Thermal stress induces tissue damage and a broad shift in regenerative signaling pathways in the honey bee digestive tract.

Honey bee colonies in the USA have suffered from increased die-off in the last few years with a complex set of interacting stresses playing a key role. With changing climate, an increase in the frequency of severe weather events, such as heat waves, is anticipated. Understanding how these changes may contribute to stress in honey bees is crucial. Individual honey bees appear to have a high capacity to endure thermal stress. One reason for this high-level endurance is likely their robust heat shock response (HSR), which contributes to thermotolerance at the cellular level. However, less is known about other mechanisms of thermotolerance, especially those operating at the tissue level. To elucidate other determinants of resilience in this species, we used thermal stress coupled with RNAseq and identified broad transcriptional remodeling of a number of key signaling pathways in the honey bee, including those pathways known to be involved in digestive tract regeneration in the fruit fly such as the Hippo and JAK/STAT pathways. We also observed cell death and shedding of epithelial cells, which likely leads to induction of this regenerative transcriptional program. We found that thermal stress affects many of these pathways in other tissues, suggesting a shared program of damage response. This study provides important foundational characterization of the tissue damage response program in this key pollinating species. In addition, our data suggest that a robust regeneration program may also be a critical contributor to thermotolerance at the tissue level, a possibility which warrants further exploration in this and other species.

HOXA5 plays tissue-specific roles in the developing respiratory system.

Hoxa5 is essential for development of several organs and tissues. In the respiratory system, loss of Hoxa5 function causes neonatal death due to respiratory distress. Expression of HOXA5 protein in mesenchyme of the respiratory tract and in phrenic motor neurons of the central nervous system led us to address the individual contribution of these Hoxa5 expression domains using a conditional gene targeting approach. Hoxa5 does not play a cell-autonomous role in lung epithelium, consistent with lack of HOXA5 expression in this cell layer. In contrast, ablation of Hoxa5 in mesenchyme perturbed trachea development, lung epithelial cell differentiation and lung growth. Further, deletion of Hoxa5 in motor neurons resulted in abnormal diaphragm innervation and musculature, and lung hypoplasia. It also reproduced the neonatal lethality observed in null mutants, indicating that the defective diaphragm is the main cause of impaired survival at birth. Thus, Hoxa5 possesses tissue-specific functions that differentially contribute to the morphogenesis of the respiratory tract.

Independent regulation of vertebral number and vertebral identity by microRNA-196 paralogs.

The Hox genes play a central role in patterning the embryonic anterior-to-posterior axis. An important function of Hox activity in vertebrates is the specification of different vertebral morphologies, with an additional role in axis elongation emerging. The miR-196 family of microRNAs (miRNAs) are predicted to extensively target Hox 3' UTRs, although the full extent to which miR-196 regulates Hox expression dynamics and influences mammalian development remains to be elucidated. Here we used an extensive allelic series of mouse knockouts to show that the miR-196 family of miRNAs is essential both for properly patterning vertebral identity at different axial levels and for modulating the total number of vertebrae. All three miR-196 paralogs, 196a1, 196a2, and 196b, act redundantly to pattern the midthoracic region, whereas 196a2 and 196b have an additive role in controlling the number of rib-bearing vertebra and positioning of the sacrum. Independent of this, 196a1, 196a2, and 196b act redundantly to constrain total vertebral number. Loss of miR-196 leads to a collective up-regulation of numerous trunk Hox target genes with a concomitant delay in activation of caudal Hox genes, which are proposed to signal the end of axis extension. Additionally, we identified altered molecular signatures associated with the Wnt, Fgf, and Notch/segmentation pathways and demonstrate that miR-196 has the potential to regulate Wnt activity by multiple mechanisms. By feeding into, and thereby integrating, multiple genetic networks controlling vertebral number and identity, miR-196 is a critical player defining axial formulae.

Gustatory receptor neurons in Manduca sexta contain a TrpA1-dependent signaling pathway that integrates taste and temperature.

Temperature modulates the peripheral taste response of many animals, in part by activating transient receptor potential (Trp) cation channels. We hypothesized that temperature would also modulate peripheral taste responses in larval Manduca sexta. We recorded excitatory responses of the lateral and medial styloconic sensilla to chemical stimuli at 14, 22, and 30 °C. The excitatory responses to 5 chemical stimuli-a salt (KCl), 3 sugars (sucrose, glucose, and inositol) and an alkaloid (caffeine)-were unaffected by temperature. In contrast, the excitatory response to the aversive compound, aristolochic acid (AA), increased robustly with temperature. Next, we asked whether TrpA1 mediates the thermally dependent taste response to AA. To this end, we 1) identified a TrpA1 gene in M. sexta; 2) demonstrated expression of TrpA1 in the lateral and medial styloconic sensilla; 3) determined that 2 TrpA1 antagonists (HC-030031 and mecamylamine) inhibit the taste response to AA, but not caffeine; and then 4) established that the thermal dependence of the taste response to AA is blocked by HC-030031. Taken together, our results indicate that TrpA1 serves as a molecular integrator of taste and temperature in M. sexta.

MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression.

MicroRNAs (miRNAs) are a class of short ( approximately 22-nt) noncoding RNA molecules that downregulate expression of their mRNA targets. Since their discovery as regulators of developmental timing in Caenorhabditis elegans, hundreds of miRNAs have been identified in both animals and plants. Here, we report a technique for visualizing detailed miRNA expression patterns in mouse embryos. We elucidate the tissue-specific expression of several miRNAs during embryogenesis, including two encoded by genes embedded in homeobox (Hox) clusters, miR-10a and miR-196a. These two miRNAs are expressed in patterns that are markedly reminiscent of those of Hox genes. Furthermore, miR-196a negatively regulates Hoxb8, indicating that its restricted expression pattern probably reflects a role in the patterning function of the Hox complex.

In Amphioxus Embryos, Some Neural Tube Cells Resemble Differentiating Coronet Cells of Fishes and Tunicates.

AbstractFor neurula embryos of amphioxus (chordate subphylum Cephalochordata), the anterior region of the neural tube was studied with transmission electron microscopy. This survey demonstrated previously unreported cells, each characterized by a cilium bearing on its shaft a protruding lateral bubble packed with vesicles. Such cilia resemble those known from immature coronet cells in other chordates-namely, fishes in the Vertebrata and ascidians and appendicularians in the Tunicata. This wide occurrence of coronet-like cells raises questions about their possible homologies within the phylum Chordata. When considered at the level of the whole cell, such homology is not well supported. For example, the fish cells are generally thought to be glia, while the tunicate cells are considered to be neurons; moreover, cytoplasmic smooth endoplasmic reticulum, which is predominant in the former, is undetectable in the latter. In contrast, a more convincing case for homology can be made by limiting comparisons to the cell apices with their modified cilia. In addition to the fine-structural similarities between fishes and tunicates already mentioned, nonvisual opsins have been found associated with the vesicles in the modified cilia of both groups. Such opsins are thought to link photoreception to endocrine output controlling behavior. Further work would be needed to test the idea that the amphioxus diencephalic cells with lateral bubble cilia might similarly be opsin rich and could provide insights into the evolutionary history of the coronet cells within the phylum Chordata.

Evolutionary assembly and disassembly of the mammalian sternum.

Evolutionary transitions are frequently associated with novel anatomical structures,1 but the origins of the structures themselves are often poorly known. We use developmental, genetic, and paleontological data to demonstrate that the therian sternum was assembled from pre-existing elements. Imaging of the perinatal mouse reveals two paired sternal elements, both composed primarily of cells with lateral plate mesoderm origin. Location, articulations, and development identify them as homologs of the interclavicle and the sternal bands of synapsid outgroups. The interclavicle, not previously recognized in therians,2 articulates with the clavicle and differs from the sternal bands in both embryonic HOX expression and pattern of skeletal maturation. The sternal bands articulate with the ribs in two styles, most clearly differentiated by their association with sternebrae. Evolutionary trait mapping indicates that the interclavicle and sternal bands were independent elements throughout most of synapsid history. The differentiation of rib articulation styles and the subdivision of the sternal bands into sternebrae were key innovations likely associated with transitions in locomotor and respiratory mechanics.3,4 Fusion of the interclavicle and the anterior sternal bands to form a presternum anterior to the first sternebra was a historically recent innovation unique to therians. Subsequent disassembly of the radically reduced sternum of mysticete cetaceans was element specific, reflecting the constraints that conserved developmental programs exert on composite structures.

Identification of chemosensory receptor genes in Manduca sexta and knockdown by RNA interference.

BACKGROUND: Insects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. Although our understanding of the molecular genetic basis for Drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. The tobacco hornworm, Manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. It is also a major agricultural pest on solanaceous crops. However, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. The ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in M. sexta would be greatly beneficial. RESULTS: Here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. We also used dsRNA feeding as a method to induce RNA interference in larval chemosensory tissues. CONCLUSIONS: We report identification of new chemosensory receptor genes including 17 novel odorant receptors and one novel gustatory receptor. Further, we demonstrate that systemic RNA interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation.

In ovo application of antagomiRs indicates a role for miR-196 in patterning the chick axial skeleton through Hox gene regulation.

Patterning of the vertebrate axial skeleton requires precise spatial and temporal control of Hox gene expression during embryonic development. MicroRNAs (miRNAs) are recently described modulators of gene activity, and members of the miR-196 and miR-10 families have been shown to target several Hox genes in vivo. Testing miRNA function in mice is complicated by potential redundancy between family members. To circumvent this, we have developed protocols for introducing modified antisense oligonucleotides (antagomiRs) in ovo during chick development. Using this approach, we identify a layer of regulatory control provided by the miR-196 family in defining the boundary of Hox gene expression along the anterior-posterior (A-P) embryonic axis. Following knockdown of miR-196, we observe a homeotic transformation of the last cervical vertebrae toward a thoracic identity. This phenotypic alteration is, in part, due to an anterior expansion of Hoxb8 gene expression and consolidates the in vivo relevance of post-transcriptional Hox gene regulation provided by miRNAs in the complex hierarchies governing axial pattering.

Hoxa5 Activity Across the Lateral Somitic Frontier Regulates Development of the Mouse Sternum.

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians.

The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development.

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

HOXA5 Participates in Brown Adipose Tissue and Epaxial Skeletal Muscle Patterning and in Brown Adipocyte Differentiation.

Brown adipose tissue (BAT) plays critical thermogenic, metabolic and endocrine roles in mammals, and aberrant BAT function is associated with metabolic disorders including obesity and diabetes. The major BAT depots are clustered at the neck and forelimb levels, and arise largely within the dermomyotome of somites, from a common progenitor with skeletal muscle. However, many aspects of BAT embryonic development are not well understood. Hoxa5 patterns other tissues at the cervical and brachial levels, including skeletal, neural and respiratory structures. Here, we show that Hoxa5 also positively regulates BAT development, while negatively regulating formation of epaxial skeletal muscle. HOXA5 protein is expressed in embryonic preadipocytes and adipocytes as early as embryonic day 12.5. Hoxa5 null mutant embryos and rare, surviving adults show subtly reduced iBAT and sBAT formation, as well as aberrant marker expression, lower adipocyte density and altered lipid droplet morphology. Conversely, the epaxial muscles that arise from a common dermomyotome progenitor are expanded in Hoxa5 mutants. Conditional deletion of Hoxa5 with Myf5/Cre can reproduce both BAT and epaxial muscle phenotypes, indicating that HOXA5 is necessary within Myf5-positive cells for proper BAT and epaxial muscle development. However, recombinase-based lineage tracing shows that Hoxa5 does not act cell-autonomously to repress skeletal muscle fate. Interestingly, Hoxa5-dependent regulation of adipose-associated transcripts is conserved in lung and diaphragm, suggesting a shared molecular role for Hoxa5 in multiple tissues. Together, these findings establish a role for Hoxa5 in embryonic BAT development.

Hox expression in the American alligator and evolution of archosaurian axial patterning.

The avian body plan has undergone many modifications, most associated with adaptation to flight and bipedal walking. Some of these modifications may be owing to avian-specific changes in the embryonic Hox expression code. Here, we have examined Hox expression in alligator, the closest living relative of birds, and an archosaur with a more conservative body plan. Two differences in Hox expression between chick, alligator, and other tetrapods correlate with aspects of alligator or bird-specific skeletal morphology. First, absence of a thoracic subdomain of Hoxc-8 expression in alligator correlates with morphological adaptations in crocodilian thoracic segments. Second, Hoxa-5, a gene required to pattern the cervical-thoracic transition, shows unique patterns of expression in chick, alligator, and mouse, correlating with species-specific morphological patterning of this region. Given that cervical vertebral morphologies evolved independently in the bird and mammalian lineages, the underlying developmental mechanisms, including refinement of Hox expression domains, may be distinct.

Detection of Gene and Protein Expression in Mouse Embryos and Tissue Sections.

Analysis of gene (mRNA and protein) expression patterns is central to the study of embryonic development. This chapter details methods for detecting mRNA and protein expression in whole-mouse embryos and in tissue sections, including mRNA in situ hybridization, immunohistochemistry, and detection of enzymatic and fluorescent protein reporters. We focus on histological methods; molecular methods of measuring gene expression (for example, RNAseq, PCR) are not included here.

HOXA5 protein expression and genetic fate mapping show lineage restriction in the developing musculoskeletal system.

HOX proteins act during development to regulate musculoskeletal morphology. HOXA5 patterns skeletal structures surrounding the cervical-thoracic transition including the vertebrae, ribs, sternum and forelimb girdle. However, the tissue types in which it acts to pattern the skeleton, and the ultimate fates of embryonic cells that activate Hoxa5 expression are unknown. A detailed characterization of HOXA5 expression by immunofluorescence was combined with Cre/LoxP genetic lineage tracing to map the fate of Hoxa5 expressing cells in axial musculoskeletal tissues and in their precursors, the somites and lateral plate mesoderm. HOXA5 protein expression is dynamic and spatially restricted in derivatives of both the lateral plate mesoderm and somites, including a subset of the lateral sclerotome, suggesting a local role in regulating early skeletal patterning. HOXA5 expression persists from somite stages through late development in differentiating skeletal and connective tissues, pointing to a continuous and direct role in skeletal patterning. In contrast, HOXA5 expression is excluded from the skeletal muscle and muscle satellite cell lineages. Furthermore, the descendants of Hoxa5-expressing cells, even after HOXA5 expression has extinguished, never contribute to these lineages. Together, these findings suggest cell autonomous roles for HOXA5 in skeletal development, as well as non-cell autonomous functions in muscle through expression in surrounding connective tissues. They also support the notion that different Hox genes display diverse tissue specificities and locations to achieve their patterning activity.

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

Development of somites and their derivatives in amphioxus, and implications for the evolution of vertebrate somites.

BACKGROUND: Vertebrate somites are subdivided into lineage compartments, each with distinct cell fates and evolutionary histories. Insights into somite evolution can come from studying amphioxus, the best extant approximation of the chordate ancestor. Amphioxus somites have myotome and non-myotome compartments, but development and fates of the latter are incompletely described. Further, while epithelial to mesenchymal transition (EMT) is important for most vertebrate somitic lineages, amphioxus somites generally have been thought to remain entirely epithelial. Here, we examined amphioxus somites and derivatives, as well as extracellular matrix of the axial support system, in a series of developmental stages by transmission electron microscopy (TEM) and in situ hybridization for collagen expression. RESULTS: The amphioxus somite differentiates medially into myotome, laterally into the external cell layer (a sub-dermal mesothelium), ventrally into a bud that forms mesothelia of the perivisceral coelom, and ventro-medially into the sclerotome. The sclerotome forms initially as a monolayered cell sheet that migrates between the myotome and the notochord and neural tube; subsequently, this cell sheet becomes double layered and encloses the sclerocoel. Other late developments include formation of the fin box mesothelia from lateral somites and the advent of isolated fibroblasts, likely somite derived, along the myosepta. Throughout development, all cells originating from the non-myotome regions of somites strongly express a fibrillar collagen gene, ColA, and thus likely contribute to extracellular matrix of the dermal and axial connective tissue system. CONCLUSIONS: We provide a revised model for the development of amphioxus sclerotome and fin boxes and confirm previous reports of development of the myotome and lateral somite. In addition, while somite derivatives remain almost entirely epithelial, limited de-epithelialization likely converts some somitic cells into fibroblasts of the myosepta and dermis. Ultrastructure and collagen expression suggest that all non-myotome somite derivatives contribute to extracellular matrix of the dermal and axial support systems. Although amphioxus sclerotome lacks vertebrate-like EMT, it resembles that of vertebrates in position, movement to surround midline structures and into myosepta, and contribution to extracellular matrix of the axial support system. Thus, many aspects of the sclerotome developmental program evolved prior to the origin of the vertebrate mineralized skeleton.

A reference gene set for chemosensory receptor genes of Manduca sexta.

The order of Lepidoptera has historically been crucial for chemosensory research, with many important advances coming from the analysis of species like Bombyx mori or the tobacco hornworm, Manduca sexta. Specifically M. sexta has long been a major model species in the field, especially regarding the importance of olfaction in an ecological context, mainly the interaction with its host plants. In recent years transcriptomic data has led to the discovery of members of all major chemosensory receptor families in the species, but the data was fragmentary and incomplete. Here we present the analysis of the newly available high-quality genome data for the species, supplemented by additional transcriptome data to generate a high quality reference gene set for the three major chemosensory receptor gene families, the gustatory (GR), olfactory (OR) and antennal ionotropic receptors (IR). Coupled with gene expression analysis our approach allows association of specific receptor types and behaviors, like pheromone and host detection. The dataset will provide valuable support for future analysis of these essential chemosensory modalities in this species and in Lepidoptera in general.