Peter Konstantinidis

Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution.

Convergent evolution in mechanical design of lamnid sharks and tunas

The evolution of ‘thunniform’ body shapes in several different groups of vertebrates, including whales, ichthyosaurs and several species of large pelagic fishes supports the view that physical and hydromechanical demands provided important selection pressures to optimize body design for locomotion during vertebrate evolution. Recognition of morphological similarities between lamnid sharks (the most well known being the great white and the mako) and tunas has led to a general expectation that they also have converged in their functional design; however, no quantitative data exist on the mechanical performance of the locomotor system in lamnid sharks. Here we examine the swimming kinematics, in vivo muscle dynamics and functional morphology of the force-transmission system in a lamnid shark, and show that the evolutionary convergence in body shape and mechanical design between the distantly related lamnids and tunas is much more than skin deep; it extends to the depths of the myotendinous architecture and the mechanical basis for propulsive movements. We demonstrate that not only have lamnids and tunas converged to a much greater extent than previously known, but they have also developed morphological and functional adaptations in their locomotor systems that are unlike virtually all other fishes.

Genetic lineage labeling in zebrafish uncovers novel neural crest contributions to the head, including gill pillar cells

At the protochordate-vertebrate transition, a new predatory lifestyle and increased body size coincided with the appearance of a true head. Characteristic innovations of this head are a skull protecting and accommodating a centralized nervous system, a jaw for prey capture and gills as respiratory organs. The neural crest (NC) is a major ontogenetic source for the ‘new head’ of vertebrates and its contribution to the cranial skeleton has been intensively studied in different model organisms. However, the role of NC in the expansion of the respiratory surface of the gills has been neglected. Here, we use genetic lineage labeling to address the contribution of NC to specific head structures, in particular to the gills of adult zebrafish. We generated a sox10:ERT2-Cre line and labeled NC cells by inducing Cre/loxP recombination with tamoxifen at embryonic stages. In juvenile and adult fish, we identified numerous established NC derivatives and, in the cranium, we precisely defined the crest/mesoderm interface of the skull roof. We show the NC origin of the opercular bones and of multiple cell types contributing to the barbels, chemosensory organs located in the mouth region. In the gills, we observed labeled primary and secondary lamellae. Clonal analysis reveals that pillar cells, a craniate innovation that mechanically supports the filaments and forms gill-specific capillaries, have a NC origin. Our data point to a crucial role for the NC in enabling more efficient gas exchange, thus uncovering a novel, direct involvement of this embryonic tissue in the evolution of respiratory systems at the protochordate-vertebrate transition.

Patterns of red muscle strain/activation and body kinematics during steady swimming in a lamnid shark, the shortfin mako (Isurus oxyrinchus).

SUMMARY The dynamics of steady swimming were examined in the shortfin mako (Isurus oxyrinchus), a member of the cartilaginous fish family Lamnidae, a family known for their morphological adaptations for high-performance locomotion and their similarity in hydromechanical design to tunas. Patterns of red muscle (RM) strain (i.e. relative length change) and activation were quantified at two axial positions (∼0.4 and 0.6L, where L is total body length), using sonomicrometry and electromyography (EMG), and correlated with simultaneous measurements of dorsal midline kinematics during steady swimming (∼0.5–1 L s–1). RM strain varied longitudinally with strain amplitudes ranging from 5.5±1.1% (s.e.m.) in the anterior to 8.7±0.9% in the posterior. We found no significant longitudinal variation in patterns of RM activation, with mean onset of activation occurring at 83–84° (90° is peak length) and offset at 200–210° at both body positions. Likewise, duty cycles were similar: 35.5±1.0% in the anterior and 32.2±1.6% in the posterior. Comparison of the timing of waves of dorsal midline curvature and predicted strain relative to measured RM strain revealed a phase shift between RM shortening and local body bending. Furthermore, when the body is bent passively, RM shortens synchronously with the surrounding white muscle (WM) and skin, as expected. During active swimming, peaks in RM strain were delayed relative to peaks in WM strain by a mean of ∼10% of the tailbeat cycle, with one individual as high as ∼17% in the anterior and nearly 50% in the posterior. The longitudinal consistency in the EMG/strain phase relationship in the mako is similar to that in the leopard shark, suggesting a consistent trend among sharks using different locomotor modes. However, unlike in the leopard shark, RM shortening in the mako is physically uncoupled from deformation of the surrounding body during steady swimming, a characteristic shared between the mako and tunas.

Same but different: ontogeny and evolution of the Musculus adductor mandibulae in the Tetraodontiformes

The morphological diversity of fishes provides a rich source to address questions regarding the evolution of complex and novel forms. The Tetraodontiformes represent an order of highly derived teleosts including fishes, such as the pelagic ocean sunfishes, triggerfishes, and pufferfishes. This makes the order attractive for comparative analyses to understand the role of development in generating new forms during evolution. The adductor mandibulae complex, the main muscle associated with jaw closure, represents an ideal model system within the Tetraodontiformes. The adductor mandibulae differs in terms of partitions and their attachment sites between members of the different tetraodontiform families. In order to understand the evolution of the jaws among the Tetraodontiformes, we investigate the development of the adductor mandibulae in pufferfishes and triggerfishes as representatives of two different suborders (Balistoidei and Tetraodontoidei) that follows two different adaptations to a durophagous feeding mode. We show that the varied patterns of the adductor mandibulae derive from similar developmental sequence of subdivision of the partitions. We propose a conserved developmental program for partitioning of the adductor mandibulae as a foundation for the evolution of different patterns of subdivisions in Tetraodontiformes. Furthermore, we argue that derived conditions in the higher taxa are realized by supplementary subdivisions and altered attachment sites. These findings support a reinterpretation of homology of different muscle partitions among the Tetraodontiformes, as muscle partitions previously thought to be disparate, are now clearly related.

The Developmental Pattern of the Musculature Associated with the Mandibular and Hyoid Arches in the Longnose Gar, Lepisosteus osseus (Actinopterygii, Ginglymodi, Lepisosteiformes)

This is the first in a planned series of studies in which we examine the cranial muscle ontogeny of exemplar taxa of actinopterygian clades to obtain a better understanding of the evolution of the cranial musculoskeletal system within the Actinopterygii. The Longnose Gar, Lepisosteus osseus, is a member of the basal actinopterygian family Lepisosteidae. Juvenile and adult gars are highly derived and anatomical characters can easily be misinterpreted, which makes a comparison with other taxa difficult. Highly complex adult structures such as the cranial skeleton and musculature are organized more simply early in development, making comparisons and homology assessments easier. Established methods such as clearing and double staining are widely used to study skeletal structures. However, methods to analyze the ontogeny of soft tissues are scarce. To study the development of the cranial musculature of L. osseus, we used a combination of 3D-reconstruction of soft tissue &mgr;CT scans and whole-mount antibody staining. The elongation of the palatoquadrate and the dentary that form the long snout of gars begins late in ontogeny. However, the adductor mandibulae complex, which separates from the mandibular muscle primordium initially as a single portion, does not follow the extension of the palatoquadrate. We also show that the ceratomandibular ligament that attaches the ceratohyal with the retroarticular process of the lower jaw is homologous with the mandibulohyoid ligament.

When Tradition Meets Technology: Systematic Morphology of Fishes in the Early 21st Century

Many of the primary groups of fishes currently recognized have been established through an iterative process of anatomical study and comparison of fishes that has spanned a time period approaching 500 years. In this paper we give a brief history of the systematic morphology of fishes, focusing on some of the individuals and their works from which we derive our own inspiration. We further discuss what is possible at this point in history in the anatomical study of fishes and speculate on the future of morphology used in the systematics of fishes. Beyond the collection of facts about the anatomy of fishes, morphology remains extremely relevant in the age of molecular data for at least three broad reasons: 1) new techniques for the preparation of specimens allow new data sources to be broadly compared; 2) past morphological analyses, as well as new ideas about interrelationships of fishes (based on both morphological and molecular data) provide rich sources of hypotheses to test with new morphological investigations; and 3) the use of morphological data is not limited to understanding phylogeny and evolution of fishes, but rather is of broad utility to understanding the general biology (including phenotypic adaptation, evolution, ecology, and conservation biology) of fishes. Although in some ways morphology struggles to compete with the lure of molecular data for systematic research, we see the anatomical study of fishes entering into a new and exciting phase of its history because of recent technological and methodological innovations. With each new advance of technology and with each new generation of researcher, systematic morphology becomes a new and vibrant science.

Parallelism and epistasis in skeletal evolution identified through use of phylogenomic mapping strategies

The identification of genetic mechanisms underlying evolutionary change is critical to our understanding of natural diversity, but is presently limited by the lack of genetic and genomic resources for most species. Here, we present a new comparative genomic approach that can be applied to a broad taxonomic sampling of nonmodel species to investigate the genetic basis of evolutionary change. Using our analysis pipeline, we show that duplication and divergence of fgfr1a is correlated with the reduction of scales within fishes of the genus Phoxinellus. As a parallel genetic mechanism is observed in scale-reduction within independent lineages of cypriniforms, our finding exposes significant developmental constraint guiding morphological evolution. In addition, we identified fixed variation in fgf20a within Phoxinellus and demonstrated that combinatorial loss-of-function of fgfr1a and fgf20a within zebrafish phenocopies the evolved scalation pattern. Together, these findings reveal epistatic interactions between fgfr1a and fgf20a as a developmental mechanism regulating skeletal variation among fishes.

The Median-Fin Skeleton of the Eastern Atlantic and Mediterranean Clingfishes Lepadogaster lepadogaster (Bonnaterre) and Gouania wildenowi (Risso) (Teleostei: Gobiesocidae)

Previous research on the osteology of the Gobiesocidae focused mostly on the neurocranium and the thoracic sucking disc (formed by the paired‐fin girdles). Little attention has been paid to the skeleton of the median fins. The dorsal‐ and anal‐fin skeleton of Lepadogaster lepadogaster and other gobiesocids (excluding Alabes, which lacks these fins) are characterized by the absence of spines, branched fin‐rays, and middle radials. In gobiesocids, the distal radials never ossify and consist of elastic hyaline‐cell cartilage. Gouania wildenowi is unique among gobiesocids in having further reductions of the dorsal‐ and anal‐fin skeleton, including a notable decrease in the size of the proximal‐middle radials in an anterior–posterior direction. Unlike L. lepadogaster, which exhibits a one‐to‐one relationship between the dorsal‐ and anal‐fin rays and proximal‐middle radials, G. wildenowi has a higher number of proximal‐middle radials than distal radial cartilages and fin rays in the dorsal and anal fins. In G. wildenowi, the dorsal‐ and anal‐fin rays do not articulate with the distal tip of the proximal‐middle radials but are instead positioned between proximal‐middle radials, which is unusual for teleosts. Previously unrecognized dorsal and ventral pads of elastic hyaline‐cell cartilage are also present in the caudal skeleton of L. lepadogaster, G. wildenowi, and all other gobiesocids examined. J. Morphol., 2010.

High-proof Ethanol Fixation of Larval and Juvenile Fishes for Clearing and Double Staining

The fixation of larval and juvenile fishes in 95% ethanol can be substituted for formalin and bears several advantages for morphological and molecular studies: 1) specimens clear and double stain rapidly and brilliantly; 2) otoliths are preserved; and 3) high-quality DNA is available from the tissues. We present merits and limitations of 70% ethanol and 95% ethanol as alternative fixatives to 4% buffered formalin. In particular, we compare our results of clearing and double staining teleost larvae and juveniles from these three fixatives and those that have been frozen at −20°C prior to the initial fixation. With our results, we can refute the long-standing notion that ethanol-fixed specimens disintegrate during clearing and staining.