Division of labor is a distinguishing characteristic of eusocial insects. To understand the proximate factors underlying caste determination, it is essential to clarify the developmental mechanisms during the differentiation of each caste. Termite soldiers have species-specific and diverse morphologies that are specialized for colony defense. Soldiers of the subfamily Nasutitermitinae (Termitidae), one of the most derived termite groups, possess a long, horn-like frontal projection (nasus), an invaginated glandular structure in the head (frontal gland), and regressed mandibles. These morphological changes occur prior to the molt into presoldiers (the preceding stage of soldiers). In Drosophila and other insects, Hox genes determine segment identities; thus they might be involved in such body-part-specific modifications during soldier differentiation. Deformed (Dfd) functions not only in the formation of the mandible and maxilla but also in other head parts (e.g., eye-antennal disc) in other insects. In this study, we examined Dfd functions in nasus/frontal gland formation and mandibular regression in Nasutitermes takasagoensis. Relative expression analyses showed that Dfd expression levels in the mouthparts were significantly higher than those in any other body parts of workers before presoldier molt. Dfd RNA interference resulted in the inhibition of mandibular regression during presoldier differentiation, but nasus and frontal gland formation were not affected. These results suggest that Dfd is involved in the determination of mandibular positional information and specific modification during presoldier differentiation in N. takasagoensis. This is the first work to show the effects of Hox genes on caste-specific morphogenesis in social insects. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1–8, 2013. © 2013 Wiley Periodicals, Inc.

The emergence of vertebrates is closely associated to the evolution of mineralized bone tissue. However, the molecular basis underlying the origin and subsequent diversification of the skeletal mineralized matrix is still poorly understood. One efficient way to tackle this issue is to compare the expression, between vertebrate species, of osteoblastic genes coding for bone matrix proteins. In this work, we have focused on the evolution of the network forming collagen family which contains the Col8a1, Col8a2, and Col10a1 genes. Both phylogeny and synteny reveal that these three paralogues are vertebrate-specific and derive from two independent duplications in the vertebrate lineage. To shed light on the evolution of this family, we have analyzed the osteoblastic expression of the network forming collagens in endochondral and intramembraneous skeletal elements of the amphibian Xenopus tropicalis. Remarkably, we find that amphibian osteoblasts express Col10a1, a gene strongly expressed in osteoblasts in actinopterygians but not in amniotes. In addition, while Col8a1 is known to be robustly expressed in mammalian osteoblasts, the expression levels of its amphibian orthologue are dramatically reduced. Our work reveals that while a skeletal expression of network forming collagen members is widespread throughout vertebrates, osteoblasts from divergent vertebrate lineages express different combinations of network forming collagen paralogues. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1–11, 2013. © 2013 Wiley Periodicals, Inc.

Fish egg envelopes consist of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among chordates. Euteleosts synthesize ZP proteins in the liver, while elopomorphs synthesize them in the ovaries. In Cypriniformes, zp genes are expressed in the ovaries. We investigated the zp genes of two Otocephalan orders: Clupeiformes (Pacific herring and Japanese anchovy) and Gonorynchiformes (milkfish), which diverged earlier than Cypriniformes. cDNA cloning of zp gene homologs revealed that Pacific herring and Japanese anchovy possessed both ovary- and liver-expressed zp genes; however, the zp genes of milkfish were only expressed in the ovaries. Molecular phylogenetic analysis showed that ovary- and liver-expressed zpc genes of two the Clupeiformes formed independent clades. Based on this, we hypothesized the evolution of teleostean zp genes, focusing on the organ expressing zp gene. As in other chordates, the original site of expression of zp genes was likely the ovary. In the early stage of teleostean evolution, the ancestral zp genes acquired the ability to express in the liver. Later, one of the two expression sites became dominant. The liver-expressed zp genes are component proteins of the egg envelope in the Euteleostei. In Otocephala, Clupeiformes possess both ovary- and liver-expressed genes that presumably participate in egg envelope formation, whereas the Gonorynchiformes and Cypriniformes have primarily preserved ovary expressed zp genes. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

The first mammalian metapodial (MP1) has periodically been argued to actually be a phalanx, because the first ray has one less element than the four posterior rays, and because the MP1 growth plate is proximal like those of all phalanges, rather than distal as in metapodials 2–5. However, growth plates are formed at both ends in non-therian tetrapod metapodials, and phylogenetic analysis demonstrates that growth plate loss is a therian synapomorphy that postdates the establishment of the mammalian phalangeal formula. These data, along with results of developmental and morphological studies, suggest that the MP1 is not a phalanx. The singular, proximal growth plates in MPs 2–5 are likely to be an adaptation to dynamic erect quadrupedal gait which was characterized by conversion of the posterior metapodials into rigid struts with the carpus/tarsus. While the adaptive significance of the reversed ossification of MP1 is less clear, we present three functional/developmental hypotheses. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

In the mature mammalian and avian central nervous systems, neuronal destructions are followed by reactive gliosis, but data on other vertebrates are rather controversial. Mammals and birds belong to different amniote groups (Synapsida and Diapsida, respectively), but exhibit common general features in their glial architecture, mainly the predominance of astrocytes. Two vertebrate groups seem to be in special positions of glial evolution: turtles (Testudiniformes) and skates and rays (Batoidea). The purely ependymoglial system of turtles seems to be the simplest one among the extant amniotes. In skates and rays, true astrocytes are preponderant glial elements, in contrast to the other “anamniotes” (and even to reptiles). We investigated stab wounds by the immunohistochemical detection of GFAP in turtles (Trachemys—formerly Pseudemys—scripta elegans), a skate (Raja clavata) and rays (Dasyatis akajei and Torpedo marmorata). Sharks (Scyliorhinus canicula) as ependymoglia-predominated chondrichthyans, and—for positive controls—rats were also studied. In the elasmobranchs, other astroglial markers: glutamine synthetase and S100 protein were also applied. Neither turtles nor elasmobranchs presented considerable astroglial reactions. Critically surveying the former reports on different vertebrates, these results complete the picture that typical post-lesion reactive gliosis is confined to mammals and birds. Analysis of the astroglial systems from phylogenetic perspective suggests that the capability of forming glial demarcation and scar formation evolved independently in mammals and birds. Predominance of astrocytes is a necessary condition but not sufficient for reactive gliosis. The intense glial reactivity of mammals and birds may be attributed to their complex cerebralization. J. Exp. Zool. (Mol. Dev. Evol.) 9999: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: XX–XX, 2013. © 2013 Wiley Periodicals, Inc.

In mice, a toothless diastema separates the single incisor from the three molars in each dental quadrant. In the prospective diastema of the embryo, small rudimentary buds are found that are presumed to be rudiments of suppressed teeth. A supernumerary tooth occurs in the diastema of adult mice carrying mutations in either Spry2 or Spry4. In the case of Spry2 mutants, the origin of the supernumerary tooth involves the revitalization of a rudimentary tooth bud (called R2), whereas its origin in the Spry4 mutants is not known. In addition to R2, another rudimentary primordium (called MS) arises more anteriorly in the prospective diastema. We investigated the participation of both rudiments (MS and R2) in supernumerary tooth development in Spry2 and Spry4 mutants by comparing morphogenesis, proliferation, apoptosis, size and Shh expression in the dental epithelium of MS and R2 rudiments. Increased proliferation and decreased apoptosis were found in MS and R2 at embryonic day (ED) 12.5 and 13.5 in Spry2^−/− embryos. Apoptosis was also decreased in both rudiments in Spry4^−/− embryos, but the proliferation was lower (similar to WT mice), and supernumerary tooth development was accelerated, exhibiting a cap stage by ED13.5. Compared to Spry2^−/− mice, a high number of Spry4^−/− supernumerary tooth primordia degenerated after ED13.5, resulting in a low percentage of supernumerary teeth in adults. We propose that Sprouty genes were implicated during evolution in reduction of the cheek teeth in Muridae, and their deletion can reveal ancestral stages of murine dental evolution. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1–14, 2013. © 2013 Wiley Periodicals, Inc.

An evolutionary developmental (evo-devo) approach to understanding the evolution, homology and development of structures has proved important for unraveling complex integrated skeletal systems through the use of modules, or modularity. An ocular skeleton, which consists of cartilage and sometimes bone, is present in many vertebrates; however the origin of these two components remains elusive. Using both palaeontological and developmental data, I propose that the vertebrate ocular skeleton is neural crest derived and that a single cranial neural crest module divided early in vertebrate evolution, possibly during the Ordovician, to give rise to an endoskeletal component and an exoskeletal component within the eye. These two components subsequently became uncoupled with respect to timing, placement within the sclera and inductive epithelia, enabling them to evolve independently and to diversify. In some extant groups, these two modules have become reassociated with one another. Furthermore, the data suggests that the endoskeletal component of the ocular skeleton was likely established and therefore evolved before the exoskeletal component. This study provides important insights into the evolution of the ocular skeleton, a region with a long evolutionary history amongst vertebrates. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1–9, 2012. © 2012 Wiley Periodicals, Inc.

Facultative endosymbionts, such as Wolbachia, perpetuate by vertical transmission mostly through colonization of the germline during embryogenesis. The remaining Wolbachia inside the embryo are internalized in progenitor cells of the somatic tissue. This perpetuation strategy triggers a cyclic bacterial bottleneck across host generations. However, throughout the host's life history (Drosophila, for example), some somatic tissues such as the Malpighian tubules (MTs) show large numbers of Wolbachia. It is assumed that Wolbachia present in the progenitor cells of the MTs are confined to this somatic tissue, implicitly considering MTs as an evolutionary dead-end for these bacteria. Nevertheless, the fact that bacteria can survive and proliferate inside MTs suggests a different fate as they may access the host's reproductive system and persist in the host population through vertical transmission. Indeed, based on the particular physiological and developmental characteristics of MT, as well as of Wolbachia, we argue the bacteria present in the MTs may constitute a secondary pool of vertically transmitted bacteria. Moreover, somatic pools of Wolbachia capable of reaching the gonads and insure vertical transmission may also provide an interesting element to the elucidation of horizontal transmission mechanisms. Finally, we also speculate that somatic pools of Wolbachia may play an important role in host fitness, namely during viral infections. In brief, we argue that the somatic pools of Wolbachia, with special emphasis on the MT subset, deserve experimental attention as putative players in the physiology and evolution of both bacteria and hosts. J. Exp. Zool. (Mol. Dev. Evol.) 320B:195–199, 2013. © 2013 Wiley Periodicals, Inc.

Well studied in mammals, amelogenesis is less known at the molecular level in reptiles and amphibians. In the course of extensive studies of enamel matrix protein (EMP) evolution in tetrapods, we look for correlation between changes in protein sequences and temporospatial protein gene expression during amelogenesis, using an evo-devo approach. Our target is the major EMP, amelogenin (AMEL) that plays a crucial role in enamel structure. We focused here our attention to an amphibian, the salamander Pleurodeles waltl. RNAs were extracted from the lower jaws of a juvenile P. waltl and the complete AMEL sequence was obtained using PCR and RACE PCR. The alignment of P. waltl AMEL with other tetrapodan (frogs, reptiles and mammals) sequences revealed residue conservation in the N- and C-terminal regions, and a highly variable central region. Using sense and anti-sense probes synthetized from the P. waltl AMEL sequence, we performed in situ hybridization on sections during amelogenesis in larvae, juveniles, and adults. We demonstrated that (i) AMEL expression was always found to be restricted to ameloblasts, (ii) the expression pattern was conserved through ontogeny, even in larvae where enameloid is present in addition to enamel, and (iii) the processes are similar to those described in lizards and mammals. These findings indicate that high variations in the central region of AMEL have not modified its temporospatial expression during amelogenesis for 360 million years of tetrapod evolution. J. Exp. Zool. (Mol. Dev. Evol.) 320B:200–209, 2013. © 2013 Wiley Periodicals, Inc.

Numerous bacteria are frequently observed in the superficial corneocytes forming the corneous layer of the soft-shelled turtle Apalona spinifera. The resistance to bacterial penetration through the living epidermis in this turtle suggests the presence of an antimicrobial barrier, possibly derived from the presence of anti-microbial peptides in the epidermis. Four beta-defensin-like peptides, named As-BD-1 to 4, have been characterized from skin tissues using molecular and bioinformatics methods. The precursor peptides contain the beta-defensin motif with the typical cysteine localization pattern. The analysis of the expression for the four different beta-defensin-like proteins show that these molecules are expressed in the skin (epidermis and dermis) of the carapace, neck, digit, and tail but are apparently not expressed in the liver or intestine under normal conditions. These data suggest that in the skin of the soft-shelled turtle there are potential effective anti-microbial peptides against epidermal bacteria. J. Exp. Zool. (Mol. Dev. Evol.) 320B:210–217, 2013. © 2013 Wiley Periodicals, Inc.

The interactive effects of contaminants and ultraviolet light (UV)-exposure on the incidence and types of abnormalities observed were measured in newly metamorphosed cane toads (Rhinella marina) from four Bermuda ponds contaminated with petrochemicals and metals. Abnormalities were compared in toadlets that were field-collected, reared in predator exclusion cages, reared in laboratory microcosms exposed to control media or corresponding pond media, and reared in laboratory microcosms exposed to UV-light and control media or media from two ponds. Percent abnormal for field-collected, cage-reared, and microcosm-reared toadlets were equivalent per site and ranged between 14% and 63%. All treatments produced similar limb abnormalities but the percentage of hind versus forelimb defects was statistically greater only in field-collected toadlets. UV-exposed control media did not induce abnormalities in larvae exhibiting no maternal effect, and did not alter the types of abnormalities observed in larvae exhibiting a maternal or latent effect. Site media treatments without UV exposure induced significant cephalic and limb abnormalities, proved additive to the observed maternal/latent effect, and produced limb defects predominantly in forelimbs. Concurrent exposure to site media and UV-light induced similar types of abnormalities but a significantly higher percentage of hind limb abnormalities (68–89%) than exposure to site media alone (7–13%). Our results suggest that the types of abnormalities expressed were principally determined by direct and/or transgenerational contaminant exposure, but that UV-light exposure caused limb abnormalities to occur primarily in the hind limbs, mirroring field observations. Our field observations also suggest that ectromelia and brachydactyly in some field-collected specimens may be predator-induced. J. Exp. Zool. (Mol. Dev. Evol.) 320B:218–237, 2013. © 2013 Wiley Periodicals, Inc.

Many reptiles, including the red-eared slider turtle (Trachemys scripta), possess a temperature-dependent sex determination (TSD) mechanism where the temperature at which the developing embryos are incubated dictates the gonadal sex of the animal. A number of mammalian gene orthologues have been identified in the sex determination/differentiation cascade of reptiles with TSD although the exact trigger(s) is not well understood. A potential early regulator of gonadal differentiation, Lhx9, controls the proliferation of gonadal cells in mice and its absence prevents gonadal development and drastically reduces the expression of Sf-1, a gene that regulates the expression of steroidogenic enzymes in the bipotential gonad. In the current study, we cloned Lhx9 from T. scripta and analyzed its expression throughout the thermosensitive period of gonad development using quantitative PCR. We examined the expression profiles of Lhx9 in embryos incubated under control conditions at male- and female-producing temperatures and with the application of exogenous 17β-estradiol or an aromatase inhibitor, Letrozole, to induce sex reversal. The T. scripta Lhx9 cDNA and predicted amino acid sequence showed high homology to those of chicken, anole, and mouse. Lhx9 was expressed at both male- and female-producing temperatures with expression levels increasing throughout the thermosensitive period. Letrozole induced sex-reversal did not alter Lhx9 expression levels. 17β-estradiol treatments appeared to inhibit or delay gonadal differentiation and resulted in lower Lhx9 expression from the presumptive gonadal ridge region. The structural homology and temporal expression pattern of Lhx9 suggests that this represents a conserved element in the gonadal differentiation cascade of T. scripta. J. Exp. Zool. (Mol. Dev. Evol.) 320B:238–246, 2013. © 2013 Wiley Periodicals, Inc.

For the past 35 years, the Cambrian fossil Pikaia gracilens was widely interpreted as a typical basal chordate based on short descriptions by Conway Morris. Recently, Conway Morris and Caron (CMC) (2012, Biol Rev 87:480–512) described Pikaia extensively, as a basis for new ideas about deuterostome evolution. This new Pikaia has characters with no clear homologues in other animals, so they could be phylogenetically uninformative autapomorphies. These characters include a dorsal organ, posterior ventral area, posterior fusiform structure, and anterior dorsal unit. Yet CMC interpret most of the unusual characters as primitive for chordates, thereby interpreting Pikaia as an even more convincing stem chordate than before. Moreover, they claim that segment (myomere) shape is a reliable guide for defining a chordate and even for assigning animals to their correct place in deuterostome phylogeny. By defining sigmoidal segments as a basal chordate character, they situate Pikaia at the base of the chordates and banish fossil yunnanozoans (which have straight segments) to a position deep within the deuterostomes. In addition, they consider amphioxus, with its conspicuously chevron-shaped segments, to be so highly derived that it is of little use for reconstructing the first chordates. We question their overemphasis on the phylogenetic value of segment shape and their marginalizing of amphioxus. We deduce that Pikaia, not amphioxus, is specialized. We performed a cladistic analysis that showed the character interpretations of CMC are consistent with their wide-ranging evolutionary scenario, but that these interpretations leave unresolved the position of Pikaia within chordates. J. Exp. Zool. (Mol. Dev. Evol.) 320B:247–271, 2013. © 2013 Wiley Periodicals, Inc.