Regional Specific Differentiation of Integumentary Organs: Regulation of Gene Clusters within the Avian Epidermal Differentiation Complex and Impacts of SATB2 Overexpression.

The epidermal differentiation complex (EDC) encodes a group of unique proteins expressed in late epidermal differentiation. The EDC gave integuments new physicochemical properties and is critical in evolution. Recently, we showed ß-keratins, members of the EDC, undergo gene cluster switching with overexpression of SATB2 (Special AT-rich binding protein-2), considered a chromatin regulator. We wondered whether this unique regulatory mechanism is specific to ß-keratins or may be derived from and common to EDC members. Here we explore (1) the systematic expression patterns of non-ß-keratin EDC genes and their preferential expression in different skin appendages during development, (2) whether the expression of non-ß-keratin EDC sub-clusters are also regulated in clusters by SATB2. We analyzed bulk RNA-seq and ChIP-seq data and also evaluated the disrupted expression patterns caused by overexpressing SATB2. The results show that the expression of whole EDDA and EDQM sub-clusters are possibly mediated by enhancers in E14-feathers. Overexpressing SATB2 down-regulates the enriched EDCRP sub-cluster in feathers and the EDCH sub-cluster in beaks. These results reveal the potential of complex epigenetic regulation activities within the avian EDC, implying transcriptional regulation of EDC members acting at the gene and/or gene cluster level in a temporal and skin regional-specific fashion, which may contribute to the evolution of diverse avian integuments.

A defect in purine nucleotide metabolism in the silkworm, Bombyx mori, causes a translucent larval integument and male infertility.

p-oily (op) is a novel mutant of Bombyx mori exhibiting translucent larval integument and male infertility. Elucidation of the causative gene of the op mutant will help understand the genetic mechanism underlying larval integument coloration and male fertility. Using polymorphisms between B. mori and B. mandarina, the op locus was narrowed down to a 375-kb region. Using RNA-seq analysis, we found that op mutants have a frameshift mutation in the KWMTBOMO13770 gene located in the 375-kb region. A database search indicated that this gene is the human cytosolic 5'-nucleotidase II gene (cN-II) homolog in Bombyx, which mediates the conversion of inosine monophosphate (IMP) to inosine, a precursor of uric acid. CRISPR/Cas9-mediated knockout mutants of the Bm-cN-II gene showed translucent integuments, and there appeared translucent larvae in the crosses between knockout moths and +/op moths. Moreover, the translucent phenotype of, and decreased uric acid content in the larval integument caused by the mutations in the Bm-cN-II gene were rescued by oral administration of inosine. These results indicated that the Bm-cN-II gene is responsible for the op phenotype and that the molecular function of the Bm-cN-II gene is the conversion of IMP to inosine. We also discuss the genetic relationship between the Bm-cN-II gene and male fertility.

The cuticular hydrocarbon profiles of honey bee workers develop via a socially-modulated innate process.

Large social insect colonies exhibit a remarkable ability for recognizing group members via colony-specific cuticular pheromonal signatures. Previous work suggested that in some ant species, colony-specific pheromonal profiles are generated through a mechanism involving the transfer and homogenization of cuticular hydrocarbons (CHCs) across members of the colony. However, how colony-specific chemical profiles are generated in other social insect clades remains mostly unknown. Here we show that in the honey bee (Apis mellifera), the colony-specific CHC profile completes its maturation in foragers via a sequence of stereotypic age-dependent quantitative and qualitative chemical transitions, which are driven by environmentally-sensitive intrinsic biosynthetic pathways. Therefore, the CHC profiles of individual honey bees are not likely produced through homogenization and transfer mechanisms, but instead mature in association with age-dependent division of labor. Furthermore, non-nestmate rejection behaviors seem to be contextually restricted to behavioral interactions between entering foragers and guards at the hive entrance.

Identification and RNAi-based function analysis of chitinase family genes in diamondback moth, Plutella xylostella.

BACKGROUND: Insect chitinases play a vital part in chitin degradation in exoskeletons and gut linings during the molting process, and therefore are considered potential targets for new insecticide designs or RNA interference (RNAi)-based pest management. Systematic functional analysis of chitinase genes has already been conducted in several insect pests, but not Plutella xylostella. RESULTS: In this study, 13 full-length chitinase transcripts were obtained in P. xylostella. Developmental and tissue-specific expression pattern analysis revealed that seven chitinase transcripts were periodically expressed during molting stage and mainly expressed in the integument or midgut, including PxCht3, PxCht5, PxCht6-2, PxCht7, PxCht8, PxCht10 and PxCht-h. RNAi-mediated knockdown of these specific expressed genes revealed that PxCht5 and PxCht10 were essential in larval molting, pupation and eclosion, and PxCht7 was indispensable only in eclosion. No significant effects were observed on insect survival or normal development when the rest chitinase transcripts were suppressed by RNAi. CONCLUSION: Our results indicated the function of P. xylostella chitinase family genes during the molting process, and may provide potential targets for RNAi-based management of P. xylostella. © 2018 Society of Chemical Industry.

Integument Development in Arabidopsis Depends on Interaction of YABBY Protein INNER NO OUTER with Coactivators and Corepressors.

Arabidopsis thaliana INNER NO OUTER (INO) is a YABBY protein that is essential for the initiation and development of the outer integument of ovules. Other YABBY proteins have been shown to be involved in both negative and positive regulation of expression of putative target genes. YABBY proteins have also been shown to interact with the corepressor LEUNIG (LUG) in several systems. In support of a repressive role for INO, we confirm that INO interacts with LUG and also find that INO directly interacts with SEUSS (SEU), a known corepressive partner of LUG. Further, we find that INO can directly interact with ADA2b/PROPORZ1 (PRZ1), a transcriptional coactivator that is known to interact with the histone acetyltransferase GENERAL CONTROL NONREPRESSIBLE PROTEIN 5 (GCN5, also known as HAG1). Mutations in LUG, SEU, and ADA2b/PRZ1 all lead to pleiotropic effects including a deficiency in the extension of the outer integument. Additive and synergistic effects of ada2b/prz1 and lug mutations on outer integument formation indicate that these two genes function independently to promote outer integument growth. The ino mutation is epistatic to both lug and ada2b/prz1 in the outer integument, and all three proteins are present in the nuclei of a common set of outer integument cells. This is consistent with a model where INO utilizes these coregulator proteins to activate and repress separate sets of target genes. Other Arabidopsis YABBY proteins were shown to also form complexes with ADA2b/PRZ1, and have been previously shown to interact with SEU and LUG. Thus, interaction with these corepressors and coactivator may represent a general mechanism to explain the positive and negative activities of YABBY proteins in transcriptional regulation. The LUG, SEU, and ADA2b/PRZ1 proteins would also separately be recruited to targets of other transcription factors, consistent with their roles as general coregulators, explaining the pleiotropic effects not associated with YABBY function.

The ABC transporter ABCH-9C is needed for cuticle barrier construction in Locusta migratoria.

ATP-binding cassette (ABC) transporters constitute a large superfamily of proteins that mediate transport of a diverse number of substrates including nutrients, lipids and xenobiotics across membranes serving a variety of developmental and physiological functions. Here, we report on the molecular properties and biological roles of the ABC transporter LmABCH-9C in the migratory locust Locusta migratoria. LmABCH-9C was expressed continuously during nymphal development in all tissues including the integument. Expression was highest just after molting. Suppression of LmABCH-9C transcript levels by RNA interference (RNAi) in nymphs provoked death during or soon after molting to the next stage. These nymphs lost weight within minutes after molting. Moreover, high humidity rescued the lethality of molted dsLmABCH-9C-injected nymphs. In histological experiments, we find that the amounts of inner-cuticular lipids are reduced in nymphs with suppressed LmABCH-9C expression. These data together indicate that LmABCH-9C is needed for lipid-dependent desiccation resistance, paralleling the function of ABCH-9C in Tribolium castaneum. Hence, the function of this ABC transporter seems to be conserved across insect species ranging from hemimetabolous (L. migratoria) to holometabolous (T. castaneum) species. In addition, we find that cuticle inward impermeability is compromised in nymphs with reduced LmABCH-9C function. In summary, consistent with the model that cuticular lipids are necessary to prevent desiccation and penetration of xenobiotics in insects, we hypothesize that LmABCH-9C is involved in the construction of a lipid-based barrier at the surface of the cuticle especially after molting to protect the animal against uncontrolled water loss and entry. Susceptibility of this ABC transporter to RNAi-mediated knockdown designates it as an excellent target for RNAi-based insect pest control.

Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model.

The integumentary organ system is a complex system that plays important roles in waterproofing, cushioning, protecting deeper tissues, excreting waste, and thermoregulation. We developed a novel in vivo transplantation model designated as a clustering-dependent embryoid body transplantation method and generated a bioengineered three-dimensional (3D) integumentary organ system, including appendage organs such as hair follicles and sebaceous glands, from induced pluripotent stem cells. This bioengineered 3D integumentary organ system was fully functional following transplantation into nude mice and could be properly connected to surrounding host tissues, such as the epidermis, arrector pili muscles, and nerve fibers, without tumorigenesis. The bioengineered hair follicles in the 3D integumentary organ system also showed proper hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches. Potential applications of the 3D integumentary organ system include an in vitro assay system, an animal model alternative, and a bioengineered organ replacement therapy.

The ß-N-acetylhexosaminidase gene family in the brown planthopper, Nilaparvata lugens.

ß-N-Acetylhexosaminidases (HEXs) are enzymes that can degrade the chitin oligosaccharides that are produced by the activity of chitinases on chitin in insects. Using bioinformatic methods based on genome and transcriptome databases, 11 ß-N-acetylhexosaminidase genes (NlHexs) in Nilaparvata lugens were identified and characterized. Phylogenetic analysis revealed a six-grouped tree topology. The O-Linked N-acetylglucosaminidase (O-GlcNAcase) group includes NlHex11, which harbours a catalytic domain that differs from that of the other 10 NlHexs. Observations of the expression of NlHexs during different developmental stages revealed that NlHex4 is expressed with periodicity during moulting. Although the tissue-specific expression patterns of most NlHexs were nonspecific, NlHex4 was found to be expressed mainly in the female reproductive system as well as in the integument. RNA interference (RNAi) demonstrated failure to shed the old cuticle only in the nymphs treated with double-stranded RNA (dsRNA) targeting NlHex4, and these nymphs eventually died; no observable morphological abnormalities were found in insects treated with dsRNAs targeting the other 10 NlHexs. Based on this study and our previous analyses, a '5 + 1 + 3' pattern of chitinolytic enzymes is proposed, in which five chitinases, one NlHEX and three chitin deacetylases are required for moulting in N. lugens. A better understanding of chitin metabolism in the hemimetabolous insect, N. lugens, would be achieved by considering three chitinolytic enzyme families: chitinase, chitin deacetylase and ß-N-acetylhexosaminidase.

Protruding structures on caterpillars are controlled by ectopic Wnt1 expression.

Spine-like or protruding structures, which may be aposematic for predators, are often observed in multiple segments of lepidopteran larvae (caterpillars). For example, the larvae of the Chinese wheel butterfly, Byasa alcinous, display many protrusions on their backs as a warning that they are toxic. Although these protrusions are formed by an integument lined with single-layered epidermal cells, the molecular mechanisms underlying their formation have remained unclear. In this study, we focused on a spontaneous mutant of the silkworm, Bombyx mori, Knobbed, which shows similar protrusions to B. alcinous and demonstrates that Wnt1 plays a crucial role in the formation of protrusion structures. Using both transgene expression and RNAi-based knockdown approaches, we showed that Wnt1 designates the position where epidermal cells excessively proliferate, leading to the generation of knobbed structures. Furthermore, in the B. alcinous larvae, Wnt1 was also specifically expressed in association with the protrusions. Our results suggest that Wnt1 plays a role in the formation of protrusions on the larval body, and is conserved broadly among diverse species in Lepidoptera.

An Immature Pachyrhinosaurus perotorum (Dinosauria: Ceratopsidae) Nasal Reveals Unexpected Complexity of Craniofacial Ontogeny and Integument in Pachyrhinosaurus.

A new specimen attributable to an immature individual of Pachyrhinosaurus perotorum (Dinosauria, Ceratopsidae) from the Kikak-Tegoseak Quarry in northern Alaska preserves a mix of features that provides refinement to the sequence of ontogenetic stages and transformations inferred for the development of the nasal boss in Pachyrhinosaurus. The new specimen consists of an incomplete nasal that includes the posterior part of the nasal horn, the dorsal surface between the horn and the left-side contacts for the prefrontal and frontal, and some of the left side of the rostrum posteroventral to the nasal horn. The combination of morphologies in the new specimen suggests either an additional stage of development should be recognized in the ontogeny of the nasal boss of Pachyrhinosaurus, or that the ontogenetic pathway of nasal boss development in P. perotorum was notably different from that of P. lakustai. Additionally, the presence of a distinct basal sulcus and the lateral palisade texture on the nasal horn of the specimen described here indicate that a thick, cornified horn sheath was present well before the formation of a dorsal cornified pad. A separate rugose patch on the nasal well posterior to the nasal horn is evidence for a cornified integumentary structure, most likely a thick cornified pad, on the posterior part of the nasal separate from the nasal horn prior to the onset of nasal boss formation in P. perotorum.

The nipple: a simple intersection of mammary gland and integument, but focal point of organ function.

Having glands that secrete milk to nourish neonatal offspring characterizes all mammals. We provide a brief overview of the development and anatomy of nipples and mammary glands in monotremes, marsupials, and marine mammals, and focus on the nipples and mammary glands in terrestrial eutherian species. We first classify eutherians into three groups: the altricial, precocial, and arboreal types based on their rearing system. We then summarize the physiology of lactation and the cell biology of nipples with specific focus on comparing these in the mouse, cow, and human, which represent the three different groups. Finally we propose that the nipple is an example of specialized epidermis. As specialized epidermis, it is dependent the underlying stroma for development and maintenance in adult life. The development of the nipple and signaling pathways that regulate its formation are described.

Description of the male, larva and nymphal stages of Cryptocellus iaci (Arachnida, Ricinulei), with an overview of tarsal sensilla and other integumental structures.

The male, larva and nymphal stages of Cryptocellus iaci Tourinho, Lo Man-Hung & Bonaldo, 2010, a species previously known only from a single female, are described based on specimens from around the type locality, in an area of both Terra Firme forest and igapó (flooded forests), at the Jufari River, Roraima State, Brazil. The specimens were illustrated using live photography, stereomicroscopy, and scanning electron microscopy, allowing us to examine and describe the large diversity of tarsal sensilla and other integumental structures in Cryptocellus and to compare them to those of the previously studied Pseudocellus. Based on the male somatic characters Cryptocellus iaci is placed in thefoedus species-group. Cryptocellus iaci has two sensilla of type 1 on the distal tarsomeres of legs III (DT III), while only one has been reported for Pseudocellus spp., suggesting a potential value in this type of character for systematic studies of the group.

Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN.

The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity.

Armigeres subalbatus (Diptera: Culicidae) prophenoloxidase III is required for mosquito cuticle formation: ultrastructural study on dsRNA-knockdown mosquitoes.

We previously suggested that Armigeres subalbatus (Coquillett) prophenoloxidase III (As-pro-PO III) might be associated with morphogenesis of larvae and pupae. Because PO and its activation system are present in the insect cuticle, and cuticle formation is a major event during pupal morphogenesis, we used ultrastructural analysis to examine the effects of As-pro-PO III knockdown on the formation of pupal and adult cuticle. Inoculation of As-pro-PO III dsRNA resulted in the incomplete formation of nascent pupal endocuticle and pharate adult cuticle, i.e., significantly fewer cuticular lamellae were deposited, the helicoidal pattern of chitin microfibrils was disorganized, and numerous electron-lucent spaces were present in the cuticular protein matrix. Similar disruptions were observed in the cuticle of adults derived from As-pro-PO III dsRNA-inoculated pupae. It has long been suggested that the quinines, generated by PO-catalyzed oxidation reactions, function as cross-linking agents; therefore, it seems reasonable to suggest that the loss of As-pro-PO III-mediated protein-protein linkages causes morphological abnormalities in the protein matrix. Our findings suggest that As-pro-PO III plays a role in cuticle formation in mosquitoes, a novel function for phenol-oxidizing enzymes.

The histological structure of glyptosaurine osteoderms (Squamata: Anguidae), and the problem of osteoderm development in squamates.

Glyptosaurinae, a fossil clade of anguid lizards, possess robust osteoderms, with granular ornamentation. In this study, the structural and histological features of these osteoderms were described in order to reconstruct their developmental pattern and further document the degree of homology that could exist between vertebrate integumentary skeletons. Glyptosaurine osteoderms have a diploe architecture and display an unusually complex structure that includes four tissue types: a core of woven-fibered bone intensely remodeled; a peripheral formation of the same tissue containing dense bundles of long Sharpey fibers; a thick basal layer of lamellar bone; and a superficial layer of a non-osseous material that belongs to the category of hypermineralized tissues such as ganoine, or enameloid and enamel tissues. The growth pattern of glyptosaurine osteoderms involved appositional processes due to osteoblast activity. In early growth stages, osseous metaplasia might have also been involved, but this possibility is not substantiated by histological observations. The superficial layer of the osteoderms must have resulted from epidermal contribution, a conclusion that would support previous hypotheses on the role of epidermal-dermal interactions in the formation of squamate osteoderms.

The ontogeny of Pseudis platensis (Anura, Hylidae): Heterochrony and the effects of larval development on postmetamorphic life.

Recent studies have described the giant tadpole, delayed metamorphic transformations, and absence of postmetamorphic growth of the skeleton of Pseudis Platensis. These features address questions about derived patterns of life cycles and the role of the heterochrony during the metamorphosis in anurans. Using anatomical methods, we provide new data on the development of reproductive, digestive and integument systems, and age inference obtained from ontogenetic series of Pseudis platensis. Our results indicate that at the end of metamorphosis, the adult skin is completely differentiated, including the calcified dermal layer; the testis has seminiferous tubules with spermatogonia, spermatocytes, and spermatids; ovarian sacs present previtellogenic ova; and the adult digestive tract is fully formed. The froglets differ from adults only in being unable to reproduce. The entire life cycle of P. platensis can occur in 4 years. In the first year, larval development, growth to adult size, and gonad differentiation are completed. Long larval development rather than size of the tadpoles seems to be involved in the absence of juvenile stages.

Elucidation of the regulation of an adult cuticle gene Acp65A by the transcription factor Broad.

Broad (BR), an ecdysone-inducible transcription factor, is a major determinant of the pupal stage. The misexpression of BR-Z1 isoform (BR-Z1) during adult development of Drosophila melanogaster prevents the expression of the adult cuticle protein 65A gene (Acp65A). We found that the proximal 237 bp of the 5' flanking region of Acp65A were sufficient to mediate this suppression. A targeted point mutation of a putative BR-Z1 response element (BRE) within this region showed that it was not involved. Drosophila hormone receptor-like 38 (DHR38) is required for Acp65A expression. We found that BR-Z1 repressed DHR38 expression and that BR's inhibition of Acp65A expression was rescued by exogenous expression of DHR38. Thus, BR-Z1 suppresses Acp65A expression by preventing the normal up-regulation of DHR38 at the time of adult cuticle formation.

Monohydroxylated polycyclic aromatic hydrocarbons inhibit both osteoclastic and osteoblastic activities in teleost scales.

AIMS: We previously demonstrated that monohydroxylated polycyclic aromatic hydrocarbons (OHPAHs) bound to a human estrogen receptor (ER) by a yeast two-hybrid assay, but polycyclic aromatic hydrocarbons did not have a binding activity. Therefore, the direct effect of 3-hydroxybenz[a]anthracene (3-OHBaA) and 4-hydroxybenz[a]anthracene (4-OHBaA) on osteoclasts and osteoblasts in teleosts was examined. As a negative control, 1-hydroxypyrene (1-OHPy), which has no binding activity to human ER, was used. MAIN METHODS: The effect of OHPAHs on osteoclasts and osteoblasts was examined by an assay system using teleost scale as each marker: tartrate-resistant acid phosphatase for osteoclasts and alkaline phosphatase for osteoblasts. Changes in cathepsin K (an osteoclastic marker) and insulin-like growth factor-I (IGF-I) (an osteoblastic marker) mRNA expressions in 4-OHBaA-treated goldfish scales were examined by using a reverse transcription-polymerase chain reaction. KEY FINDINGS: In both goldfish (a freshwater teleost) and wrasse (a marine teleost), the osteoclastic activity in the scales was significantly suppressed by 3-OHBaA and 4-OHBaA, although 1-OHPy did not affect the osteoclastic activity. In reference to osteoblasts, the osteoblastic activity decreased with both 3-OHBaA and 4-OHBaA and did not change with the 1-OHPy treatment. However, 17beta-estradiol (E(2)) significantly increased both the osteoclastic and osteoblastic activities in the scales of both goldfish and wrasse. The mRNA expressions of both cathepsin K and IGF-I decreased in the 4-OHBaA-treated scales but increased in the E(2)-treated scales. SIGNIFICANCE: The current data are the first to demonstrate that 3-OHBaA and 4-OHBaA inhibited both osteoclasts and osteoblasts and disrupted the bone metabolism in teleosts.

Drosophila DHR38 nuclear receptor is required for adult cuticle integrity at eclosion.

DHR38 is the only Drosophila member of the NR4A subclass of vertebrate nuclear receptors, which have been implicated in multiple biological pathways, including neuronal function, apoptosis, and metabolism. Although an earlier study identified three point mutations in DHR38, none of these were shown to be a null allele for the locus, leaving it unclear whether a complete loss of DHR38 function might uncover novel roles for the receptor. Here we show that a specific DHR38 null allele, DHR38(Y214), leads to fully penetrant pharate adult lethality, similar to the most severe phenotype associated with the EMS-induced mutations. DHR38(Y214) mutants display minor effects on ecdysone-regulated transcription at the onset of metamorphosis. In contrast, cuticle gene expression is significantly reduced in DHR38(Y214) mutant pupae. These studies define the essential functions of DHR38 and provide a genetic context for further characterization of its roles during development.

Impact of the insect growth regulator diflubenzuron on biochemical composition of cuticle of the shrimp Penaeus kerathurus.

Diflubenzuron (Dimilin 25WP) is an insecticide of the class of benzoylphenylurea (BPUs) and is widely used in the control of insect pest in agriculture and forestry. Typically, these chemistries prevent the moulting process by inhibiting chitin formation and thereby causing abnormal cuticular deposition and abortive moulting in insect and crustacean species. In a previous study, we have shown that diflubenzuron could cause a modification in the lamellar ultrastructural, particularly in the membranous layers, in the non-target organism, the caramote prawn or triple-grooved shrimp Penaeus kerathurus (Forskal, 1775) (Decapoda, Peneidae). Therefore, the aim of this study was to evaluate under laboratory conditions the potential side-effects of diflubenzuron on the biochemical composition of the cuticle of treated P. kerathurus. Diflubenzuron was added to the rearing seawater at a final concentration of 1 microg/L) and newly-ecdysed adult shrimps were exposed for 10 days, i.e. until stage C during the molting cycle. The most typical treatment symptoms were a significantly reduced thickness of both the principal and membranous layers, and reduced amounts of chitin in the cuticle. These symptoms help in explaining the structural alterations observed in treated cuticles, and confirm the primary mode of action of diflubenzuron to inhibit chitin biosynthesis.