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Paraffin histology is one of the most important and commonly-used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs' correspondents, embryologist Wilhelm His, Sr., learned of Klebs' experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His's paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His's protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His's protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol.Die paraffinbasierte histologische Untersuchung ist eines der wichtigsten angewendeten Laborverfahren in der diagnostischen Histopathologie. Die Entwicklung der Paraffineinbettung wird oft dem Pathologen Edwin Klebs zugeschrieben. Klebs orientierte sich an den Arbeiten von Stricker, der Embryonen in eine Mischung aus heißem Tristearin und weißem Bienenwachs einbettete, und experimentierte mit Paraffin, um Tumorgewebe einzubetten. Er verwarf den Ansatz aber rasch, da Paraffin das Gewebe nicht infiltrierte. Klebs stand im Briefwechsel mit dem Embryologen Wilhelm His Senior, der von dessen Experimenten erfuhr und beschloss, sich an der Paraffineinbettung zu versuchen. His entwässerte Hühnerembryonen in Alkohol, klärte sie mit Lavendelöl und träufelte heißes Paraffin darüber. Dieses Verfahren ermöglichte ihm die Anfertigung guter Schnitte. In der vorliegenden Arbeit haben wir His’ Protokoll der Paraffineinbettung nachvollzogen, um zu ermitteln, ob His tatsächlich die Infiltrationseinbettung mit Paraffin gelungen ist. Wir befolgten das Protokoll aus seiner 1868 erschienenen Monografie. Anhand der Anweisungen von His gelang es uns nicht, Schnitte mit der von ihm illustrierten Qualität herzustellen. In der Regel zerfiel das Gewebe beim Schneiden wegen schlechter Wachsinfiltration. Brauchbare Schnitte wurden nur bei Abwandlung von His’ Protokoll durch das Wiedereinschmelzen der eingebetteten Embryos in frischem Paraffin erreicht. Möglicherweise ist es uns nicht gelungen ist, das Protokoll von His detailgetreu zu replizieren, oder das Protokoll war unvollständig. Nach unserer Einschätzung hat His wahrscheinlich die Infiltrationseinbettung mit Paraffin erfunden und perfektioniert, aber kein vollständiges Protokoll veröffentlicht.

Paraffin histology is one of the most important and commonly used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs' correspondents, embryologist Wilhelm His, Sr., learned of Klebs' experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His's paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His's protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His's protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol.Die paraffinbasierte histologische Untersuchung ist eines der wichtigsten angewendeten Laborverfahren in der diagnostischen Histopathologie. Die Entwicklung der Paraffineinbettung wird oft dem Pathologen Edwin Klebs zugeschrieben. Klebs orientierte sich an den Arbeiten von Stricker, der Embryonen in eine Mischung aus heißem Tristearin und weißem Bienenwachs einbettete, und experimentierte mit Paraffin, um Tumorgewebe einzubetten. Er verwarf den Ansatz aber rasch, da Paraffin das Gewebe nicht infiltrierte. Klebs stand im Briefwechsel mit dem Embryologen Wilhelm His Senior, der von dessen Experimenten erfuhr und beschloss, sich an der Paraffineinbettung zu versuchen. His entwässerte Hühnerembryonen in Alkohol, klärte sie mit Lavendelöl und träufelte heißes Paraffin darüber. Dieses Verfahren ermöglichte ihm die Anfertigung guter Schnitte. In der vorliegenden Arbeit haben wir His’ Protokoll der Paraffineinbettung nachvollzogen, um zu ermitteln, ob His tatsächlich die Infiltrationseinbettung mit Paraffin gelungen ist. Wir befolgten das Protokoll aus seiner 1868 erschienenen Monografie. Anhand der Anweisungen von His gelang es uns nicht, Schnitte mit der von ihm illustrierten Qualität herzustellen. In der Regel zerfiel das Gewebe beim Schneiden wegen schlechter Wachsinfiltration. Brauchbare Schnitte wurden nur bei Abwandlung von His’ Protokoll durch das Wiedereinschmelzen der eingebetteten Embryos in frischem Paraffin erreicht. Möglicherweise ist es uns nicht gelungen ist, das Protokoll von His detailgetreu zu replizieren, oder das Protokoll war unvollständig. Nach unserer Einschätzung hat His wahrscheinlich die Infiltrationseinbettung mit Paraffin erfunden und perfektioniert, aber kein vollständiges Protokoll veröffentlicht.

Background: The outflow tract of crocodilians resembles that of birds and mammals as ventricular septation is complete. The arterial anatomy, however, presents with a pulmonary trunk originating from the right ventricular cavum, and two aortas originating from either the right or left ventricular cavity. Mixing of blood in crocodilians cannot occur at the ventricular level as in other reptiles but instead takes place at the aortic root level by a shunt, the foramen of Panizza, the opening of which is guarded by two facing semilunar leaflets of both bicuspid aortic valves. Methods: Developmental stages of Alligator mississipiensis, Crocodilus niloticus and Caiman latirostris were studied histologically. Results and Conclusions: The outflow tract septation complex can be divided into two components. The aorto-pulmonary septum divides the pulmonary trunk from both aortas, whereas the interaortic septum divides the systemic from the visceral aorta. Neural crest cells are most likely involved in the formation of both components. Remodeling of the endocardial cushions and both septa results in the formation of bicuspid valves in all three arterial trunks. The foramen of Panizza originates intracardially as a channel in the septal endocardial cushion.

Fossorial talpid moles use their limbs predominantly for digging, which explains their highly specialized anatomy. The humerus is particularly short and dorsoventrally rotated, with broadened distal and proximal parts where muscles attach and which facilitate powerful abductive movements. The radius and ulna are exceptionally robust and short. The ulna has an expanded olecranon process. The femur is generalized, but the fused tibia-fibula complex is short and robust. To understand the developmental bases of these specializations, we studied expression patterns of four 5' Hox genes in the fossorial Iberian mole (Talpa occidentalis). These genes are known to play major roles in patterning the developing limb skeleton in the mouse, with which comparisons were made (Mus musculus, C57BL/6Jico strain). We find that HoxA9 expression is spatially expanded in the developing stylopodial area in the mole forelimb, compared to the less specialized mouse forelimb and mole hind limb. HoxD9 expression does not extend into the thoracic body wall in the mole forelimb in contrast to the mouse, and is also reduced in the presumptive zeugopodium in mole forelimb, compared to mouse. Expression of HoxD11 is upregulated in the mole in the postaxial area of the hind limb zeugopod, compared to the mouse. On the other hand, HoxD13 is downregulated in the postaxial zeugopodial area in the forelimb of the mole, compared to the mouse. The differences in the expression patterns of these 5' Hox genes between Talpa and Mus are an indication of the developmental changes going hand in hand with anatomical digging adaptations in the mole adult.

Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (&alpha, neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to &alpha, neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with &alpha, neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit &alpha, neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of &alpha, neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.

Talpid moles across all northern continents exhibit a remarkably large, sickle-like radial sesamoid bone anterior to their five digits, always coupled with a smaller tibial sesamoid bone. A possible developmental mechanism behind this phenomenon was revealed using molecular markers during limb development in the Iberian mole ( Talpa occidentalis ) and a shrew ( Cryptotis parva ), as shrews represent the closest relatives of moles but do not show these conspicuous elements. The mole's radial sesamoid develops later than true digits, as shown by Sox9 , and extends into the digit area, developing in relation to an Msx2 -domain at the anterior border of the digital plate. Fgf8 expression, marking the apical ectodermal ridge, is comparable in both species. Developmental peculiarities facilitated the inclusion of the mole's radial sesamoid into the digit series; talpid moles circumvent the almost universal pentadactyly constraint by recruiting wrist sesamoids into their digital region using a novel developmental pathway and timing.

In this study, we subjected eels from Lake Balaton (Hungary) to a swimming period of 1 week and 2 or 6 weeks. Most eels were silver and were 13–21 years old. Time dependent changes in morphometrical parameters and developmental characteristics of the oocytes were determined. Already after 1 week of swimming, the gonadal mass increased and oocytes became larger, filled with large numbers of lipid droplets. After 2 and 6 weeks of swimming we found in addition a significant enlargement of the eyes, which is a sign of sexual maturation. In contrast to the resting eels, that had oocytes in the primary growth phase (stage 1–2); the swimming eels had oocytes in stage 3; the cortical alveolus or lipid droplet stage. The results indicate that lipid mobilisation induced by swimming is a requirement for the natural incorporation of lipid droplets in the oocytes, a crucial step in oocyte maturation. As the Balaton eels responded stronger to swimming than young farmed eels, it is suggested that older eels are more sensitive for maturation triggers.

Objectives Pharyngeal arches develop in the head and neck regions, and give rise to teeth, oral jaws, the hyoid bone, operculum, gills, and pharyngeal jaws in teleosts. In this study, the expression patterns of genes in the sonic hedgehog (shh), wnt, ectodysplasin A (eda), and bone morphogenetic protein (bmp) pathways were investigated in the pharyngeal arches of Haplochromis piceatus, one of the Lake Victoria cichlids. Furthermore, the role of the shh pathway in pharyngeal arch development in H. piceatus larvae was investigated. Methods The expression patterns of lymphocyte enhancer binding factor 1 ( lef1 ) , ectodysplasin A receptor ( edar ), shh , patched 1 ( ptch1 ) , bmp4, sp5 transcription factor ( sp5 ), sclerostin domain containing 1a ( sostdc1a ), and dickkopf 1 ( dkk1 ) were investigated in H. piceatus larvae by in situ hybridization. The role of the shh pathway was investigated through morphological phenotypic characterization after its inhibition. Results We found that lef1 , edar, shh, ptch1, bmp4 , dkk1 , sostdc1a , and sp5 were expressed not only in the teeth, but also in the operculum and gill filaments of H piceatus larvae. After blocking the shh pathway using cyclopamine, we observed ectopic shh expression and the disappearance of ptch1 expression. After six weeks of cyclopamine treatment, an absence of teeth in the oral upper jaws and a poor outgrowth of premaxilla, operculum, and gill filaments in juvenile H. piceatus were observed. Conclusions These results suggest that the shh pathway is important for the development of pharyngeal arch derivatives such as teeth, premaxilla, operculum, and gill filaments in H. piceatus .

Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.

Due to a lack of distinctive morphological characters, swift taxonomy and phylogeny has always been an area of disagreement. To shed more light on this subject, we reconstructed swift(let) phylogeny based on 1143 bp of mitochondrial cytochrome-b DNA sequence. Although this is not the first attempt to reconstruct swift phylogeny using molecular data, our results show higher support for many of the branches due to our much longer sequences. However, placement of Hydrochous is still unexpected. Implementation of more conservative genetic regions and sampling of more taxa could solve this problem. Most importantly, the Collocaliini resolve as a monophyletic group. The internal structure of the group shows that non-echolocating Collocalia and echolocating Aerodramus form two distinct clades. This is in congruence with earlier classifications based on morphological characters, but in contrast with more recent classifications. (C) 2003 Elsevier Science (USA). All rights reserved.

Birdsong is one of the main models in sexual selection studies. Most investigations focused on oscines in which male song and female preference learning occur. Yet, some non-oscines are well suited for such studies as well. In Columbidae song learning does not occur. However, like in oscines, song is involved in inter- and intra-sexual interactions. Surprisingly, experimental evidence of female song-based preferences are still largely lacking in this group. We conducted playback tests on wild-caught Streptopelia decaocto females. We tested for the sexual function of song by playing decaocto songs versus songs of an unrelated species. We then investigated female responses to trill. Although they do not produce this trait, males react more strongly to artificially trilled decaocto songs, i.e. to conspecific songs in which a trill from a S. roseogrisea song has been inserted, than to normal decaocto songs. We also tested female responses to S. roseogrisea songs. Females flew more often and with a shorter latency during decaocto songs than during wren songs, suggesting that species recognition occurred, but we found no evidence of preference for trilled decaocto songs. The low activity observed during the experiment might have obscured actual preferences. Nevertheless, females consistently reacted more to normal decaocto songs than to trilled decaocto or roseogrisea songs. This contrasts with the strong responses for trilled decaocto songs and the virtual lack of reaction to S. roseogrisea songs observed in males. Thus, female collared doves, as receivers, may have different characteristics from males and impose constraints on the evolution of song.

During cardiogenesis the epicardium, covering the surface of the myocardial tube, has been ascribed several functions essential for normal heart development of vertebrates from lampreys to mammals. We investigated a novel function of the epicardium in ventricular development in species with partial and complete septation. These species include reptiles, birds and mammals. Adult turtles, lizards and snakes have a complex ventricle with three cava, partially separated by the horizontal and vertical septa. The crocodilians, birds and mammals with origins some 100 million years apart, however, have a left and right ventricle that are completely separated, being a clear example of convergent evolution. In specific embryonic stages these species show similarities in development, prompting us to investigate the mechanisms underlying epicardial involvement. The primitive ventricle of early embryos becomes septated by folding and fusion of the anterior ventricular wall, trapping epicardium in its core. This folding septum develops as the horizontal septum in reptiles and the anterior part of the interventricular septum in the other taxa. The mechanism of folding is confirmed using DiI tattoos of the ventricular surface. Trapping of epicardium-derived cells is studied by transplanting embryonic quail pro-epicardial organ into chicken hosts. The effect of decreased epicardium involvement is studied in knock-out mice, and pro-epicardium ablated chicken, resulting in diminished and even absent septum formation. Proper folding followed by diminished ventricular fusion may explain the deep interventricular cleft observed in elephants. The vertical septum, although indistinct in most reptiles except in crocodilians and pythonidsis apparently homologous to the inlet septum. Eventually the various septal components merge to form the completely septated heart. In our attempt to discover homologies between the various septum components we aim to elucidate the evolution and development of this part of the vertebrate heart as well as understand the etiology of septal defects in human congenital heart malformations.

Objective Genes involved in bone and tissue remodelling in the vertebrates include matrix metalloproteinase-9 (mmp-9), receptor activator of necrosis factor κ-β (rank), cathepsin-k (Ctsk) and tartrate-resistant acid phosphatase (TRAcP). We examine whether these markers are expressed in cells of zebrafish embryos of 1-5 days post fertilization. We also examine adult scales, which are known to contain mature osteoclasts, for comparison. Materials and Methods In this experimental study, in situ hybrdisation, histochemistry and serial plastic and paraffin sectioning were used to analyse marker expression. Results We found that mmp-9 mRNA, TRAcP enzyme and Ctsk YFP protein were expressed in haematopoietic tissues and in the cells scattered sparsely in the embryo. Ctsk and rank mRNA were both expressed in the branchial skeleton and in the developing pectoral fin. In these skeletal structures, histology showed that the expressing cells were located around the developing cartilage elements, in the parachondral tissue. In a transgenic zebrafish line with YFP coupled to Ctsk promoter, Ctsk expressing cells were found around pharyngeal skeletal elements. To see whether we could activate osteoclasts, we exposed prim-6 zebrafish embryos to a mixture of 1 µM dexamethasone and 1 µM vitaminutes D3. These compounds, which are known to trigger osteoclastogenensis in cell cultures, lead to an increase in intensity of Ctsk YFP expression around the skeletal elements. Conclusion Our findings show that cells expressing a range of osteoclast markers are present in early larvae and can be activated by the addition of osteoclastogenic compounds.

Research on the Nile crocodile and five birds shows that limb evolution is shaped by an interplay between natural selection and developmental constraints, the outcome varying between different digits, and between embryos and adults. The archosaurs are a group of land vertebrates that included the dinosaurs; birds and crocodiles are their only living representatives. Throughout their history, archosaurs show a persistent pattern of digit loss from the ancestral condition of five digits per limb. In all cases, however, rudiments for digits persist in the embryo even though they are not present in the adult, and the most persistent is digit five. Looking at embryology in crocodiles and five bird species, Michael Richardson and colleagues show that from the Nile crocodile (where adults have all five digits) to the emu (with a rudimentary wing containing just one digit) the signs for digit five are always present, if transiently. The authors suggest that this persistence might be a developmental constraint resulting from the association between digit five and the zone of polarizing activity, the region of the embryonic limb that governs digit development as a whole. Evolution involves interplay between natural selection and developmental constraints1,2,3. This is seen, for example, when digits are lost from the limbs during evolution1,3,4. Extant archosaurs (crocodiles and birds) show several instances of digit loss3,5,6 under different selective regimes, and show limbs with one, two, three, four or the ancestral number of five digits. The ‘lost’ digits sometimes persist for millions of years as developmental vestiges7,8,9,10. Here we examine digit loss in the Nile crocodile and five birds, using markers of three successive stages of digit development. In two independent lineages under different selection, wing digit I and all its markers disappear. In contrast, hindlimb digit V persists in all species sampled, both as cartilage, and as Sox9- expressing precartilage domains, 250 million years after the adult digit disappeared. There is therefore a mismatch between evolution of the embryonic and adult phenotypes. All limbs, regardless of digit number, showed similar expression of sonic hedgehog (Shh). Even in the one-fingered emu wing, expression of posterior genes Hoxd11 and Hoxd12 was conserved, whereas expression of anterior genes Gli3 and Alx4 was not. We suggest that the persistence of digit V in the embryo may reflect constraints, particularly the conserved posterior gene networks associated with the zone of polarizing activity (ZPA11). The more rapid and complete disappearance of digit I may reflect its ZPA-independent specification, and hence, weaker developmental constraints. Interacting with these constraints are selection pressures for limb functions such as flying and perching. This model may help to explain the diverse patterns of digit loss in tetrapods. Our study may also help to understand how selection on adults leads to changes in development.

Introduction Few areas of evolutionary investigation have benefited so much from the integration of molecular and morphological studies as that of the evolution of limbs (e.g. Shubin et al . 2009). Much effort has been concentrated on understanding the transition from ‘fins to limbs’ (Westoll 1943; Coates 1995; Shubin 1995; Shubin et al . 1997, 2004, 2006; Coates et al . 2002; Johanson et al . 2007; Larsson 2007) – for a recent summary of different aspects see Hall (2007) – but also major advances have been made concerning the origin and development of particularly specialized limbs such as those of bats (Sears et al . 2006; Sears 2008), whales (Thewissen et al . 2006), or their absence in snakes (Cohn and Tickle 1999). In fact, tetrapod limbs exhibit a stunning morphological diversity, reflecting their recruitment for a wide variety of functions ranging from walking to watch-making, often deployed as multipurpose structures serving in locomotion and support, personal hygiene, fighting, mating, manipulation and communication. The reconstructed ground pattern of extant tetrapods may be viewed as a limb with a proximal stylopod (humeral/femoral region), a zeugopod (radial and ulnar/tibial and fibular region) and a distal, pentadactylous autopod (carpal/tarsal region), which contains the proximal mesopodium (wrist and ankle bones) and the terminal acropodium (the digits). With the notable exception of limbless tetrapods, most tetrapod limbs conservatively retain this pattern (Goodwin and Trainor 1983; Holder 1983). Based on these components, the diversity of skeletal morphology in tetrapod limbs is generated largely by differential growth and proportional changes of the common elements. Regarding the acropodia, hypodactyly, hyperphalangy and modifications of branching patterns involve various developmental mechanisms (Motani 1999; Hamrick 2001b, 2002; Richardson and Oelschlager 2002; Fedak and Hall 2004; Sears et al . 2007; Rolian 2008; Weisbecker and Nilsson 2008; Cooper and Dawson 2009).

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.

We recently reconstructed the troublesome swiftlet phylogeny using cytochrome- b mitochondrial DNA sequences. The relationship of the giant swiftlet ( Hydrochous gigas ) with swiftlets of the genus Aerodramus was, however, unresolved. In an attempt to clarify this issue, we now incorporated mitochondrial 12S rRNA and nuclear β-fibrinogen intron 7 nuclear DNA sequences with the cyt- b sequences of six swiftlet, two swift, and one hummingbird outgroup species. A partition homogeneity (PH) test, used to determine the congruence of phylogenetic signal between two sets of sequences, suggested that cyt- b and Fib7 sequences were incongruent and therefore should not be combined. However, further analyses revealed that the apparent incongruence was probably due to the high amount of variation in cyt- b sequences. Separate and combined analyses of the three sequences unambiguously placed H. gigas as the sister-group of Aerodramus and supported monophyly of the swiftlets. These results were supported by analyses of combined NADH dehydrogenase subunit-2 (ND2) and cyt- b sequences of H. gigas in combination with sequences previously published by other workers. Recently, it was shown that the pygmy swiftlet ( C. troglodytes )—in our phylogenetic analyses consistently placed with other, non-echolocating, Collocalia species—is in fact able to echolocate. Echolocation thereby lost its value to distinguish between different swiftlet genera. Furthermore, the phylogenetic distribution of echolocation can be explained either by its single evolution at the base of the swiftlets, with subsequent loss, or by independent evolution in Aerodramus and C. troglodytes . Because yet unpublished data suggest that only the auditory nuclei in swiftlet brains show adaptations to echolocation, the latter explanation seems the more likely one.

Background Talpid moles show many specializations in their adult skeleton linked to fossoriality, including enlarged hands when compared to the feet. Heterochrony in developmental mechanisms is hypothesized to account for morphological evolution in skeletal elements. Methods The temporal and spatial distribution of SOX9 expression, which is an early marker of chondrification, is analyzed in autopods of the fossorial Iberian mole Talpa occidentalis, as well as in shrew (Cryptotis parva) and mouse (Mus musculus) for comparison. Results and discussion SOX9 expression is advanced in the forelimb compared to the hind limb in the talpid mole. In contrast, in the shrew and the mouse, which do not show fossorial specializations in their autopods, it is synchronous. We provide evidence that transcriptional heterochrony affects the development of talpid autopods, an example of developmental penetrance. We discuss our data in the light of earlier reported pattern heterochrony and later morphological variation in talpid limbs. Conclusion Transcriptional heterochrony in SOX9 expression is found in talpid autopods, which is likely to account for pattern heterochrony in chondral limb development as well as size variation in adult fore- and hind limbs.

It is generally assumed that the characteristic deregionalized body plan of species with a snake-like morphology evolved through a corresponding homogenization of Hox gene expression domains along the primary axis. Here, we examine the expression of Hox genes in snake embryos and show that a collinear pattern of Hox expression is retained within the paraxial mesoderm of the trunk. Genes expressed at the anterior and most posterior, regionalized, parts of the skeleton correspond to the expected anatomical boundaries. Unexpectedly however, also the dorsal (thoracic), homogenous rib-bearing region of trunk, is regionalized by unconventional gradual anterior limits of Hox expression that are not obviously reflected in the skeletal anatomy. In the lateral plate mesoderm we also detect regionalized Hox expression yet the forelimb marker Tbx5 is not restricted to a rudimentary forelimb domain but is expressed throughout the entire flank region. Analysis of several Hox genes in a caecilian amphibian, which convergently evolved a deregionalized body plan, reveals a similar global collinear pattern of Hox expression. The differential expression of posterior, vertebra-modifying or even rib-suppressing Hox genes within the dorsal region is inconsistent with the homogeneity in vertebral identity. Our results suggest that the evolution of a deregionalized, snake-like body involved not only alterations in Hox gene cis-regulation but also a different downstream interpretation of the Hox code.