Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Germline specification and axis determination in viviparous and oviparous pea aphids: conserved and divergent features.

Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

Morpho-regulation in diverse chicken feather formation: Integrating branching modules and sex hormone-dependent morpho-regulatory modules.

Many animals can change the size, shape, texture and color of their regenerated coats in response to different ages, sexes, or seasonal environmental changes. Here, we propose that the feather core branching morphogenesis module can be regulated by sex hormones or other environmental factors to change feather forms, textures or colors, thus generating a large spectrum of complexity for adaptation. We use sexual dimorphisms of the chicken to explore the role of hormones. A long-standing question is whether the sex-dependent feather morphologies are autonomously controlled by the male or female cell types, or extrinsically controlled and reversible. We have recently identified core feather branching molecular modules which control the anterior-posterior (bone morphogenetic orotein [BMP], Wnt gradient), medio-lateral (Retinoic signaling, Gremlin), and proximo-distal (Sprouty, BMP) patterning of feathers. We hypothesize that morpho-regulation, through quantitative modulation of existing parameters, can act on core branching modules to topologically tune the dimension of each parameter during morphogenesis and regeneration. Here, we explore the involvement of hormones in generating sexual dimorphisms using exogenously delivered hormones. Our strategy is to mimic male androgen levels by applying exogenous dihydrotestosterone and aromatase inhibitors to adult females and to mimic female estradiol levels by injecting exogenous estradiol to adult males. We also examine differentially expressed genes in the feathers of wildtype male and female chickens to identify potential downstream modifiers of feather morphogenesis. The data show male and female feather morphology and their color patterns can be modified extrinsically through molting and resetting the stem cell niche during regeneration.

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 Yes-Associated Protein (YAP) and Insulin-Like Growth Factor 1 Receptor (IGF-1R) Signaling Loop Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma.

The role of a YAP-IGF-1R signaling loop in HCC resistance to sorafenib remains unknown. METHOD: Sorafenib-resistant cells were generated by treating naïve cells (HepG2215 and Hep3B) with sorafenib. Different cancer cell lines from databases were analyzed through the ONCOMINE web server. BIOSTORM-LIHC patient tissues (46 nonresponders and 21 responders to sorafenib) were used to compare YAP mRNA levels. The HepG2215_R-derived xenograft in SCID mice was used as an in vivo model. HCC tissues from a patient with sorafenib failure were used to examine differences in YAP and IGF-R signaling. RESULTS: Positive associations exist among the levels of YAP, IGF-1R, and EMT markers in HCC tissues and the levels of these proteins increased with sorafenib failure, with a trend of tumor-margin distribution in vivo. Blocking YAP downregulated IGF-1R signaling-related proteins, while IGF-1/2 treatment enhanced the nuclear translocation of YAP in HCC cells through PI3K-mTOR regulation. The combination of YAP-specific inhibitor verteporfin (VP) and sorafenib effectively decreased cell viability in a synergistic manner, evidenced by the combination index (CI). CONCLUSION: A YAP-IGF-1R signaling loop may play a role in HCC sorafenib resistance and could provide novel potential targets for combination therapy with sorafenib to overcome drug resistance in HCC.

Folding Keratin Gene Clusters during Skin Regional Specification.

Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate ß-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather ß-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.

Whole-mount identification of gene transcripts in aphids: protocols and evaluation of probe accessibility.

In situ hybridization has become a powerful tool for detecting the temporal and spatial distribution of gene transcripts in prokaryotes and eukaryotes. We report an efficient protocol for whole-mount identification of the expression of mRNAs in the parthenogenetic pea aphid Acyrthosiphon pisum, an emerging model organism with a growing accumulation of genome sequencing data. In addition to steps common for most animal in situ hybridization protocols, we describe processing methods specific to aphids, the accessibility of antisense riboprobes of different lengths in whole-mounted aphids, and signal intensity versus probe lengths. To find substrate combinations that clearly contrast single and double in situ signals in A. pisum, we tested our protocols using riboprobes constructed from two conserved germline markers, Apvasa and Apnanos, and examined colocalized signals in the germaria and developing oocytes. Finally, we propose conditions for stringent permeabilization that may be applied to tissues deep within the aphid embryo.

Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers.

The germarium, oocytes and embryos of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum are contained within a single ovariole. This species provides an excellent model for studying how maternally-inherited germ plasm is specified and how it is transferred to primordial germ cells. Previous studies have shown that germ cells are first segregated at the embryonic posterior after formation of the blastoderm. We used two cross-reacting antibodies against the conserved germline markers Vasa and Nanos, which specifically identified these presumptive germ cells, to investigate whether germ cells were determined during early development. We observed randomly-distributed weak expression of Vasa signals in the developing oocyte but no localization in the oocyte segregated from the germarium. Localized Vasa was not apparent until it was detected at the posterior in the embryo undergoing the second nuclear division. Nanos, on the other hand, was localized to a nuage-like structure surrounding the nucleus in the developing and segregated oocytes. At the beginning of the oocyte maturation division, Nanos localization shifted to the posterior and could be identified in successive stages until it was incorporated into the germ cells. Taken together, our results suggest that germ plasm is specified in the developing oocyte and that Nanos is an earlier germline marker than Vasa. Germ cells stained for Vasa remained at a dorsal location in the egg during mid-development and then were guided into abdominal segments A1 to A6 during germ-band retraction. We infer that germ cells coalesce with segmented gonadal mesoderm during this period.

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum.

BACKGROUND: Germline specification in some animals is driven by the maternally inherited germ plasm during early embryogenesis (inheritance mode), whereas in others it is induced by signals from neighboring cells in mid or late development (induction mode). In the Metazoa, the induction mode appears as a more prevalent and ancestral condition; the inheritance mode is therefore derived. However, regarding germline specification in organisms with asexual and sexual reproduction it has not been clear whether both strategies are used, one for each reproductive phase, or if just one strategy is used for both phases. Previously we have demonstrated that specification of germ cells in the asexual viviparous pea aphid depends on a preformed germ plasm. In this study, we extended this work to investigate how germ cells were specified in the sexual oviparous embryos, aiming to understand whether or not developmental plasticity of germline specification exists in the pea aphid. RESULTS: We employed Apvas1, a Drosophila vasa ortholog in the pea aphid, as a germline marker to examine whether germ plasm is preformed during oviparous development, as has already been seen in the viviparous embryos. During oogenesis, Apvas1 mRNA and ApVas1 protein were both evenly distributed. After fertilization, uniform expression of Apvas1 remained in the egg but posterior localization of ApVas1 occurred from the fifth nuclear cycle onward. Posterior co-localization of Apvas1/ApVas1 was first identified in the syncytial blastoderm undergoing cellularization, and later we could detect specific expression of Apvas1/ApVas1 in the morphologically identifiable germ cells of mature embryos. This suggests that Apvas1/ApVas1-positive cells are primordial germ cells and posterior localization of ApVas1 prior to cellularization positions the preformed germ plasm. CONCLUSIONS: We conclude that both asexual and sexual pea aphids rely on the preformed germ plasm to specify germ cells and that developmental plasticity of germline specification, unlike axis patterning, occurs in neither of the two aphid reproductive phases. Consequently, the maternal inheritance mode implicated by a preformed germ plasm in the oviparous pea aphid becomes a non-canonical case in the Hemimetabola, where so far the zygotic induction mode prevails in most other studied insects.

Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.

RNA in situ hybridization (ISH), including chromogenic ISH (CISH) and fluorescent ISH (FISH), has become a powerful tool for revealing the spatial distribution of gene transcripts in model organisms. Previously, we developed a robust protocol for whole-mount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomic model. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow, using confocal microscopy, clearer visualization of target messenger RNAs (mRNAs) - including those subcellularly localized and those with spatially overlapping expression. We find that Fast dye-based substrate fluorescence (SF), tyramide signal amplification (TSA), and TSA Plus all enable identifying gene expression thanks to multiplex amplification of fluorescent signals. By contrast, methods of direct fluorescence (DF) do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plus for most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvas1 and Appiwi2 in the germ cells. For detection of multiple gene targets using double FISH, we recommend: (i) TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ cells, as proteinase K treatment can be omitted; and (ii) SF/TSA Plus for other gene targets such as Apen1 and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due to signal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid. We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from which useful insights into development and evolution may be obtained.

Developmental expression of Apnanos during oogenesis and embryogenesis in the parthenogenetic pea aphid Acyrthosiphon pisum.

Among genes that are preferentially expressed in germ cells, nanos and vasa are the two most conserved germline markers in animals. Both genes are usually expressed in germ cells in the adult gonads, and often also during embryogenesis. Both nanos-first or vasa-first expression patterns have been observed in embryos, implying that the molecular networks governing germline development vary among species. Previously we identified Apvasa, a vasa homologue expressed in germ cells throughout all developmental stages in the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum. In asexual A. pisum, oogenesis is followed by embryogenesis, and both occur within the ovarioles. In order to understand the temporal and spatial distribution of nanos versus vasa during oogenesis and embryogenesis, we isolated a nanos homologue, Apnanos, and studied its expression. In adults, Apnanos is preferentially expressed in the ovaries. In early embryos, Apnanos transcripts are localized to the cytoplasm of cellularizing germ cells, and soon thereafter are restricted to the newly segregated germ cells in the posterior region of the cellularized blastoderm. These results strongly suggest that the Apnanos gene is a germline marker and is involved in germline specification in asexual A. pisum. However, during the middle stages of development, when germline migration occurs, Apnanos is not expressed in the migrating germ cells expressing Apvasa, suggesting that Apnanos is not directly associated with germline migration.

Apvasa marks germ-cell migration in the parthenogenetic pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea).

In the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum, germline specification depends on the germ plasm localized to the posterior region of the egg chamber before the formation of the blastoderm. During blastulation, germline segregation occurs at the egg posterior, and in early gastrulation germ cells are pushed inward by the invaginating germ band. Previous studies suggest that germ cells remain dorsal in the embryo in subsequent developmental stages. In fact, though, it is not known whether germ cells remain in place or migrate dynamically during katatrepsis and germ-band retraction. We cloned Apvasa, a pea aphid homologue of Drosophila vasa, and used it as a germline marker to monitor the migration of germ cells. Apvasa messenger RNA (mRNA) was first restricted to morphologically identifiable germ cells after blastoderm formation but that expression soon faded. Apvasa transcripts were again identified in germ cells from the stage when the endosymbiotic bacteria invaded the embryo, and after that, Apvasa mRNA was present in germ cells throughout all developmental stages. At the beginning of katatrepsis, germ cells were detected at the anteriormost region of the egg chamber as they were migrating into the body cavity. During the early period of germ-band retraction, germ cells were separated into several groups surrounded by a layer of somatic cells devoid of Apvasa staining, suggesting that the coalescence between migrating germ cells and the somatic gonadal mesoderm occurs between late katatrepsis and early germ-band retraction.