returned to normal as they matured. Furthermore, supplementing Vtg1-deficient zebrafish with dietary PC and DHA partly ameliorated the impaired female reproductive capacity and larval development during early phases. This study indicates that, DHA and PC carried by Vtg1 are crucial for female fecundity, and affect embryo and larval development through maternal-nutrition effects. This is the first study elucidating the nutrient and physiological functions of Vtg1 and the underlying biochemical mechanisms in fish reproduction and development.