Novel developmental bases for the evolution of hypobranchial muscles in vertebrates.

BACKGROUND: Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx1, originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic. RESULTS: Here we characterize the expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark (gnathostome; jawed vertebrates). We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A, do not resemble the somitic expression of mammalian Lbx1. Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A, whereas Lbx1 marks the rostral HBM and pectoral fin muscle. CONCLUSIONS: We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of Lbx genes would have been a prerequisite.

In vitro characterisation of calcium influx across skin and gut epithelia of the Pacific hagfish, Eptatretus stoutii.

Although hagfishes osmoconform, concentrations of calcium in their extracellular fluids are maintained at levels lower than those of seawater. Ultimately, calcium homeostasis is a product of relative rates of influx and efflux into the animal, but little is known regarding these processes in hagfish. Using in vitro approaches, calcium influx across gut and skin epithelia of the Pacific hagfish, Eptatretus stoutii, was characterised. Analysis of concentration-dependent kinetics showed that accumulation into skin tissue was linear at mucosal calcium concentrations up to 10 mM. However, movement into the serosal compartment was saturable, with a maximal transport rate of 59 nmol cm-2 h-1 and an affinity (substrate concentration to give half maximal transport rate) of 1139 µM. Calcium accumulation into gut tissue and uptake into the gut serosal compartment was also saturable, with maximal transport rates of 5.1 and 2.8 nmol cm-2 h-1, and affinities of 6417 and 3327 µM, respectively. A strong correlation between epidermal and intestinal calcium tissue uptake was noted in assays where tissues were taken from the same individual. Intestinal calcium influx was inhibited by nickel, but not significantly affected by other putative inorganic (lanthanum, zinc) or organic (diltiazem, verapamil) transport blockers. Inhibitors had no effect on skin calcium handling. These data indicate that both gut and skin epithelia mediate calcium influx in hagfishes, albeit via distinct mechanisms and with differences in responsiveness to potential modifying factors.

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates.

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)-a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species-a hagfish, a lamprey and a shark-encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates.

Evolution of the Vertebrate Cranium: Viewed from Hagfish Developmental Studies.

Our knowledge of vertebrate cranium evolution has relied largely on the study of gnathostomes. Recent evolutionary and developmental studies of cyclostomes have shed new light on the history of the vertebrate skull. The recent ability to obtain embryos of the hagfish, Eptatretus burgeri, has enabled new studies which have suggested an embryonic morphological pattern (the "cyclostome pattern") of craniofacial development. This pattern is shared by cyclostomes, but not by modern jawed vertebrates. Because this pattern of embryonic head development is thought to be present in some stem gnathostomes (ostracoderms), it is possible that the cyclostome pattern represents the vertebrate ancestral pattern. The study of cyclostomes may thus lead to an understanding of the most ancestral basis of craniofacial development. In this review, we summarize the development of the hagfish chondrocranium in light of the cyclostome pattern, present an updated comparison of the cyclostome chondrocranium, and discuss several aspects of the evolution and development of the vertebrate skull.

The origin of developmental mechanisms underlying vertebral elements: implications from hagfish evo-devo.

The origins of the vertebral elements and the underlying developmental mechanisms have so far remained unclear, largely due to the unusual axial skeletal morphology of hagfish, one of two extant jawless vertebrate clades. Hagfish axial supporting tissue is generally believed to consist of the notochord and cartilaginous fin rays only. However, careful investigations of whether vertebral elements are truly absent in hagfish are scarce, and it is also unclear whether the axial skeletal morphology of the hagfish is an ancestral or a derived condition. To address these questions, we re-examined the axial skeletal morphology of the Japanese inshore hagfish (Eptatretus burgeri). Based on a report published a century ago which implied the existence of vertebral elements in hagfish, we conducted anatomical and histological analyses of the hagfish axial skeletal systems and their development. Through this analysis, we demonstrate that hagfish possesses sclerotome-derived cartilaginous vertebral elements at the ventral aspect of the notochord. Based on (i) molecular phylogenetic evidence in support of the monophyly of cyclostomes (hagfish and lampreys) and jawed vertebrates (gnathostomes), and (ii) the morphology of the vertebral elements in extant gnathostomes and cyclostomes, we propose that the embryos of the common ancestor of all vertebrates would have possessed sclerotomal cells that formed the segmentally arranged vertebral elements attached to the notochord. We also conclude that the underlying developmental mechanisms are likely to have been conserved among extinct jawless vertebrates and modern gnathostomes.

Development of the chondrocranium in hagfishes, with special reference to the early evolution of vertebrates.

Recent molecular phylogenetic analyses have shown that the modern jawless vertebrates, hagfishes and lampreys, are more closely related to each other than to the other vertebrates, constituting a monophyletic group, the cyclostomes. In terms of their developmental morphology as well, it is possible to identify an embryonic pattern in hagfish embryos that is common to cyclostomes but not shared by jawed vertebrate embryos. On the basis of this pan-cyclostome embryonic pattern, we describe the developmental sequence of the chondrocranium and associated structures in the hagfish species Eptatretus burgeri and E. atami. Our aim was to establish homologies of the skeletal elements among cyclostomes by comparison of the developmental patterns with a lamprey, Lethenteron reissneri, to characterize further the cyclostome morphotype and its diversification in early vertebrate evolution. We show that the hagfish and lamprey chondrocrania can be compared perfectly at the level of modules corresponding to the craniofacial primordia constituting the cyclostome morphotype. In the adult anatomy, however, there are many instances in which homology cannot be established at the level of single skeletal elements, mainly because of the apparently highly apomorphic nature of the hagfish cranium. Even at the craniofacial modular level, the chondrocrania of cyclostomes and those of jawed vertebrates display very few primary homologies and are therefore very difficult to compare. We also discuss the problem of the homology of a neurocranial element, the trabecula.

Relationships between plasma concentrations of sex steroid hormones and gonadal development in the brown hagfish, Paramyxine atami.

The relationship between sex steroid hormone profiles in plasma and gonadal function in hagfish is poorly understood. In the present study, plasma concentrations of estradiol, testosterone, and progesterone were examined with respect to gonadal development, sexual differences, and possible function of atretic follicles in the brown hagfish, Paramyxine atami, using a time-resolved fluoroimmunoassay. Plasma concentrations of these three hormones were low in juveniles of both sexes. In females, plasma estradiol showed a significant correlation with ovarian development, with the highest concentrations in late vitellogenic adults. Plasma testosterone and progesterone also increased significantly in non-vitellogenic adult females; however, plasma testosterone showed no significant differences among adult females at different ovarian developments, while plasma progesterone was significantly lower in late vitellogenic adults than it was in non-vitellogenic adults. Vitellogenic females that possessed atretic follicles showed significantly lower concentrations of all three hormones than females that only possessed normal follicles. In males, no significant differences were found in plasma estradiol or testosterone levels among groups of different developmental stages of the testis, while plasma progesterone showed a clear inverse relationship with testicular development. Thus, differences were found in plasma sex steroid hormone profiles between male and female P. atami. Moreover, plasma estradiol showed a significant correlation with ovarian development, which suggests that estradiol is involved in the regulation of ovarian development. The present study also revealed that steroid hormone production was strongly suppressed in females that possessed atretic follicles in their ovaries.

Morphometics and gonadal development of the hagfish Eptatretus cirrhatus in New Zealand.

Hagfishes have been the target of commercial fisheries in many areas of the world, with the catch processed for leather and for human consumption. A fishery has been operating in New Zealand waters for the last six years, harvesting the bearded hagfish, Eptatretus cirrhatus. The fishery has thus far been unregulated. Based on samples collected dockside over a two-year period, this report expands the morphometric database for this species, provides information on the size and weight of the harvested animals, determines the sizes at the onset of gonadal development and the minimum sizes at sexual maturation for males and females, and indicates that E. cirrhatus, like most other hagfish species, has no specific breeding season. Although females appear in the population at smaller sizes, the sex ratio for mature animals is 1:1 and the sizes of the largest males and females are comparable. The changes observed in sex ratio as a function of TL suggest differences in the timing and rates of gonadal development in females versus males rather than protogyny. Based on the size of the eggs, the number of eggs per female, the proportion of the population that contains large eggs, and the number of postovulatory females, it is clear that E. cirrhatus, like other hagfish species, are potentially vulnerable to overexploitation.

Evolution of the regionalization and patterning of the vertebrate telencephalon: what can we learn from cyclostomes?

The telencephalon, the most anterior part of the vertebrate central nervous system (CNS), is a highly diversified region of the vertebrate body. Its evolutionary origin remains elusive, especially with regard to the ancestral state of its architecture as well as the origin of telencephalon-specific neuron subtypes. Cyclostomes (lampreys and hagfish), the sister group of the gnathostomes (jawed vertebrates), serve as valuable models for studying the evolution of the vertebrate CNS. Here, we summarize recent studies on the development of the telencephalon in the lamprey. By comparing detailed developmental studies in mammals, we illustrate a possible ancestral developmental plan underlying the diversification of the vertebrate telencephalon and propose possible approaches for understanding the early evolution of the telencephalon.

[Processes of scale formation in fish and Agnatha].

The formation of the scale cover in ancient fish and Agnatha was analyzed using paleontology data on some forms and by studying recent species according to the geochronological principle and morphological--genetic coupling approach. The histogenesis of true scale cover was transformed simultaneously but independently from dermal denticles, when the first process was accompanied by reinforcement of the bones and formation of head-body armor and dermal-like external elements of the skullcap. The scale cover is an independent organ system and is characterized by self-supporting conservative ancestral mechanisms of development.

Evolutionary crossroads in developmental biology: cyclostomes (lamprey and hagfish).

Lampreys and hagfish, which together are known as the cyclostomes or 'agnathans', are the only surviving lineages of jawless fish. They diverged early in vertebrate evolution, before the origin of the hinged jaws that are characteristic of gnathostome (jawed) vertebrates and before the evolution of paired appendages. However, they do share numerous characteristics with jawed vertebrates. Studies of cyclostome development can thus help us to understand when, and how, key aspects of the vertebrate body evolved. Here, we summarise the development of cyclostomes, highlighting the key species studied and experimental methods available. We then discuss how studies of cyclostomes have provided important insight into the evolution of fins, jaws, skeleton and neural crest.

Study on the accumulation of heavy metals in shallow-water and deep-sea hagfishes.

Hagfish, the plesiomorphic sister group of all vertebrates, are scavengers, and many of them live at depths reaching thousands of meters. They are caught for use as food and serve as a substitute for leather in crafts in Asian hagfisheries. At present, the amount of various pollutants present in hagfishes from bioaccumulation through the food chain is unknown. To understand the bioaccumulation characteristics of heavy metals in deep-sea scavengers, selected heavy metals, including iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), mercury (Hg), and lead (Pb), were analyzed and compared in two hagfish species, Paramyxine nelsoni (Pn) (found at approximately 200 m) and Myxine formosana (Mf) (found at approximately 850 m) caught in southwestern Taiwanese waters. Hagfish muscle (PnM and MfM) and liver (PnL and MfL) samples were lyophilized, and their metal levels were then analyzed using an inductively coupled plasma mass spectrometer. The metals with the highest levels in Pn tissues included Cu and As (PnL > MfL and PnM > MfM); in contrast, those that were higher in Mf tissues were Cd, Hg (both MfL > PnL and MfM > PnM), and Zn (MfM > PnM). Multivariate analyses, i.e., principle component analysis and partial least squares for discriminant analysis of metal levels were able to clearly separate these four tissue types into two groups corresponding to the two species: Pn and Mf. The present data also show differences in the levels of certain heavy metals in these tissues of the two hagfish species. These differences might have resulted not only from depth-related environmental factors but also from different species' accumulation characteristics. Fe, Cu, and Hg concentrations were much higher in hagfish muscle than have been found in other fishes from adjacent polluted regions, and Hg was approximately 10- to 100-fold higher in hagfish muscles. Public health issues related to the consumption of hagfish are also discussed.

An alternative model for the structural mechanics of hagfish slime threads.

Hagfish slime threads are an interesting intermediate filament (IF) system in that, in contrast to hair, they appear to be a series system of IFs, consisting of rods and terminal domains (TDs), without matrix. The protein composition also differs from hair. Published data show that the wet stress-strain curve consists of four regions: I, increasing low modulus; II, plateau; III increasing higher modulus; IV decreasing modulus to break. Beyond 34% extension, there is plastic deformation, which contrasts with the elastic deformation of most biological fibres. The paper considers two explanations, one published by Fudge et al. [D.S. Fudge, K.H. Gardner, V.T. Forsyth, C. Riekel, J.M. Gosline, Biophys. J. 85 (2003) 2015] and one new, of deformation in the four regions. The former regards the TDs as elastic, entropic coils, which extend in region I. The latter regards the TDs as having a plastic, energy-dependent extension in regions III and IV, which is comparable to the drawing of polyester fibres. A rough theoretical model of jumps over energy barriers gives a similar prediction. Twist is suggested as a mechanism for overall thread cohesion. Experimental and theoretical ways forward to a greater understanding of the structural mechanics of hagfish threads are suggested. The behaviour of the total thread/mucus/water system is discussed and some speculations on the defensive mechanisms that have evolved are presented.

Evolution of a core gene network for skeletogenesis in chordates.

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1-3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.

Hagfish in the New Zealand fjords are supported by chemoautotrophy of forest carbon.

Forest litter is often considered to be a minor energy source to marine communities due to its refractory nature. Large volumes of forest litter are deposited in the New Zealand fjords, and likely recycled into available energy by microbial activity. In this study we used evidence from stable isotope analyses to test whether recycled carbon from chemoautotrophs was an important contributor to the diet of hagfish (Eptatretus cirrhatus). We then analyzed fatty acid biomarkers from the chemoautotrophic clam Solemya parkinsoni and E. cirrhatus to further discriminate the contribution of marine, terrestrial, and chemoautotrophic sources. Bulk isotopic signatures of E. cirrhatus varied considerably (delta13C, from -29.2 per thousand to -16.7 per thousand; delta15N, from -2.8 per thousand to +15.5 per thousand; delta34S, from -21.7 per thousand to +16.7 per thousand) and indicated that a significant percentage of organic matter (38-51%) originated from chemoautotrophs (delta13C, -31.3 per thousand +/- 0.1 per thousand [mean +/- SE]; delta15N, -5.7 per thousand +/- 0.2 per thousand; delta34S, -32.per thousand +/- 3.8 per thousand). Fatty acid biomarkers were depleted in 13C, particularly cis-vaccenic acid (18:1omega7: delta13C, -39.0 per thousand) indicating specific microbial origins of carbon. A high proportion of forest litter in sediments, coupled with isotopic and fatty acid biomarker results, indicates that terrestrial organic matter is a dominant contributor to this marine benthic system. This study demonstrates a clear linkage between terrestrial and marine ecological processes.

Genes coding for intermediate filament proteins closely related to the hagfish "thread keratins (TK)" alpha and gamma also exist in lamprey, teleosts and amphibians.

The "thread keratins (TK)" alpha and gamma so far have been considered highly specialized intermediate filament (IF) proteins restricted to hagfish. From lamprey, we now have sequenced five novel IF proteins closely related to TKalpha and TKgamma, respectively. Moreover, we have detected corresponding sequences in EST and genomic databases of teleosts and amphibians. The structure of the TKalpha genes and the positions of their deduced amino acid sequences in a phylogenetic tree clearly support their classification as type II keratins. The genes encoding TKgamma show a structure typical for type III IF proteins, whereas their positions in phylogenetic trees favor a close relationship to the type I keratins. Considering that most keratin-like sequences detected in the lancelet also exhibit a gene structure typical for type III IF proteins, it seems likely that the keratin gene(s) originated from an ancient type III IF protein gene. According to EST analyses, the expression of the thread keratins in teleost fish and amphibians may be particularly restricted to larval stages, which, in conjunction with the observed absence of TKalpha and TKgamma genes in any of the available Amniota databases, indicates a thread keratin function closely related to larval development in an aquatic environment.

Seasonal concentrations of reproductive steroids in the gonads of the Atlantic hagfish, Myxine glutinosa.

Changes in gonadal morphology, gonadal estradiol, and progesterone were examined in Atlantic hagfish, Myxine glutinosa, during a period of 17 months, beginning in April, 2001. Atlantic hagfish were captured from the ocean on a monthly basis. A total of 60 hagfish were divided into three different size classes of twenty hagfish each (small 20-35 cm, medium 35-45 cm, large 45-55+cm) and transported to the University of New Hampshire for sampling. Overall, in the medium and large size hagfish, estradiol and progesterone had significantly elevated peaks in January, 2001. There were significant increases in estradiol concentrations in January, with relatively low fluctuations in levels for the rest of the sampling period. Progesterone concentrations increased significantly in January, 2002, in medium and large hagfish, and remained elevated until June and April, 2002, for the two size classes respectively. The majority of hagfish sampled were females or hermaphrodites; few true males were identified in any of the samples. The number of females with large eggs increased following the estradiol peak in January and hermaphrodites with mature sperm were identified in the July, 2002, sample. These data represent the first evidence for a seasonal reproductive cycle in M. glutinosa and only the second seasonal reproductive cycle identified in any hagfish species.