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Comparative pelvic development of the axolotl (Ambystoma mexicanum) and the Australian lungfish (Neoceratodus forsteri): conservation and innovation across the fish-tetrapod transition
Catherine Anne Boisvert1, 2Email author,
Jean MP Joss3 and
Per E Ahlberg2
EvoDevo20134:3
© Boisvert et al.; licensee BioMed Central Ltd. 2013
Received: 14 September 2012
Accepted: 3 December 2012
The fish-tetrapod transition was one of the major events in vertebrate evolution and was enabled by many morphological changes. Although the transformation of paired fish fins into tetrapod limbs has been a major topic of study in recent years, both from paleontological and comparative developmental perspectives, the interest has focused almost exclusively on the distal part of the appendage and in particular the origin of digits. Relatively little attention has been paid to the transformation of the pelvic girdle from a small unipartite structure to a large tripartite weight-bearing structure, allowing tetrapods to rely mostly on their hindlimbs for locomotion. In order to understand how the ischium and the ilium evolved and how the acetabulum was reoriented during this transition, growth series of the Australian lungfish Neoceratodus forsteri and the Mexican axolotl Ambystoma mexicanum were cleared and stained for cartilage and bone and immunostained for skeletal muscles. In order to understand the myological developmental data, hypotheses about the homologies of pelvic muscles in adults of Latimeria, Neoceratodus and Necturus were formulated based on descriptions from the literature of the coelacanth (Latimeria), the Australian Lungfish (Neoceratodus) and a salamander (Necturus).
In the axolotl and the lungfish, the chondrification of the pelvic girdle starts at the acetabula and progresses anteriorly in the lungfish and anteriorly and posteriorly in the salamander. The ilium develops by extending dorsally to meet and connect to the sacral rib in the axolotl. Homologous muscles develop in the same order with the hypaxial musculature developing first, followed by the deep, then the superficial pelvic musculature.
Development of the pelvic endoskeleton and musculature is very similar in Neoceratodus and Ambystoma. If the acetabulum is seen as being a fixed landmark, the evolution of the ischium only required pubic pre-chondrogenic cells to migrate posteriorly. It is hypothesized that the iliac process or ridge present in most tetrapodomorph fish is the precursor to the tetrapod ilium and that its evolution mimicked its development in modern salamanders.
Fish-tetrapod transition
Pelvic girdle
Heterochrony
Extant phylogenetic bracketing method
Evolutionary novelty
Around 395 million years ago, the first tetrapods (four-legged vertebrates) appeared, having evolved from lobe-finned fish [1, 2]. This fish-tetrapod transition was marked by many morphological transformations and ecological adaptations ranging from the evolution of fingers and toes [3–5] to new modes of respiration, hearing [6, 7] and locomotion [8, 9]. One of the major changes in locomotory habit is that of a shift from fish principally using their pectoral fins and lateral undulation to swim to tetrapods relying much more heavily on their hindlegs to swim and walk [10]. This shift from 'front-wheel drive' to 'back wheel drive' locomotion was enabled by the evolution of a large, weight-bearing pelvic girdle in tetrapods. In lobe-finned fishes, the pelvic girdle is composed of a crescentric pubis often connected through cartilage at the midline but lacking an ilium to connect it and, consequently, the whole fin, to the vertebral column [11]. In tetrapods, not only is an ilium present and fused to the vertebral column through a sacral rib, but an ischium is also present posterior to the pubis. The pubis and ischium from both halves of the girdle are fused along their midlines and, hence, the girdle is weight-bearing [2]. Tetrapod legs are also oriented laterally compared to the posterior orientation of fish pelvic fins. This reflects the orientation of the acetabulum, which is located on the lateral face of the pelvis in tetrapods but at the posterior end of the pelvis in fish. The morphology of Paleozoic lobe-finned fish pelves is known from descriptions of Eusthenopteron[12] and other fish members of the tetrapod stem group [13, 14], as well as from stem dipnoans [15] and a porolepiform [11], and that of the earliest tetrapods by descriptions of Acanthostega[16] and Ichthyostega[8, 17]. This provides a good picture of the general morphology on either side of the transition (Figure 1) but little information as to how the transformation occurred (Figure 1). In order to elucidate this, the pelvic girdle of the transitional fish Panderichthys was studied [18] but it is very fish-like and unfortunately does little to answer the following questions: Is the iliac process present in Eusthenopteron a precursor of the ilium of tetrapods? How did the ilium become connected to the sacral rib? How did the ischium evolve? And how did the acetabulum move during the transition?
Phylogeny spanning the fish-tetrapod transition, showing stepwise transformation of the pelvic morphology. In lungfishes (Dipnoi) there is no iliac ramus, but a low ridge that can be homologized with the iliac process of other tetrapodomorph fishes. In the extant Neoceratodus the homologues of tetrapod iliac musculature attach to a low ridge anterodorsal to the acetabulum. In fish members of the tetrapod stem group (exemplified here by the rhizodont Gooloogongia and the osteolepiform Eusthenopteron), overall pelvic morphology is similar to that of lungfishes but an iliac process is present. In the stem tetrapods Acanthostega and Ichthyostega an ischium is present and overall pelvic morphology approaches that of extant salamanders such as Ambystoma. The boundary between ishium and pubis is approximate in stem tetrapods and axolotl as the elements are not separated by sutures. Anterior to the left. Phylogeny based on [4], Griphognathus and Neoceratodus redrawn from [15], Gooloogongia from [46], Eusthenopteron from [12], Acanthostega from [16], Ichthyostega from [17], Ambystoma from Figure 2, Mus musculus from [47]. All reconstructions by CAB.
In the absence of more informative fossils, we can use the Extant Phylogenetic Bracketing method [19] as a basis for framing hypotheses about how the transformation occurred. By comparing the development of the pelvic girdle in modern representatives of groups on either side of the transition, we can determine which aspects are similar and, thus, likely to have been conserved from fish to tetrapod, and which appear to be tetrapod innovations. This approach not only illuminates the evolution of pelvic development, but provides clues for the interpretation of the adult morphologies of transitional fossils. Heterochronies in developmental sequences have proven useful in understanding evolutionary change and are, thus, of particular interest in this context [20, 21]. The Australian lungfish (Neoceratodus forsteri) is a morphologically conservative member of the Dipnoi and is the only lobe-finned fish available for developmental studies. Fortunately, its pelvic girdle is very similar to that of Paleozoic dipnoans [15] and a good representation of the general lobe-finned fish pelvic shape (Figure 1). The Mexican axolotl (Ambystoma mexicanum) is a commonly used laboratory animal, which, like other salamanders, has a pelvic morphology very similar to that of early tetrapods. In both species, a cartilaginous anterior process (pubic process in Neoceratodus and ypsiloid cartilage in Ambystoma) is present. This process is absent, or arguably unpreserved, in both Devonian lungfish and in Devonian tetrapods, and so cannot be assumed to be homologous in the two groups. However, the pubis and acetabulum are uncontroversially homologous [22] and will be used here as landmarks for the developmental comparison.
The skeletal components of the pelvic girdle do not exist in isolation, but are intimately linked both developmentally and functionally to the muscles that attach there. In order to understand the transformation of the pelvis at the fish-tetrapod transition, it is thus important to consider not just the skeleton but also the musculature. Accordingly, we present here a comparison of muscular development in Neoceratodus and Ambystoma, as a complement to the skeletal study. Establishing a robust comparative framework for the muscular data is, however, a more complicated matter than for the skeletal data because the musculature of the pelvis and hind limb is far more complex than the skeleton and the mapping of homologies between taxa correspondingly more difficult. Establishing muscle homologies has been an important focus of comparative anatomy in the 19th and early 20th century [23–26] and its importance is now being put back into focus as a way of understanding and explaining evolutionary change [27, 28]. Despite the adult pelvic musculature having been described for two of the living sarcopterygians, the coelacanth Latimeria chalumnae[29] and the Australian lungfish Neoceratodus forsteri[15], as well as for several salamanders [23, 24, 30] and several studies having been published on hindlimb muscle homologies [25, 28], no attempt has been made to establish detailed muscular homologies across the fish-tetrapod transition. We based our mapping of muscle homologies on close representatives of the taxa used in the developmental study. As a representative for the axoltol, we used the mudpuppy Necturus maculosus[31] because of its close phylogenetic position and similarities to Ambystoma mexicanum[32, 33]. We present a detailed comparison of the three musculatures, developed from published descriptions, with a set of proposed homologies (Tables 1, 2, 3, 4, 5). The proposed homologies are based on the points of origin and insertion of the muscles as well as their function, following established principles for such comparisons [25, 27, 28].
Comparison of adductor muscles in the coelacanth (Latimeria), Australian lungfish (Neoceratodus) and mudpuppy (Necturus)
Latimeria chalumnae
Neoceratodus forsteri
Necturus maculosus
Superficial adductor, main bundle « Abaisseur superficiel, faiseau principal » (Ventral)
O:posterior border of the mesial hypophysis
I: fascia attaching to the base of lepidotrichia
Superficial ventromesial adductor (Dorsal and ventral)
O: Median posteroventral margin of the pelvis and the superficial ventromesial adductor from the other side (for the mesialmost fibres).
I: Distal medial process on the first axial element and on radials
Ischioflexorius (Adductor) (Ventral)
O: caudal end of the ischium
I: Fascia of the distal end of the shank.
Deep adductor « Abaisseur profond » (Ventral)
O: Middle of the pubis and posterior part of the pubic ramus.
I: through tendons onto the fascia of the superficial adductor, main bundle
Superficial ventro-lateral adductor (Ventral)
O: Median posteroventral margin of the pelvis
I: Distal medial process on the first axial element and radials
Puboischio-tibialis (Adductor) (Ventral)
O: Ventral and caudal part of the pubis, most of the ischium
I: Proximal end of the tibia
Pronators 1+2+3 (Dorsal)
O: very middle of the pubis on dorsal side
I: 3 first preaxial radials and 10 first preaxial lepidotrichia
Deep ventral adductor depressor (Dorsal)
O: Posterodorsal and posteroventral faces of the pelvis
I: Base of first fin element
Ischiofemoralis (Adductor) (Ventral)
O: Ischium I:Proximal end of the femur
Fin adductor «Adducteur de la nageoire» (Dorsal)
O :Arcuate ridge on the dorsal side
I: Fascia of the 5th pronator and base of the 9th and 10th lepidotrichia.
Dorsomesial adductor levator (dorsal)
O: Arcuate ridge (posterior dorsal part, anterior to the acetabulum)
I: Proximal and subsequent fin elements
Mesial adductor (Dorsal)
O: Muscles of fin elements from one side
I: Muscles of fin elements from the other side
Puboischio-femoralis externus (Adductor) (Ventral)
O: ventral surface of the girdle/Pubis and ischium
I: Proximal end of the femur
Comparison of abductor muscles in the coelacanth (Latimeria), Australian lungfish (Neoceratodus) and mudpuppy (Necturus)
Superficial abductor, secondary bundle « Élévateur superficiel, faiseau secondaire »
O: posterior internal region of the lateral hypophysis
Superficial ventromesial abductor (Dorsal and ventral)
O: Ventrolateral face pelvis
Pubotibialis (Adductor) (Ventral)
O: lateral edge of the pubic cartilage
I: Base of the 6th and 7th preaxial lepidotrichia
I: Proximal lateral face pelvic fin
Superficial ventrolateral abductor (Dorsal and ventral)
O: posterior fascia of the body myotomes
Caudofemoralis (Adductor) (Ventral)
O: Caudal vertebra
I: Dorsolateral edge proximal axial elements of the fin
I: Proximal end femur
Superficial abductor, main bundle « Élévateur superficiel, faiseau principal »
O: Posterior medial face of the lateral hypophysis.
Deep ventral abductor depressor (Dorsal and ventral)
O: Ventrolateral process on the pelvis
Puboischio femoralis internus (Abductor) (Ventral)
O: Internal surface of the pubic cartilage and ischium
I: Fascia at the base of lepidotrichia.
I: Ventral process at the distal end of the first axial element.
I: along most of the femur
Deep abductor "Élévateur profond"
O: lateral sides and base of the anterior pubic ramus I: through tendons to the fascia of the superficial abductor
Dorsolateral abductor levator (Dorsal)
O: Swelling on the posterodorsal surface of the pelvisI: Proximal and subsequent fin elements
Iliotibialis (Dorsal surface of the thigh) (Abductor) (Dorsal)
O: base of the iliumI: extend over the knee as a tendon, inserts on the tibia
Ilioextensorius (Abductor) (Dorsal)
I: base of the ilium O: extend over the knee as a tendon, inserts on the tibia
Pelvic abductor or supinator, second layer, group 5 "Supin, couche 2, groupe 5, Abducteur pelvien"
O: Middle of the mesial hypophysis I: following the preaxial border, inserting directly onto the six first preaxial lepidotrichia
Iliofibularis (Abductor) (Dorsal)
O: base of the iliumI: inserts on the fibula
Iliofemoralis (Abductor) (Dorsal)
O: base of the iliumI: caudal edge of the femur
Comparison of adductor/abductor muscles in the coelacanth (Latimeria), Australian lungfish (Neoceratodus) and mudpuppy (Necturus)
Radial flexors (Adductor and abductor)
O: fin elements I: fin elements
Shank flexors and shank extensors (adductor and abductor respectively)
O: Distal end of the femur
I: proximal and distal end of the tibia and fibula.
Comparison of supinator and pronator muscles in the coelacanth (Latimeria), Australian lungfish (Neoceratodus) and mudpuppy (Necturus)
Fourth pronator "4 ième pronateur " (dorsal)
O: posterior edge of the process on the fourth axial element I: Base of the last few preaxial lepidotrichia
Lepidotrichial flexors (Dorsal))
O: Lateral sides of fin elements I: Lepidotrichia
Fifth pronator "5ième pronateur" (dorsal)
O: posterior edge of the process on all postaxial elements surrounding the fourth axial elementI: base of all post-axial lepidotrichia
Supinator, second layer, group 3. "Supin couche 2, groupe 3"
O:Posterior edge of all four axial elements.I: Preaxial radials and preaxial lepidotrichia.
Lepidotrichial flexors (Ventral)
Supinator, second layer, 4th group "Supin, couche 2, groupe 4"
O: Posterior edge of the fourth axial element. I: Postaxial lepidotrichia
Supinator, second layer, group 3 "Supin, couche 2, groupe 3"
O: Posterior edge of all four axial elements.I: Preaxial radials and preaxial lepidotrichia.
Radial-axial (Ventral and dorsal )
O: All axial elementsI : All radial elements
Supinator, first layer. "Supin, couche1 "
O: postaxial region between the base of the mesial hypophysis (anteriorly) and the arcuate ridge (posteriorly)I: through a tendon to the preaxial radials and preaxial lepidotrichia
Supinator, second layer, group 2 "Supin couche 2, groupe 2 "
O : postaxial side of the arcuate ridgeI: Preaxial radials and preaxial lepidotrichia.
Popliteus (pronator and supinator) (Ventral)
O: Underside of the femur near the insertion of the puboischiofemo-ralis externus)
Comparison of hypaxial musculature in the coelacanth (Latimeria), Australian lungfish (Neoceratodus) and mudpuppy (Necturus)
Hypaxial muscle "Muscle de l'hyposome "
O: tip of the pubic ramus I: Hypaxial musculature
Hypaxial muscles do not attach to the pelvic girdle
Rectus abdominis is not attached to the pelvic girdle
Ischiocaudalis (tail flexion) (Ventral)
O: Caudal vertebrae
I: Caudal end of ischium
Caudopuboischioti-bialis (tail flexion) (Ventral)
O: Puboischiotibialis
Salamander and lungfish larvae
Albino larvae of the Mexican axolotl (A. mexicanum) were purchased from the Ambystoma genetic stock center at the University of Kentucky, USA. They were fixed in paraformaldehyde overnight and stored in 100% methanol. The youngest larvae of the series used in this article were staged using the extended table of development developed by Nye et al. [34]. Given the absence of an adequate development table, older larvae were staged according to total length (in cm). Larvae of the Australian lungfish (N. forsteri) were raised in captivity from eggs collected in the lungfish spawning ponds at Macquarie University (protocols approved by the Macquarie University Animal Ethics Committee, approval # 2003/001). The embryos were left to hatch and develop for approximately five months, being fed on brine shrimp and bloodworms (for older larvae). The youngest larvae used in this study were staged using the developmental table developed by Kemp [35]. Fish older than stage 55 (latest stage of the table) were staged according to pelvic fin length and were given a stage number corresponding to the same developmental progress as between stages 54 and 55. The larvae were euthanized with Tricaine (MS-222) and fixed overnight in 4% paraformaldehyde with a pH of 7.4.
Alcian blue and Alizarin red staining
No acid, Kimmel protocol
Lungfish were cleared and stained using a protocol without acid developed by the Kimmel Laboratory (University of Oregon, USA) for zebrafish and modified by Catherine Anne Boisvert. The larvae were eviscerated and then washed in Tris/MgCl2 before being transferred to Alcian stain solution (0.02% alcian in 71% EtOH and 25 mM MgCl2 in Tris pH 7.5 aqueous solution) for a period ranging from three to seven days. The specimens were then rehydrated through a series of ethanol in 100 mM Tris pH 7.5 and 25 mM MgCl2 for 30 minutes each, after which they were bleached in 3% H2O2 and 0.5% KOH for 20 to 22 hours with a change of solution. Muscles were digested away at room temperature in a solution of 1% pancreatin in 35% saturated sodium borate for 17 to 51 hours. The specimens were washed for one hour in 25% glycerol and 0.1% KOH and then stained for bone in a solution of 0.02% Alizarin stain in 10% glycerol and 0.5% KOH for two to four days. Excess stain was removed by placing them in a solution of 50% glycerol and 0.5% KOH for at least a day after which they were stored in 100% glycerol with a few crystals of thymol to avoid fungal growth. All steps were carried out on a gyrating platform at a low setting.
Zebrafish protocol for optical tomography
Axolotls of stages 54, 55 and 1.5 cm were cleared and stained following a protocol developed by Silke Berger from the Currie Laboratory (Australian Regenerative Medicine Institute, Monash University) for Optical Tomography on zebrafish and modified by Catherine Anne Boisvert. The larvae were eviscerated and dehydrated before being bleached in a mixture of formamide, SSC (sodium chloride citrate) and H2O2 under a light source for 20 to 25 minutes. They were then washed in PBS, dehydrated to 75% ethanol and stained in Alcian stain solution (EtOH, glacial acetic acid and 0.01 mg/ml Alcian blue) for 22 hours. The specimens were washed in 80% EtOH/Tris/MgCl2 and rehydrated and washed in dH2O. They were then stained for 11 to 24 hours in Alizarin stain (0.1 mg/ml in 0.5% KOH aqueous solution) and washed in PBS. They were then transferred to an increasing series of glycerol in PBS and stored in 100% glycerol with a few crystals of thymol. All steps were carried out on a gyrating platform at a low setting.
Taylor and VanDyke protocol
Ambystoma mexicanum specimens of sizes 2.0 cm, 2.5 cm, 3.0 cm, 3.5 cm and 4.0 cm were cleared and stained according to a protocol developed by Taylor and VanDyke [36] and modified by Catherine Anne Boisvert. The salamanders were eviscerated and washed in 80% EtOH/Tris/MgCl2 before being stained in Alcian blue stain (0.3 mg/ml Alcian stain in 80% EtOH and glacial acetic acid) for three days. They were neutralized in a saturated solution of sodium borate and bleached for one hour and forty minutes in a solution of 0.5% KOH and H2O2. Muscles were removed in a solution of 2.25 mg/ml trypsin in saturated sodium borate. They were then stained in Alizarin red solution (0.1 mg/ml in 0.5% KOH aqueous solution) for two to three days, rinsed in dH2O and transferred to an increasing series of glycerol in water. They were stored in 100% glycerol with a few crystals of thymol. All steps were carried out on a gyrating platform at a low setting.
Klymkowsky and Hanken protocol
Neoceratodus larvae from stages 50 and 51 were stained as whole-mounts according to a protocol modified from Klymkowsky and Hanken [37]. The larvae were refixed overnight in Dent's fixative and bleached for 29 hours in Dent's bleach. The specimens were then rehydrated and washed in 'saline cocktail' (PBS, 0.4% Triton X-100) before being blocked in 'serum cocktail' (PBS, 0.4%Triton X-100, 2% bovine serum albumin (BSA), 5% dimethylsulfoxide (DMSO)) for one hour. The specimens were then incubated with the primary antibody against skeletal muscle (Hybridoma gene bank 12/101, 3.7 mg/ml IgG1) diluted 1:50 in 'serum cocktail' for five days at room temperature. They were then washed and re-blocked in 'serum cocktail' overnight. The larvae were then incubated in the secondary antibody (488 goat anti-mouse Alexa antibody by Molecular Probes/Invitrogen; 2 mg/ml) diluted 1:150 in 'serum cocktail' for two days at room temperature in the dark. They were then washed in 'serum cocktail' and 'saline cocktail' before being dehydrated to 100% methanol and transferred to an increasing series of BABB (benzyl alcohol/benzyl benzoate). All steps were carried out on a gyrating platform at a low setting.
Currie Laboratory protocol
The remainding stages of Ambystoma and Neoceratodus larvae were immunostained according to a protocol developed by Silke Berger and adapted by CAB. The pelvic region was dissected out, skinned and eviscerated. The specimens were bleached in an aqueous solution of H2O2, formamide and SSC. Specimens were permeabilized by a trypsin treatment (0.25% trypsin in PBST) and acetone cracking. Specimens were then washed in PBST and blocked for six hours in PBS containing 1% BSA and 1% DMSO. They were then incubated in the primary antibody against skeletal muscle (Hybridoma gene bank 12/101, 38 μg/ml IgG1) diluted 1:10, washed in PBS/BSA/DMSO and incubated in Alexa Fluor 568 goat anti-mouse IgG1(γ1) 2 mg/ml (Molecular Probes/Invitrogen A21124) diluted 1:150 in PBS/BSA/DMSO. They were then washed in PBS/BSA/DMSO, then in PBS over a day before being embedded in 1.5% low melting point agarose (BDH Electran, VWR 444152 g) in PBS. The blocks were left to solidify and dry at 4°C in the dark, were trimmed and slowly dehydrated to 100% methanol before being cleared through an increasing series of BABB. Most steps were carried out on a gyrating platform at a low setting.
All specimens were examined with a Leica MZ FLIII dissecting microscope and photographed using a Leica DFC 490 camera and the Leica Fire cam program. Fluorescent samples were examined with a mercury lamp and Leica GFP filters.
Development of the pelvic girdle in the Australian lungfish
Kemp's staging table for Neoceratodus[35] stops at stage 55 and no staging table currently exists for older stages. Older larvae have been given a stage starting at number 56 according to general growth. These stage numbers are indicated between single quotation marks to differentiate them from the published staging table [35]. At stage 49, the pelvic girdle is already present as two narrow bands of cartilage curving mesially but not meeting at the midline (Figure 2A). In the following stages, the pelvic cartilage gradually thickens (Figure 2B). During stages 52 and 53, the pelvis gradually elongates anteriorly and the two halves start fusing at the midline (Figure 2C). During stages 54 and 55, the halves of the pelvic girdle progressively fuse anteriorly at the midline but not between the acetabula. A gap is visible between the acetabula until it closes at stage '56' and thickens at stage '59' (Figure 2D). At stage '60', the pubic process appears as a small triangular projection much thinner than the rest of the pelvis. At stage '61', the pubic process is much longer and forms the extremity of a large triangle formed by the pelvis itself (Figure 2E and F). At this stage, cartilage anterior to the acetabula is thick and a crescentric arch of strongly chondrified tissue is visible, bridging the left and right sides of the pelvic girdle. This morphology is very similar to that of a larger juvenile as described by Young et al. [15].
Comparative pelvic development in the Australian lungfish (left) and the axolotl (right). Cleared and stained larvae of Neoceratodus forsteri (A-F) and of Ambystoma mexicanum (G-L) showing development of the pelvic girdle. Cartilage is stained blue and bone in red. Neoceratodus: A) Stage 49, B) Stage 51, C) Stage 53, D) 'Stage 59', E) 'Stage 60', F) 'Stage 61' All in ventral view. Ambystoma mexicanum: G) Stage 54, H) 1.5 cm, I) 1.5 cm, J) 2.0 cm, K) 3.0 cm, L) 3.5 cm. Anterior to the left. The anterior process and ypsiloid cartilage are in grey to denote that they are non homologous structures. Ac, acetabulum; Ant pr, anterior process; Fe, femur; Il, ilium; Is, ischium; Pb, pubis; Sr, sacral rib; Vc, vertebral column; Yp c, ypsiloid cartilage.
Development of the pelvic girdle in the axolotl
Stage 54 [34] marks the beginning of pelvic development in the axolotl with the appearance of the pubis as a condensation anterior to the future acetabulum (indicated as the area between the pubic condensation and the femur) (Figure 2G). An anlage of the femur is also present at this stage posterior to the pubis condensation.(Figure 2Glateral). In the least developed 1.5 cm larva, the ischium has developed posterior to the pubic cartilage (Figure 2Hventral). The pubis is still only a small condensation of cells at the acetabulum but the ilium is now present (Figure 2Hlateral). In another 1.5 cm larva, the pubis is larger, having extended anteriorly from the acetabulum and towards the midline as a thick condensation. The ischium is also longer, reaching farther posteriorly (Figure 2Iventral). The ilium is longer, projecting postero-dorsally towards the vertebral column (Figure 2Ilateral) but not reaching it. In 2.0 cm larvae, the pubis is much larger and almost complete anteriorly (Figure 2Jventral). It is pierced by the obturator foramen on either side but the halves do not meet in the middle. The ischium is also larger but not complete; each half of the pelvic girdle is still triangular in shape, the anterior extremity being widest. At this stage, the ilium is much longer but far from reaching the vertebral column (Figure 2Jlateral). Cartilage of the pubis, ischium and ilium continue to thicken and grow in 3.0 cm larvae but there is little change in overall shape. Both sides of the pelvic girdle are still unfused and roughly triangular in shape. The major difference is the development of the sacral rib and the elongation of the ilium towards it (Figure 2K). In the most developed larvae of the series (a 3.5 cm larva), the ypsiloid cartilage is complete and both sides of the pubis and ischium are almost fused (Figure 1Lventral). The ilium almost connects with the sacral rib and has started ossifying (white zone close to its base) but is not calcified yet, preventing the Alizarin red from binding (Figure 1Llateral).
A schematic comparative representation of pelvic development in Neoceratodus and Ambystoma, aligned using the acetabulum as a fixed landmark, is shown in Figure 3.
Schematic representation of pelvic development in the Australian lungfish and the axolotl. Each panel compares pelvic development of the Australian lungfish (left side of each panel) and the axolotl (right side). The grey line marks the position of the acetabulum. A) Beginning of pelvic development through cartilaginous condensations at the acetabula. B) The condensations extend anteriorly for the lungfish (pubis) and posteriorly (ischium) and slightly anteriorly (pubis) for the axolotl. C) The pubis of the lungfish continues to extend anteriorly and the pubis of the axolotl grows anteriorly. D) The pubis of the lungfish is now fused at the midline and is triangular shaped. The axolotl pubis is complete anteriorly and the ischium is complete posteriorly. E) Anterior growth of the lungfish pelvic process and appearance of the ypsiloid cartilage in the axolotl. F) Adult morphology of the pelvic girdle in both species. Anterior is at the top.
Comparison of the pelvic musculature in adult Latimeria, Neoceratodus and Necturus
Table 1 displays equivalences of pelvic muscles in Latimeria, Neoceratodus and Necturus. Comparison of the musculature of Neoceratodus and Necturus is relatively straightforward and most muscles present in Neoceratodus can be associated with one or several present in Necturus. This is not the case for Latimeria, where text in italics indicates an incomplete equivalence. Often, the point of insertion is much more distal than in Neoceratodus or Necturus and it is often on lepidotrichia rather than endoskeletal fin elements. In some instances this may be the result of an incomplete dissection and it is possible that some of the muscles written in italics actually are equivalent to those of Neoceratodus and Necturus noted in plain text on the same row. However, these identifications should be regarded as tentative until a re-examination of Latimeria is made. For all tables, the names of the muscles were taken from their original description and have been directly translated from French for Latimeria.
Adductor muscles
All adductor muscles in Latimeria insert very distally on the fin, making each one an incomplete equivalent to those of Neoceratodus and Necturus. However, equivalences between Neoceratodus and Necturus are easily established. Only two muscles do not have equivalents. The mesial adductor of Neoceratodus was described by Young et al. [15] as linking muscles of both fins from either side. This is unique for Neoceratodus. In Necturus, the puboischiofemoralis externus, a superficial adductor originating from the pubis and ischium and inserting at the base of the femur, cannot be equated with any muscle present in Neoceratodus.
Abductor muscles
Abductor muscles equivalences are displayed in Table 2. Again, all muscles described in Latimeria insert very distally on the fin, either on lepidotrichia or on muscles inserting onto lepidotrichia. An incomplete equivalence can be established between the pelvic abductor of Latimeria and the iliofibularis of Necturus with no equivalent in Neoceratodus. The iliofemoralis of Necturus originating at the base of the ilium and inserting on the caudal edge of the femur does not have an equivalent in Latimeria or Neoceratodus.
Adductor/Abductor muscles
Table 3 presents muscles that either have an adductor or abductor function. The radial flexors of Neoceratodus can be equated to the shank flexors and extensors of Necturus.
Supinators and pronators
Many supinators and pronators have been described for Latimeria and most of them can be equated to lepidotrichial flexors and radial-axials present in Neoceratodus. Lepidotrichial flexors do not have equivalents in Necturus since lepidotrichia have been lost during the fin-limb transition and are, therefore, absent in all tetrapods. The radial-axials of Neoceratodus and their equivalents in Latimeria cannot be directly equated to muscles in Necturus, lacking pre- and post-axial radials in a 'fish configuration', but given that fish distal radials are precursors to digits [3, 38], it is possible that those muscles were the precursors of tetrapod digit musculature. The only supinator muscle in Latimeria that has no equivalent in Neoceratodus is the supinator of the second layer, second group which originates from the pelvic girdle and inserts on preaxial radials and preaxial lepidotrichia. The popliteus of Necturus, wrapping around the knee, does not have direct equivalents in Latimeria or Neoceratodus since this muscle is specific to the tetrapod configuration of a knee joint.
Hypaxial musculature
Table 5 displays hypaxial muscles attaching to the pelvic girdle. No complete equivalences can be established despite the fact that Latimeria, Neoceratodus and Necturus all have a specific pelvic component of the hypaxial musculature. In Latimeria, the hypaxial muscle inserts onto the tip of the pubic ramus but neither the hypaxial musculature in Neoceratodus nor the rectus abdominis insert on the pelvic girdle. Two hypaxial muscles inserting on the ischium are present in Necturus but not in Latimeria and Neoceratodus. These are involved in tail movement and are specific to tetrapods in more or less elaborated ways [39].
Development of the pelvic musculature in the Australian lungfish
At stage 50, there is no trace of pelvic musculature. Only the hypaxial musculature is visible in lateral and ventral views (Figure 4A). At stage 51, the deep ventral abductor depressor (D. V. abd. depr.), the deep ventral adductor depressor (D. V. add. depr.) and the dorsomesial adductor levator (Dm add. lev.) appear (Figure 4B) as short and thin muscles. These muscles are longer and thicker (Figure 4C) at stage 52 and are accompanied by radial flexors (R.f.) over five or six axial elements and radial-axials (R.-A.) on the pre-axial side of the second element. Radial flexors proximal on the fin are more developed than the ones more distal and these muscles seem to develop in a proximo-distal direction following the development of axial elements. The deep ventral abductor depressor continues to develop anteriorly at stage 54 and the superficial ventrolateral adductor (S. vl. add.) is now present between the dorsal side of the pelvis and the postaxial edge of the fin (Figure 4D). No new muscles appear at stage 56. However, the dorsomesial adductor levator is more developed and its fibers extend more distally. The radial flexors are also longer, and the radial-axials extend on all six axial elements pre-axially and appear for the first time on post-axial radials (Figure 4E). At stage 61, proximal muscles (D. V. abd. depr. and Dm. add. lev.) are fully developed and begin being covered by the superficial ventromesial abductor (S. vm. abd.), which originates at a very anterior position on the pelvic girdle (Figure 4F). Radial-axials on the post-axial side of the fin are now present through its entire length. Lepidotrichial flexors (L.f.) are present through the entire pre-axial side of the fin but are only covering the second and third elements post-axially. By stage 63, all pelvic muscles have appeared except for the mesial adductor (Figure 4G). The superficial ventromesial abductor is more developed and reaches farther anteriorly onto the pelvic girdle. The dorsolateral abductor levator (Dl. Abd. lev.) can be seen as a swelling on the pre-axial side of the fin at the level of the first axial element (Figure 4Gl). The superficial ventrolateral abductor (S. vl. abd.) is now present, visible as thin fibers reaching to the body myotomes (Figure 4Gld) and in ventral view, in the middle of the post-axial muscle bundles (Figure 4Gv). Radial-axials are completely covered by radial flexors and lepidotrichial flexors, now well developed both on the pre and post-axial sides of the fin.
Pelvic musculature development in the Australian lungfish. Immunostained larvae of Neoceratodus forsteri showing the developing pelvic musculature. All stages were incubated in a primary antibody against skeletal muscle. A and B were visualized through a secondary anti-mouse 488 Alexa antibody and C and D were visualized with a secondary anti- IgG1(γ1) 568 Alexa antibody. v: ventral view and l: lateral view. A) Stage 50, B) Stage 51, C) Stage 52, D) Stage 54, E) Stage '56', F) Stage '61', G) Stage '63'. Dl. Abd. lev., dorsolateral abductor levator; Dm. add. lev., dorsomesial adductor levator; D. V. abd. depr., deep ventral abductor depressor; D. V. add. depr., deep ventral adductor depressor; L.f., Lepidotrichia flexors; R.-A., radial-axials; R. f., radial flexors; S. vl. abd., superficial ventrolateral abductor; S. vl. add., superficial ventrolateral adductor; S. vm. abd., superficial ventromesial abductor. Anterior to the left.
Development of the pelvic musculature in the axolotl
The very first stage of this series is at the pelvic bud stage 55 [34] when only the rectus abdominis (R. a.) and the caudofemoralis (Cfe) are present, both of which are extensions of the hypaxial musculature (Figure 5A). Those muscles flank the emerging limb bud and at the following stage (1.5 cm), the ischiofemoralis (Isfe), the puboischiofemoralis internus (Pisfe int) and the iliofemoralis are all present (Figure 5Bl) (all muscles inserting into the limb). At 2.0 cm, the caudofemoralis muscle now extends all the way to the base of the femur and is a lot thicker. The puboischiotibialis (Pist) is now covering the ischiofemoralis and the caudopuboischiotibialis (Cpist) is now visible, linking the puboischiotibialis to bands of muscles extending towards the caudal vertebrae (Figure 5Cl). Fibers of the iliofemoralis now extend caudally, suggesting that its point of origin on the ilium extends dorsally in connection with the dorsal extension of the iliac cartilage (Figure 2). Shank flexors (Figure 5Cl) and extensors (Figure 5Cv) are now present, as well as the pubotibialis (pt), visible in the middle of the thigh in the ventral view. The 2.5 cm larva is very similar to the previous stage except for the distal leg muscles, which are missing along with the rest of the leg in this individual, having fallen victim to cannibalism (a common behavior in axolotl). The puboischiotibialis is thicker and extends farther anteriorly, the iliofemoralis extends a little more dorsally and the caudopuboischiotibialis can be clearly seen connecting the puboischiotibialis to muscle fibers extending to the caudal vertebrae (Figure 5Dl). In the ventral view, the puboischiofemoralis externus (Pisfe ext) is now present, its origin overlapping with that of the puboischiofemoralis internus (Figure 5Dv). Due to species variation in the pelvic shape, fibers of the puboischiofemoralis externus are more parallel to the hypaxial musculature than those of Necturus but can be distinguished from it by its posterior point of origin. The ischioflexorius (Isfl) is probably present at this stage as a very faint band of muscle originating from the ischium and extending distally on the post-axial side of the thigh (Figure 5Dvd). The last stage of this series is very similar to 2.5 cm. At 3.0 cm, the ischioflexorius is thicker and more visible and shank extensors and flexors as well as the popliteus are present (Figure 5Ed). All other muscles seem to have reached their adult conformation (Figure 5E) but the ischiocaudalis, the iliotibialis, ilioextensorius and iliofibularis cannot be seen in any view. They are all small, deep muscles, which may be covered by more superficial muscles, or they might be absent altogether in Ambystoma.
Pelvic musculature development in the axolotl. Immunostained larvae of Ambystoma mexicanum showing the developing pelvic musculature. All stages were incubated in a primary antibody against skeletal muscle and were visualized with a secondary anti-IgG1(γ1) 568 Alexa antibody. v, ventral view and l, lateral view; ld, lateral view, detail; d, dorsal view. A) Stage 55, B) 1.5 cm, C) 2.0 cm, D) 2.5 cm, *E) 3.0 cm. Cfe, caudofemoralis; Cpist, caudopuboischiotibialis; Ilfe, iliofemoralis; Isfe, ischiofemoralis; Isfl, ischioflexorius; Pisfe ext, puboischiofemoralis externus; Pisfe int, puboischiofemoralis internus; Pist, puboischiotibialis; Pop, popliteus; Pt, pubotibialis; R.a., rectus abdominis; S. e., shank extensors; S. f., shank flexors. Anterior to the left. *Shank extensors and flexors are absent because the distal part of the limb has been bitten of by another individual.
Comparative pelvic development and hypotheses of pelvic evolution
One of the major questions about the evolution of the tetrapod pelvic girdle during the fish-tetrapod transition has been the apparent shift in position of the acetabulum. In sarcopterygian fish, the fin articulation is located posteriorly on the pelvic girdle [11, 12, 15] whereas in early tetrapods, it is lateral [16, 17] (Figure 1). This seems to imply that the acetabulum has moved across the lateral face of the pelvis during the transition. However, the fact that the acetabular region is the first part of the pelvis to develop in both Neoceratodus and Ambystoma suggests that the acetabulum should instead be regarded as a fixed landmark (grey line, Figure 3). Such a change of perspective makes the evolutionary transformation of the pelvis much easier to understand. As shown in the descriptions above, the principal difference in early pelvic development between salamander and lungfish is that in the salamander, the pubis grows anteriorly and the ischium posteriorly from the acetabular region, whereas in lungfish the pubis grows anteriorly and the ischium is absent; in other words, chondrogenic cells proliferate both anteriorly and posteriorly in the salamander, whereas in the lungfish they only proliferate anteriorly. The presence of an ischium and the lateral position (and orientation) of the acetabulum are, therefore, developmentally coupled morphological states, presumably resulting from a change in molecular signalling in the immediate ancestors to tetrapods: there is no need to postulate a 'migration' of the acetabulum. Figure 3 schematizes pelvic development in lungfish and axolotl with this hypothesis in mind. The grey line represents the position of the acetabula for both species.
Another major question about the evolution of the pelvic girdle in tetrapods concerns the origin of the ilium and its relationship, if any, to the presence of an iliac process in Eusthenopteron, Goologongia and other fish members of the tetrapod stem group [11, 12, 14, 15]. The fact that the ilium of salamanders slowly extends dorsally during development, only contacting the sacral rib at a late stage when the pelvis is more or less fully formed, means that the ilium passes through a protracted developmental stage when it closely resembles the iliac process of these fishes (Figure 1). Together with the wide phylogenetic distribution of the iliac process, which suggests that it is a general character for the 'fish' part of the tetrapod stem group [11, 12, 14, 15], this provides strong circumstantial evidence for the homology of the two structures.
Muscle homologies in Coelacanth, Lungfish and a salamander, and the evolution of pelvic musculature
Here, the muscles of Necturus are described as a model for basal salamanders. There are almost twice as many muscles originating from the pelvic girdle in salamanders as in lungfish (13 versus 7) but six of these muscles are either originating or inserting on the ischium, which is a purported novelty in tetrapods. Very few pelvic muscles present in Necturus could not be associated with a muscle present in Neoceratodus. Among the exceptions is the puboischiofemoralis externus, originating mostly on the anterior process of the pubis, which is probably not homologous to the pubic process of Neoceratodus. However, the puboischiofemoralis is the only muscle with an ischiatic origin that could not be compared to a muscle in Neoceratodus. In all other cases, muscles originating from the mesial surface of the pelvic girdle in Neoceratodus had the same function and were very similar in terms of insertion point to muscles originating from the ischium in Necturus. This suggests that the ischium originated as an inflation of the posteromesial face of the pubis, created by increased proliferation of chondrogenic cells posterior to the acetabulum, an interpretation that also fits well with the data from skeletal development (Figures 2, 3). If the ischium is, in fact, a posterior projection of the pubis, the muscle groups paired up in Tables 1, 2, 3, 4 and 5 are likely to be truly homologous and the muscles themselves would not have changed substantially during the fish-tetrapod transition. This is consonant with the results from the comparative study of muscle development (see below). As for muscles originating from the ilium in Necturus, almost all seem to correspond to muscles originating from the posterodorsal surface of the pelvic girdle in Neoceratodus. Most known pelvic girdles of fossil tetrapodomorph fish have a lateral ridge or process on their posterodorsal surface [11, 12, 14, 15]. Most of these ridges are very slight but given the fact that muscles originating on the ilium in Necturus originate at the very base of it, a homologous muscle could in principle originate from a small process situated at the same position on the pelvic girdle of a fish. This gives further support to the hypothesis that the iliac process of Eusthenopteron and other fish members of the tetrapod stem group is the precursor to the ilium of tetrapods.
Comparative muscular development
In both Ambystoma and Neoceratodus, the hypaxial musculature develops first, followed by deep musculature originating from the pelvic girdle and inserting proximally onto the fin/limb. All muscles that have been equated in the tables above develop in the same order except for the caudofemoralis, which is the first pelvic muscle to develop in axolotls. Its homologue, the superficial ventrolateral abductor, develops last. This suggests a large heterochronic shift in the appearance of this muscle. The other, less dramatic, exception is the ischioflexorius, which develops slightly earlier in sequence than its counterpart, the superficial ventromesial adductor. The deep ventral abductor depressor, equivalent to the puboischiofemoralis internus, and the deep ventral adductor depressor, equivalent to the ischiofemoralis is the first homologous pair to appear. The dorsomesial adductor levator then appears in Neoceratodus and the iliofemoralis, in Ambystoma. These muscles have not been homologized on the basis of origin and insertion points but their developmental sequence might indicate some homology. The dorsomesial adductor levator of lungfish develops in the same sequence as the iliofemoralis and the radial-axial develop simultaneously to the caudopuboischiotibialis. Homologous pairs of slightly more superficial muscles appear next: the radial flexors/shank flexors and the superficial ventrolateral adductor/puboischiotibialis. The first of the superficial muscles to form in Ambystoma is the puboischiofemoralis externus, which was not homologized to any muscle in lungfish and is followed by the ischioflexorius. Its homologue (superficial ventromesial adductor) develops slightly later in lungfish, after the appearance of the superficial ventromesial abductor (synchronous with the development of the pubotibialis). This heterochronic shift might be functional in nature or an artefact of the whole mount staining method where discerning incompletely differentiated muscles can be difficult. Lepidotrichal flexors and the popliteus develop last. This might be due to the superficial nature of those muscles. Despite large differences in pelvic morphology, development of both the cartilage and muscles of these species is thus very similar. One important difference is that while the order of appearance is the same, there is more lag between consecutive muscle appearances in Neoceratodus. While several muscles appear at once in Ambystoma, new muscles appear one by one in Neoceratodus.
Further insight can be gained from the direction in which muscles develop. For example, the iliofemoralis is one of the first pelvic muscles to appear in 1.5 cm long Ambystoma larvae, the stage at which the ilium starts to extend dorsally (Figure 2Ilateral). This muscle continues to extend dorsally, presumably following the ilium. The same thing is true for muscles attaching to the pubis: in both Neoceratodus and Ambystoma, muscles originating from the pubis extend anteriorly through development, presumably following the anterior extension of the pubic cartilage. Similarly, radial-axial and lepidotrichial flexors of the fins of Neoceratodus start to develop pre-axially before developing post-axially, in a manner resembling that of the appearance of radials on the fin [38]. In summary, the sequence and mode of development of the pelvic musculature appears to be substantially conserved between Neoceratodus and Ambystoma, suggesting that the morphological transformation from pelvic fin to tetrapod hind limb was accomplished without major heterochronic reorganization of muscle development.
Despite large differences in pelvic morphology, the development of the pelvic girdle in Neoceratodus and Ambystoma is strikingly similar. Most pelvic muscles can be homologized between the two species and homologous muscles develop in the same order. Deep muscles develop first, followed by some muscles unique to either lungfish or salamanders and superficial muscles develop last. The only exception to this gradual development from deep to superficial is the appearance of the caudofemoralis of the axolotl at the very beginning of development whereas the superficial ventrolateral abductor of the lungfish develops last, along with the rest of the superficial musculature. The caudofemoralis originates from caudal vertebrae and is involved in tail motion. Its early appearance might be due to the very large reliance on undulatory motion of larval axolotls. Its early development might also be a consequence of the derived mode of development recently described for tetrapod pelvic appendages [40]. In lungfish, pelvic fin musculature develops from both a myotomal extension from the body wall and the migration of somite derived mesenchymal cells. However, in tetrapods, including Ambystoma, pelvic limb muscle formation is solely dependent on somite derived mesenchymal cells; it is argued that this mode of development would allow for earlier initiation and easier heterochronic reorganization of muscle development.
With regards to the skeleton, if we consider the acetabulum to be a fixed landmark, the evolution of the ischium simply required pre-chondrogenic cells of the pubis to migrate posteriorly as well as anteriorly in order to form it. This would explain why very similar muscles originating from the inside of the pubis in the lungfish originate from the ischium in the salamander. The ischium does not ossify in neotenic forms, such as the axolotl. However, in several salamander taxa, the ischium ossifies whilst the pubis remains unossified (Personal observation, CAB). This is reminiscent of the condition in early tetrapods [2] and might have initially been caused by the greater reliance on muscles originating from the ischium such as the puboischiotibialis. Ossification would have occurred in the ischium as a result of muscular tension which is known to promote ossification [41, 42]. It would be interesting to combine the results of this study and recent biomechanical studies [8, 43] to test those hypotheses. As for the origin of the ilium, some very similar muscles originating from the posterodorsal surface of the pelvis in Neoceratodus originate from the base of the ilium in Ambystoma, suggesting that the lateral ridge or process present in most tetrapodomorph fish is the precursor of the tetrapod ilium (Figure 1). This precursor would then have extended dorsally to eventually reach and articulate with the sacral rib, as observed by the development of both iliac cartilage and muscles. In order to test those hypotheses, the origin and insertion points of the muscles of Latimeria, Neoceratodus and Ambystoma should be verified using three-dimensional models generated from undissected computed tomography (CT)-scanned specimens. This will provide a more robust comparative morphological framework against which to analyze triple-stained (double immunostained for muscles and nerves plus alcian stained for cartilage) growth series of Neoceratodus and Ambystoma by optical tomography (OPT), which allows for three-dimensional three-color visualization. As a complement to these techniques for investigatng extant morphologies, the application of propagation phase contrast synchrotron microtomography (PPC-SRμCT) to the study of fossil bone histology is opening up new possibilities for detecting muscle attachments on the pelvic bones of fossil fishes and early tetrapods [44]. We expect that data extracted by these novel methods will cast a great deal of light on the transformation of the pelvic appendage at the fish-tetrapod transition and perhaps help resolve the question of the origin of tetrapod locomotion raised in several recent publications [8, 43, 45].
Lungfish and Axolotl pelvic girdle
Ac:
Acetabulum
Ant pr:
Anterior process
Fe:
Il:
pubis
Sr:
Sacral rib
Vc:
Vertebral column
Yp c:
ypsiloid cartilage
Lpm:
Lungfish pelvic musculature
Dl. Abd. lev:
dorsolateral abductor levator
Dm. add. lev:
dorsomesial adductor levator
D. V. abd. depr:
deep ventral abductor depressor
D. V. add. depr:
deep ventral adductor depressor
L.f:
lepidotrichia flexors
S vl abd:
Superficial ventrolateral abductor
S vl add:
superficial ventrolateral adductor
S. vm. abd:
superficial ventromesial abductor
R.-A:
radial-axials
R. f:
Radial flexors
Apm:
Axolotl pelvic musculature
Cfe:
caudofemoralis
Cpist:
caudopuboischiotibialis
Ilfe:
iliofemoralis
Isfe:
ischiofemoralis
Isfl:
ischioflexorius
Pisfe ext:
Puboischiofemoralis externus
Pisfe int:
Puboischiofemoralis internus
Pist:
Puboischiotibialis
Pop:
Popliteus
Pt:
Pubotibialis
R.a:
Rectus abdominis
S. e:
Shank extensors
S. f:
Shank flexors.
We would like to thank Rolf Ericsson and Elizabeth Eyre for the collection and care of egg and larvae of Neoceratodus. CAB would like to thank Laura Muzinic of the Ambystoma genetic stock center for raising and shipping a large quantity of older larvae of Ambystoma. She would also like to thank Silke Berger, Peter Currie and Rob Bryson-Richardson for providing protocol and technical advice. CAB would like to thank Daniel Snitting for proofreading this manuscript. We thank Rui Boliqueime Martins Diogo and two anonymous reviewers for constructive comments on the manuscript. CAB is currently supported by a Human Frontiers Science Program Cross-Disciplinary Fellowship (LT000130/2009 L) and two Australian Research Council Discovery Project Grants (DP1096002 and DP110101127). Part of this work was carried out while CAB was a PhD student at Uppsala University; at that time, she was supported by Uppsala University and the Swedish Research Council, through a grant to PEA. At present, PEA is supported by a Wallenberg Scholarship provided by the Knut and Alice Wallenberg Foundation. JMPJ is supported by the Australian Research Council. The monoclonal antibody 12/101 developed by Jeremy P. Brockes was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA.
Authors' original submitted files for images
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13227_2012_77_MOESM1_ESM.tiff Authors' original file for figure 1
CAB planned and performed the experiments, collected the data, prepared the manuscript and the figures. PEA contributed to the conception and design of the experiments and to the analysis and interpretation of the data. JMPJ contributed materials and all authors contributed, read and approved the final manuscript.
Australian Regenerative Medicine Institute, Monash University, Wellington Road, Clayton, VIC, 3800, Australia
Subdepartment of Evolution and Development, Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, Uppsala, 752 36, Sweden
School of Biological Sciences, Macquarie University, Sydney, NSW, 2010, Australia
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Walthall JC, Ashley-Ross MA: Postcranial myology of the California newt, Taricha torosa. Anat Rec A Discov Mol Cell Evol Biol. 2006, 288A: 46-57. 10.1002/ar.a.20279.View ArticleGoogle Scholar
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Wiens JJ, Bonett RM, Chippindale PT: Ontogeny discombobulates phylogeny: paedomorphosis and higher-level salamander relationships. Syst Biol. 2005, 54: 91-110. 10.1080/10635150590906037.View ArticlePubMedGoogle Scholar
Boisvert CA: Vertebral development of modern salamanders provides insights into a unique event of their evolutionary history. J Exp Zool B. 2009, 312: 1-29.View ArticleGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The following sections describe general issues you may encounter while using the Jive 5/SBS/Clearspace connector and attempts to provide the best course of action to resolve them.
Permissions in Jive are flexible. They support users and groups, use inheritance to propagate through sub-communities, and support a hierarchy of precedence so an explicit permission always overrides an inherited one, whether it is denied or allowed.
Since permissions in CES only support denied and allowed entries, a denied entry always overrides an allowed entry for a user or a group, with no regard to whether the allowed permission was inherited or explicitly set. For that reason, when group permissions are used in Jive, in some cases these groups have to be expanded and permissions have to be set on group members to make sure CES permissions mirror those from Jive.
Example: Consider the following situation.
Group A contains three members: member 1, member 2 and member 3.
For a given Jive community, Group A is denied the View Documents permission through an inherited permission but member 1 is explicitly allowed the View Document permission.
When you look at the final permissions in CES, you see that member 1 is effectively allowed on any document from that community and that member 2 and member 3 are denied. There will be no denied permission set on Group A since doing so would deny all members from the group, including member 1.
Therefore, expanding groups and setting permissions on group members rather than on the groups themselves is not effective in CES until a refresh operation is performed.
Modifications made to permissions in Jive do not impact the last modification date of objects affected by the permission modification. Since incremental refresh is using this last modification date to retrieve objects to update, the permission modification is not detected.
When Jive permissions are frequently modified, schedule a source refresh weekly or more often to keep the index permissions synchronized with Jive permissions.
The Jive 5/SBS/Clearspace connector keeps a local cache of all active Jive users for faster access and to improve indexing performances. New Jive users are detected by incremental refresh and added to CES only when this local cache of users expires and is refreshed.
By default, the user cache is refreshed every 24 hours but can be refreshed more often by adding the UserCacheLifeSpan parameter to your source and setting its value (in minutes) accordingly (see Modifying Hidden Jive 5/SBS/Clearspace Source Parameters).
Note: The UserCacheLifeSpan parameter is also available on the security provider and its value should always match the value set on its associated source (see Configuring a Jive 5/SBS/Clearspace Security Provider). If a security provider is used by more than one source, make sure the UserCacheLifeSpan parameter on the security provider matches the lowest value of this parameter among all its associated sources.
The connector needs to keep track of items that were deleted from Jive in order for incremental refresh to keep the index up-to-date. Every time a incremental refresh run completes, items that were deleted more than two weeks ago are removed from the deleted history.
If incremental refresh was disabled on a source for a period greater than two weeks and you have more than one source performing incremental refresh on the same Jive server, you should perform a source refresh on the source where incremental refresh was disabled to make sure your index is fully up-to-date.
If the web server hosting Jive is configured so that SSL is enabled for Jive, you should always use the secure starting address.
This is the typical error message returned by Jive when an internal error occurs on the Jive server. While there is not much that you can do to prevent these errors from occurring, it is recommended to restart the Jive service if the errors occur frequently.
A PermGen related error means the Jive server was temporarily out of memory. If this error occurs repeatedly, ensure your Jive JVM settings follow the recommendation and, if required, increase the PermGen Heap space to at least 512 MB (see the Jive document Adjusting the Java Virtual Machine (JVM) Settings).


Maura Derrane admits that getting older means that it is easier to gain weight and with her return to TV just weeks away it is something that she is quite conscious of.
As one of the most recognisable- and beautiful- TV presenters in Ireland a lot of focus is on her day-to-day and that has resulted in some criticism from viewers, but she is able to rise above it. Even though some people may be more sensitive.
Asked if she was nervous about her weight and getting her figure right before going back on air, she said: "Well, you probably need to drop half a stone. I came back from holiday in Italy and I said that my weighing scales were definitely broken [laughs]. The older you get, the easier it is to put on weight and that is just a reality of life."
Earlier this year, the Galway woman was targeted by trolls over an outfit she wore on Today with Maura and Dáithí .
However, instead of getting down about it she worries about society as a whole.
"They must have a lot of time on their hands because I wouldn't have that much time to be trolling around. This year, I was just more annoyed than anything because I was wearing one of the most expensive outfits I have worn on air and it was just very regular. I wasn't wearing hot pants or some sort of crazy top, it was just a regular outfit.
"I worry about people like that and I worry that society is going in the wrong direction. I worry about the desensitization of us as people and that we think that we can say what we want without repercussions.
"Would you say such negative things to people in the street? No of course not. A lot of people who make comments should really look into the mirror themselves," she said in this month's issue of RSVP Magazine.
These comments seem to be coming from women as well and the mum-of-one think that it is shocking that it is women attacking other women.
"I have always said that women are the worst, women can be so bitchy towards other women. With the Me Too movement growing legs this year, women should look inwardly and not blame men.
"Standing together in a campaign is well and good but people need to mean what they are saying. #Metoo? It should be #lookintothemirror," she adds.


text:
Q: Wrong DateTime field (+1 hour) in a Telerik RadGrid with Odata client binding I'm using a RadGrid (but I think the problem is not with the grid itself) with client binding to a service with Type = Odata, ResponseType = JSON.
With Fiddler, when I see the Raw Detail of the call to my service I can see: "LogDate": "/Date(1417796043643)/"
Correct me if I'm wrong, this number is the DateTime converted to the milliseconds from 01/01/1970.
All the algorithm I found googling around (and the telerik grid too) are converting this number to: 05 December 2014 17:14:03.
But in my DB the value of  'LogDate'  field of the table where the service is reading from, is 16:14:03!!
As far as I understood the "Date" javascript object takes in input an UTC Date and automatically adds the 'offset' of my timezone ( I'm GMT+1 ) and this is where the +1 hour comes from! (maybe? :D)
Hoping I explained myself well. Any solution? Maybe an event where i can intercept the JSON response (OnRowCreating?) and manupulate myself?
Thanks!

redpajama_set_name: RedPajamaStackExchange


text:
Facebook Reveals Details Of Its Cryptocurrency Libra
On the 18th of June, Facebook revealed details of its brand new cryptocurrency called Libra. An alternative to cash, this cryptocoin can be used to buy things or send money to people with close to no fees. The cryptocurrency is going to be launched by an association called The Libra Association, which consists of a group of companies interested in getting Libra out into the world.
You can use Libra to buy or cash out your Libra at local stores like grocery stores and through third party wallet apps. To make using Libra an easier task, Facebook owned Calibra Wallet will be built into WhatsApp and Messenger, thereby simplifying transaction processes. Although certain countries have banned the use of cryptocurrencies, Facebook is trying to break new ground with the launch of Libra.
While Facebook is launching Libra, the social media giant is not going to be in complete control of the cryptocoin. The coin will be controlled by a consortium consisting of its founding members, Visa, Uber and Andreessen Horowitz. The three companies have invested at least $ 10 million each into the creation of this cryptocurrency.
Customers interested in holding or transferring the newly acquired token will be given multiple options to do what they wish. To further simplify matters, Facebook will let customers access transactions through this cryptocurrency via a special app designed for iOS and Android.
To protect the identity of its users while making transactions through the new app, Facebook won't require you to share personal details. "The advent of the internet and mobile broadband has empowered billions of people globally to have access to the world's knowledge and information, high fidelity communication, and a wide range of lowercost, more convenient services," the Libra Association said in a paper announcing the cryptocurrency.
Libra will be made available to the world during the first half of 2020. As of now, Facebook is focusing on building relationships in international waters.
Stay tuned for more updates.
Related Topics:2019 Latest Technology NewsCryptocurrency LibraFacebook Announces Libra Cryptocurrencyfacebook latest newsFacebook LibraFacebook Libra ProjectFacebook New CryptocurrencyFacebook Reveals Cryptocurrency LibraFeaturedNew Libra Cryptocurrencystartup stories
Binny Bansal Sells ₹531 Crore Worth Flipkart Shares To Walmart
Facebook Invests In Indian Startup Meesho

redpajama_set_name: RedPajamaCommonCrawl


The Thermo Scientific™ HAAKE™ Viscotester™ iQ rheometer — the new Quality Control rheometer was designed to support the demands of new rheological tasks in quality control. The Viscotester iQ rheometer enables fast, reliable and precise rheological measurements with maximum ease of operation.
Find out all about the next generation of Quality Control rheometers and how it helps you in our webinar.


text:
* Gulp => manifest.yyyyMMddhhmmss.json
{
    files: [
        { path: './am/services/cuid.js', hash: 'dkls9kekd94ekkjf9999k' }
    ]
}
* Gulp => publish to CDN, only add "new" files.
* Each filename ends with hash:
    * to cachebust cdn
    * to allow multiple version of the app run side by side (A - B testing etc.)
* The app will always have only one version of the file
    * To preserve space
    * only one version of the app can be available on a device at one time.
* Per user settings: User.AppVersion: string => yyyyMMddhhmmss
* Per sync return User.AppVersion if changed, update AppVersion
* When all files are updated, updat client User.AppVersion.
* When file does not exist:
    * Clear System registration
    * System.import it.
    * update file ondisk

redpajama_set_name: RedPajamaGithub


text:
package com.antsoft.ant.property.defaultproperty;

import java.awt.*;
import java.awt.event.*;
import java.io.File;
import java.util.*;
import javax.swing.*;
import javax.swing.event.ChangeListener;
import javax.swing.event.ChangeEvent;

import com.antsoft.ant.util.*;

public class WebPanel extends JPanel {
  private JComboBox browserCombo;
  private Vector allPaths;
  private DefaultComboBoxModel model;
  private JButton addBtn, removeBtn;
  private JFrame parent;
  private JCheckBox internalCbx;
  private boolean isInternal = true;

  public WebPanel(JFrame parent) {
    this.parent = parent;
    try  {
      jbInit();
    }
    catch (Exception ex) {
      ex.printStackTrace();
    }
  }

  void jbInit() throws Exception {
    setLayout(new BorderLayout());
    setBorder(BorderList.etchedBorder5);

    ActionListener al = new ActionEventHandler();

    internalCbx = new JCheckBox("Use internal JavaDoc Viewer");
    internalCbx.setSelected(true);
    internalCbx.addChangeListener(new ChangeListener(){
      public void stateChanged(ChangeEvent e){
        if(internalCbx.isSelected()) {
          isInternal = true;
          browserCombo.setEnabled(false);
        }
        else {
          isInternal = false;
          browserCombo.setEnabled(true);
        }
      }});

    JPanel internalP = new JPanel(new BorderLayout());
    internalP.add(internalCbx, BorderLayout.WEST);

    //label
    //JLabel webLbl = new JLabel("WebBrowser Path");
    //JPanel webLblP = new JPanel(new BorderLayout());
    //webLblP.add(webLbl, BorderLayout.WEST);

    //combo box
    allPaths = new Vector(2, 1);
    model = new DefaultComboBoxModel(allPaths);
    browserCombo = new JComboBox(model);
    browserCombo.setPreferredSize(new Dimension(330,20));
    browserCombo.setEnabled(false);

    //button
    addBtn = new JButton("Add");
    addBtn.setActionCommand("ADD");
    addBtn.addActionListener(al);

    removeBtn = new JButton("Remove");
    removeBtn.setActionCommand("REMOVE");
    removeBtn.addActionListener(al);

    JPanel btnP = new JPanel(new FlowLayout(FlowLayout.LEFT));
    btnP.add(addBtn);
    btnP.add(removeBtn);

    //combobox panel
    JPanel comboP = new JPanel(new BorderLayout());
    comboP.add(browserCombo, BorderLayout.CENTER);

    JPanel p = new JPanel(new FlowLayout(FlowLayout.LEFT));
    p.setPreferredSize(new Dimension(350,100));
    BlackTitledBorder border = new BlackTitledBorder("WebBrowser Path");
    p.setBorder(border);
    p.add(comboP);
    p.add(btnP);

    Box box = Box.createVerticalBox();
    box.add(internalP);
    box.add(p);

    JPanel centerP = new JPanel(new BorderLayout());
    centerP.add(box, BorderLayout.NORTH);
    centerP.add(new JPanel(),BorderLayout.CENTER);

    add(centerP, BorderLayout.CENTER);
    add(new JPanel(), BorderLayout.NORTH);
    add(new JPanel(), BorderLayout.WEST);
    add(new JPanel(), BorderLayout.EAST);
    add(new JPanel(), BorderLayout.SOUTH);
  }

  public boolean isInternalUse(){
    return this.isInternal;
  }

  public void setInternal(boolean flag){
    this.isInternal = flag;
    this.internalCbx.setSelected(flag);
  }

  public void addBtnSelected(){
    ExtensionFileFilter filter = null;

    if(OSVerifier.getOS().equals(OSVerifier.MS_WINDOW)){
      filter = new ExtensionFileFilter("exe");
    }

    JFileChooser filechooser = new JFileChooser();
    if(filter != null) filechooser.setFileFilter(filter);

    filechooser.setPreferredSize(new Dimension(400, 300));
    filechooser.setApproveButtonText("Select");
    filechooser.setApproveButtonToolTipText("Select WebBrowser Path");
    filechooser.setBorder(BorderList.lightLoweredBorder);
    filechooser.setDialogTitle("WebBrowser path");

    if(filechooser.showOpenDialog(parent) == JFileChooser.APPROVE_OPTION) {
      File f = filechooser.getSelectedFile();
      if(model.getIndexOf(f.getAbsolutePath()) == -1){
        model.addElement(f.getAbsolutePath());
        model.setSelectedItem(f.getAbsolutePath());
      }
    }
  }

  public void removeBtnSelected(){
    if(model.getSize() > 0)
    model.removeElement(browserCombo.getSelectedItem());
  }

  /**
   * return current web browser path
   *
   * @return current web browser path
   */
  public String getSelectedBrowserPath(){
    String item = (String)model.getSelectedItem();
    if(item == null) return null;

    StringBuffer ret = new StringBuffer();
    StringTokenizer st = new StringTokenizer(item, "\\", true);
    while (st.hasMoreTokens()) {
      String token = st.nextToken();
      if(token.indexOf(" ") != -1){
        token = makeDosStyle(token);
        ret.append(token);
      }
      else{
        ret.append(token);
      }
    }
    return ret.toString();
  }

  public String getSelectedBrowserPathToSave(){
    return (String)model.getSelectedItem();
  }

  private String makeDosStyle(String path){
    String ret = null;
    ret = path.substring(0, 6);
    ret = ret + "~1";
    return ret;
  }

  public void setSelectedBrowserPath(String path){
    if(model.getIndexOf(path) != -1){
      model.setSelectedItem(path);
    }
  }

  public Vector getAllBrowserPaths(){
    Vector ret = new Vector(2, 1);
    for(int i=0; i<model.getSize(); i++){
      ret.addElement(model.getElementAt(i));
    }
    return ret;
  }

  public void setAllBrowserPaths(Vector allPaths){
    model = new DefaultComboBoxModel(allPaths);
    browserCombo.setModel(model);
  }

  class ActionEventHandler implements ActionListener{
    public void actionPerformed(ActionEvent e){
      String cmd = e.getActionCommand();
      if(cmd.equals("ADD")){
        addBtnSelected();
      }
      else if(cmd.equals("REMOVE")){
        removeBtnSelected();
      }
    }
  }
}


Mark Watney is stranded….on Mars. That in itself was enough to grab my interest. It makes the idea of being stranded on a desert island almost a non event.
After a huge sand storm and an equally huge misunderstanding by his crew mates Mark is left to die. Unless he can find a way to survive for about four years in the totally hostile environment he finds himself in.
This takes sci-fi to the extreme, it shows very cleverly how the answers to seemingly insurmountable problems can be achieved by applying science and the resourcefulness of man. Mark's engineering skills are tested to the limit as is his determination not to give in to the impossible odds stacked against him.
The amount of research that must have gone into this book is incredible. Although a lot of the technicalities were way over my head it didn't get in the way of my enjoyment of the book at all. It's very interesting, amazingly descriptive and detailed and gives a really visual picture of what Mars would be like and how unforgiving it is.
Every time Mark finds an ingenious solution to one problem another one arises, it's very gripping and so full of suspense. And that's no mean feat considering for most of the time it was Mark's narration with no dialogue except when the focus switched to the US and China for a short while. Mark is very intelligent and creative and tells his story with humour and wit through his, mostly daily, log entries. A very likeable and real personality.
I enjoyed the narration very much, R C Bray was Mark Watney with all the emotional reactions you would expect in such a bizarre and nerve-rackingly tense situation.


Planet Earth is the home planet of the Humans and capital world of the Confederacy of Humanity.
Earth, (also known as Gaia, Sol Prime or Terra) is the third planet in the Sol System and the homeworld of humanity, plus the first natural known E-Class world to humans. Earth has one natural satellite, Luna and is the nominal center of the Confederacy of Humanity. It's capitals are Washington D.C, and Geneva.
As of 200000, the Earth has undergone major upheavals, but is currently a paradise like it was at the start of the Fourth and Fifth Age. Many green megacities can be seen from orbit spread across the planet, a few even in the ocean. Most farming on Earth is accomplished via either massive farming towers built in placed where there could be no cities before (e.g. Sahara Desert) or large floating farms on the oceans, but there are still some foodstuffs imported from other colonies.
The Earth is greener than it was back in the 21st century thanks to the population crash and subsequent ruralization of most of the large cities on Earth. Each building and home of each terrestrial city and megacity now is required to have small plants and/or grass growing on their roofs as well as in their lawns. There were even a few cities, such as Detroit, Michigan and Dhaka, Bangladesh that were completely abandoned and taken down in order to let nature reclaim the land. Those cities had since been rebuilt greener.
Most of the energy on Earth comes from a mixture of solar energy, fusion energy, wind energy, hydroelectric energy and geothermal energy as well as a few others.
Being the homeworld of humanity the global population of 30 billion makes Earth the largest populated body in the entire system, and that doesn't include the large amount of habitats and space elevator cities in orbit. Most of the population live in the megacities, but some still live in parts of reclaimed nature. Earth is the most diverse world culturally. Earth is nearing overpopulation like its twin Venus. There are a few megacities that can be seen from orbit. London ,Paris, Berlin, Moscow, New York, Washington DC, Delhi, Mumbai, Shanghai, Beijing, Tokyo, Jakarta, Singapore, Lagos, Sao Paulo, Madrid, Dubai, Rome, St. Petersburg and Atlantic city.
Earth has one of the most diverse climates in the entire system. Cold enough at the poles to freeze water and warm enough at the equator to fry an egg on a metallic surface, each region of Earth has a unique biosphere able to support a variety of plant and animal life.
Earth never had any rings until the year 13728 CE. In celebration of Vega becoming Earth's new North Star, a petition went through the terrestrial government, pleading for a beautification type project, as Solarians (which is what all inhabitants of the Sol System called themselves at the time) were obsessed with physical beauty. Many of asteroids and comets were diverted and crashed into each other. A few smaller asteroids were placed in orbit in order to keep the rings from degrading and trillions of nanites were released to construct the rings. The nanites then rained down after completing their tasks, forming the most brilliant meteor shower seen on Earth in millenia. They have since remained stable.
After the War of the Decamillenial Transition and the Split of the Confederacy, Earth was left with only one billion inhabitants, mostly the richer who used automation to live and survive. The descendants of those evacuated began to slowly come back, although the majority had made new worlds for themselves, most notably three copies of Earth using Terrashaping. By the year 21000, Earth's megacities had been repopulated. By the year 25000, the population remained steady at around 30 billion, mostly within the megacities once again, although the land was allowed time to fallow and regrow.


text:
William E. Schuyler, Jr.
William E. Schuyler Jr. was born on February 3, 1914, in Washington, D.C., and resided in the Washington area for his entire career. His mother operated a patent information business. He received a bachelor's degree in electrical engineering from Catholic University and a law degree from Georgetown University.
He was a law firm partner and litigator who represented clients in district court patent infringement trials throughout the United States. He argued many appeals in regional Courts of Appeals in the era before the Court of Appeals for the Federal Circuit was established. He taught a law school course in patent litigation for more than 20 years.
President Richard Nixon appointed Schuyler commissioner of patents, and he took the oath of office on May 7, 1969.
He obtained approval for the United States to host a diplomatic conference in Washington to negotiate the Patent Cooperation Treaty, which allowed patent applicants to start the process of obtaining patents in multiple countries by filing a single international application. The diplomatic conference was one of the largest on intellectual property rights ever held in the United States, with 77 countries and 22 international organizations represented. The United States and other countries signed the treaty at the end of the conference on June 17, 1970.
U.S. industry donated generously to entertain the visiting diplomats at the conference. A highlight was a one-day break to visit the Kennedy Space Center in Florida. About 200 delegates and guests departed on a chartered Boeing 707 from Dulles airport and returned the same day. It was less than a year after the first moon landing.
Other treaties signed or ratified during Schuyler's term included the treaty establishing the World Intellectual Property Organization, ratified in 1970, the Stockholm revision of the 1883 Paris Convention for the Protection of Industrial Property, also ratified in 1970, and the Strasbourg Agreement on international patent classification, signed in 1971.
In 1970, for the first time, the office received more than 100,000 patent applications. By 1971, with patent and trademark fees set by statute and therefore frozen, the portion of the office supported by fee income had fallen to 50 percent.
Schuyler experimented with examiners using dictating machines with support from a typing pool. Later he introduced a printed form for some examiner "actions" with the forms filled out in handwriting. The office established a Disclosure Document Program to allow inventors to deposit papers in the office describing their inventions while they were deciding whether to file a patent application, but usage was low.
In 1970 the office was one of the first large customers of the Government Printing Office to adopt a new printing system to replace hot metal printing, in which patent text was keyboarded onto magnetic tapes. A side benefit was a database of tapes to use for other purposes.
Patent printing fell behind in 1971. A few hundred patent numbers were voided, breaking the numbering sequence followed since 1836. Some patents were issued on Wednesdays, the first time in the 20th century that patents were issued on any day but Tuesday.
During Schuyler's tenure the office accelerated efforts to use automation for efficiency and quality in patent searching and subject matter classification. The largest effort was Project POTOMAC, later abandoned in favor of programs that did not require research and development.
Schuyler started an Office of Technology Assessment and Forecast that began disseminating information on trends in technological activity. According to the 1971 Commissioner's Annual Report, it was an effort "to take advantage of the wealth of statistical information generated by the Patent Office as a byproduct of examining and issuing patents."
Schuyler resigned on August 26, 1971. After he returned to private life he was called on to represent the United States in additional international meetings, in at least one instance with the rank of ambassador. He practiced law for several years. He died July 25, 2007, in Rockville, Maryland, at age 93.
Stacy V. Jones, The Patent Office (1971).
USPTO, Annual Reports of the Commissioner of Patents (1969-71).
USPTO, The Story of the U.S. Patent and Trademark Office (1988).


A warm welcome to the Holistic Chiro-Practice Berlin. Chiropractic is a free of pain, effective and pleasant method of treatment to relieve blockages of the spine and joints.
Chiropractors are looking holistically at the interplay of the spine, nerves, joints and musculature in order to ease pain in a fast and, at the same time, gentle way. Further, it is a major principle of chiropractic to identify root causes by holistic diagnosis, in order to elevate your mobility and well-being in the long term.
We are happy to advise you on if and how we may best help you. Please call us and we will take time for you.
Our chiropractors have completed full-time academic studies, which guarantees the precision and quality of our manual treatments. With all that, your well-being is our main concern.
As a chiropractor and non-medical practitioner, Jan is guiding his patients effectively and holistically on the path of well-being.
As a chiropractor, his main concern is putting the patient in the center of attention.
Patricia Peer is a Chiropractor and joined our team in the fall 2018.
Your treatment success when suffering from pain is scientifically proven. By the union of modern medicine, a holistic approach and the ideas of man, chiropractic makes mobility, balance and vitality possible with lasting effects. At the beginning of the treatment our chiropractors will find out, together with you, how to realize a new way of life to improve the quality of your everyday life. For this, we are alway making use of creative and individual concepts.
The human being is a complex system. In the model of chiropractic we are always having a positive influence on all levels of being.
A chiropractor completes full-time academic studies lasting 5-7 years, guaranteeing the precision and quality of their manual treatment.
You as a human being are always in the center of our attention. You will always get the time to fully explain all your ailments and problems.
Jahrelang hatte ich Probleme mit meiner Wirbelsäule. Vor den Behandlungen durch Jan dachte ich daß sich Stress bei mir immer negativ auf die Wirbelsäule auswirkt. Doch schon nach den ersten Behandlungen gingen meine Probleme zurück und kamen auch bei späteren erhöhtem Stress nicht wieder. Ich habe das erste Mal seit einem Jahrzehnt einen so langen Zeitraum ohne Schmerzen verbracht. Durch Jans professionelle Behandlung sowie dem ganzheitlichen Ansatz kann er tiefer liegende Probleme erkennen und erfolgreich behandeln. Wer freundliche und vor allem kompetente Hilfe sucht ist bei Jan Müller genau richtig.
Jan has helped our daughter so much! She trains five days a week in ballet and her body was really out of alignment from the intensity of her classes. Jan is always so good to find the root issue and help put her back together again. We're so thankful for him!
Ich habe Jan Müller gesucht, ich habe Ihn gefunden. Ich habe Ihn um Hilfe gebeten und nach unserem ersten Gespräch wusste ich, er kann nicht nur zuhören, er ist auch anempfindend. Ich fühlte mich ernst genommen, freundschaftlich angenommen und da war Vertrauen und Harmonie. Nach wenigen Sitzungen spürte ich Linderung, Klarheit im Kopf, zurückgeschenkte Lebensfreude und Hoffnung auf völlige Schmerzfreiheit. Ich kann wieder mit meinem Körper umgehen, und ich erfahre eine unermessliche Weite in meinem Inneren. Dafür bin ich sehr dankbar.


When should a building material be sampled?
If you suspect an area within your home or place of work contains asbestos materials, you would be right to be cautious and to take every care before you disturb it. In addition, many homebuyers' surveys now highlight the potential for specific materials such as artex to contain asbestos.
If requested we will send one of our team around to the property to take a sample from a specified area. This is a relatively straight forward task and does not pose any danger to the occupants of the building. After the sample has been analysed we will contact the client with the result and if required, provide free impartial advice on how to deal with the material should any asbestos be found during the analysis.
We also work regularly with insurance companies to deliver a fast, efficient and impartial sampling service to their valued customers.
Standard turnaround is 3 to 5 days however same day analysis can be undertaken with prior notice.


When: April 22 & 23, 8 p.m.
When: April 22 and 23, 8:00 p.m.
Selections for Spring, Momentum Dance Co.
Location: Heritage Crossing District, Main Street & Irving Blvd.
When: April 23, 11 a.m. - 5 p.m.
Books and Authors and Teens, oh my!
When: April 23, 8 a.m. - 5 p.m.