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Standardization and reduction of variation is key to behavioural screening of animal models in toxicological and pharmacological studies. However, individual variation in behavioural and physiological phenotypes remains in each laboratory population and can undermine the understanding of toxicological and pharmaceutical effects and their underlying mechanisms. Here, we used zebrafish (ABTL-strain) larvae to explore individual consistency in activity level and emergence time, across subsequent days of early development (6–8 dpf). We also explored the correlation between these two behavioural parameters. We found inter-individual consistency over time in activity level and emergence time, but we did not find a consistent correlation between these parameters. Subsequently, we investigated the impact of variation in activity level on the effect of a 1% ethanol treatment, suitable for our proof-of-concept case study about whether impact from pharmacological treatments might be affected by inter-individual variation in basal locomotion. The inter-individual consistency over time in activity level did not persist in this test. This was due to the velocity change from before to after exposure, which turned out to be a dynamic individual trait related to basal activity level: low-activity individuals raised their swimming velocity, while high-activity individuals slowed down, yielding diametrically opposite response patterns to ethanol exposure. We therefore argue that inter-individual consistency in basal activity level, already from 6 dpf, is an important factor to take into account and provides a practical measure to improve the power of statistical analyses and the scope for data interpretation from behavioural screening studies.

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 explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. Results Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus, Heterodon nasicus, Rhabdophis subminiatus; Homalopsidae – Homalopsis buccata; Lamprophiidae - Malpolon monspessulanus, Psammophis schokari, Psammophis subtaeniatus, Rhamphiophis oxyrhynchus; and Viperidae – Bitis atropos, Pseudocerastes urarachnoides, Tropidolaeumus subannulatus, Vipera transcaucasiana. These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms. Conclusions We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator–prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline.

Background Venom has evolved in parallel in multiple animals for the purpose of self-defense, prey capture or both. These venoms typically consist of highly complex mixtures of toxins: diverse bioactive peptides and/or proteins each with a specific pharmacological activity. Because of their specificity, they can be used as experimental tools to study cell mechanisms and develop novel medicines and drugs. It is therefore potentially valuable to explore the venoms of various animals to characterize their toxins and identify novel toxin-families. This study focuses on the annotation and exploration of the transcriptomes of six scorpion species from three different families. The transcriptomes were annotated with a custom-built automated pipeline, primarily consisting of Basic Local Alignment Search Tool searches against UniProt databases and filter steps based on transcript coverage. Results We annotated the transcriptomes of four scorpions from the family Buthidae, one from Iuridae and one from Diplocentridae using our annotation pipeline. We found that the four buthid scorpions primarily produce disulfide-bridged ion-channel targeting toxins, while the non-buthid scorpions have a higher abundance of non-disulfide-bridged toxins. Furthermore, analysis of the “unidentified” transcripts resulted in the discovery of six novel putative toxin families containing a total of 37 novel putative toxins. Additionally, 33 novel toxins in existing toxin-families were found. Lastly, 19 novel putative secreted proteins without toxin-like disulfide bonds were found. Conclusions We were able to assign most transcripts to a toxin family and classify the venom composition for all six scorpions. In addition to advancing our fundamental knowledge of scorpion venomics, this study may serve as a starting point for future research by facilitating the identification of the venom composition of scorpions and identifying novel putative toxin families. Electronic supplementary material The online version of this article (10.1186/s12864-019-6013-6) contains supplementary material, which is available to authorized users.

The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 μg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.

Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.

The first PLA(2) (LsPA-1) from L. stenophrys snake venom was purified to homogeneity using three chromatographic steps and had its complete primary structure determined. An average molecular mass of 13,870.3 kDa was determined by mass spectrometry and a 3.3-fold increase in the PLA(2) activity was observed for LsPA-1 as compared to the whole venom. Multiple alignment of PLA(2) from Lachesis spp. snakes suggested the existence of two geographical clades for this genus in the New World, which is in accordance with morphological, behavioral and mtDNA data obtained by others. Phospholipases A(2) from Crotalus spp. snake venoms were similarly distributed into two groups. Intergroup analysis indicated that most amino acid substitutions were observed in the amino- and carboxy-terminal regions of the molecules in each clade. Both regions have been suggested to play important roles in determining the biological properties of PLA(2) from snake venoms. The dendogram derived for PLA(2) from Lachesis and Crotalus snakes highlighted the phylogenetic relationships between these two genera in the New World.

The study of drug-driven biochemical changes is important in order to determine the biomarkers associated with a specific compound activity in an individual biological system. Rodent models have been widely used to study the metabolic changes induced by psychostimulants in a cell, tissue or whole organism. However these models are not suitable for large-scale, high-throughput screening. Here, we used zebrafish embryos to study the metabolic effects of the cannabinoid receptor type 1 (CB1) agonist (−)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol (∆9-THC) and antagonist (AM251). The zebrafish embryos were exposed to ∆9-THC and AM251 at 24 h post fertilization (hpf) for 96 h. Proton nuclear magnetic resonance based metabolomic results show an increase in the level of choline, betaine, taurine, adenosine triphosphate and glucose upon exposure to ∆9-THC. The levels of excitatory neurotransmitters (glutamate and glutamine) increased at lower doses of ∆9-THC, whereas toxic dose resulted in reduction of glutamate. In contrast to ∆9-THC, AM251 caused a dose dependent reduction of betaine, choline, taurine and also reduce the level of glutamate and glutamine. Interestingly, both compounds induce the production of the dopamine precursors, phenylalanine and tyrosine at higher doses. These findings suggest that CB1 receptor is involved in the regulation of metabolites, which might be involved in the neurotransmission of zebrafish embryos. Furthermore, our results show that zebrafish embryo can be successfully used to provide a detailed overview of general effects of drug on the overall metabolome of an intact organism.

Among extant reptiles only two lineages are known to have evolved venom delivery systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded Lizard). Evolution of the venom system is thought to underlie the impressive radiation of the advanced snakes (2,500 of 3,000 snake species). In contrast, the lizard venom system is thought to be restricted to just two species and to have evolved independently from the snake venom system. Here we report the presence of venom toxins in two additional lizard lineages (Monitor Lizards and Iguania) and show that all lineages possessing toxin-secreting oral glands form a clade, demonstrating a single early origin of the venom system in lizards and snakes. Construction of gland complementary-DNA libraries and phylogenetic analysis of transcripts revealed that nine toxin types are shared between lizards and snakes. Toxinological analyses of venom components from the Lace Monitor Varanus varius showed potent effects on blood pressure and clotting ability, bioactivities associated with a rapid loss of consciousness and extensive bleeding in prey. The iguanian lizard Pogona barbata retains characteristics of the ancestral venom system, namely serial, lobular non-compound venom-secreting glands on both the upper and lower jaws, whereas the advanced snakes and anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard) have more derived venom systems characterized by the loss of the mandibular (lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians and anguimorphs form a single clade provides overwhelming support for a single, early origin of the venom system in lizards and snakes. These results provide new insights into the evolution of the venom system in squamate reptiles and open new avenues for biomedical research and drug design using hitherto unexplored venom proteins.

It is postulated widely that changes in developmental timing (i.e., heterochrony) represent a major mechanism of evolutionary change. However, it is only with recent methodological advances that changes in the order in which development proceeds (sequence heterochrony) can be identified and quantified. We apply these techniques to examine whether heterochrony in the early embryonic (organogenetic) period has played an important role in the diversification of mammals. Although we find clear instances of sequence heterochrony in mammals, particularly between eutherians and marsupials, the majority of mammalian lineages that we could examine (those within the major clades Euarchontoglires and Laurasiatheria) show few or no heterochronic changes in the 116 events examined (e.g., Artiodactyla, Euarchonta, Fereuungulata, Glires, Primates, Rodentia). This is in contrast with the timing shifts reported between and within other tetrapod clades. Our results suggest that sequence heterochrony in embryonic stages has not been a major feature of mammalian evolution. This might be because mammals, and perhaps amniotes in general, develop for an extended time in a protected environment, which could shield the embryos from strong diversifying selection. Our results are also consistent with the view that mammal embryos are subject to special developmental constraints. Therefore, other mechanisms explaining the diversity of extant mammals must be sought.

Development involves a series of developmental events, separated by transformations, that follow a particular order or developmental sequence. The sequence may in turn be arbitrarily subdivided into contiguous segments (developmental stages). We discuss the properties of developmental sequences. We also examine the differing analytical approaches that have been used to analyse developmental sequences in an evolutionary context. Ernst Haeckel was a pioneer in this field. His approach was evolutionary and he introduced the idea of sequence heterochrony (evolutionary changes in the sequence of developmental events). Despite the availability of detailed developmental data (e.g. Franz Keibel’s ‘Normal Tables’), Haeckel was unable to undertake a quantitative analysis of developmental data. This is now possible through computer-based analytical techniques such as event-pairing, which can extract important biological information from developmental sequences by mapping them onto established phylogenies. It may also yield data that can be used in phylogeny reconstruction, although the inherent ‘non-independence’ of the data may make this invalid. In future, the methods discussed here may be applied to the analysis of patterns of gene expression in embryos, or adapted to studying gene order on chromosomes.

&p.1: Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression ‐ the zootype. Haeckel’s drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage ‐ thought to correspond to a conserved stage ‐ show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel’s drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is difficult to reconcile with the idea of a phyogenetically-conserved tailbud stage, and suggests that at least some developmental mechanisms are not highly constrained by the zootype. Our study also highlights the dangers of drawing general conclusions about vertebrate development from studies of gene expression in a small number of laboratory species. &kwd:words Morphogenesis · Developmental biology · Comparative anatomy · Comparative study · Embryology&bdy

If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.

Snakes have graceful, elongated bodies containing hundreds of vertebrae. This extreme of morphology stems from evolutionary changes in a developmental clock that regulates body patterning.

Two studies of fruitflies suggest that although development relies on a diverse toolkit of genes, the evolution of physical characteristics might be powered by variation in just a few of these tools.

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.