Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation.

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.

Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation.

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent expression in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for expression control and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA and protein expression.

The effect of Drosophila attP40 background on the glomerular organization of Or47b olfactory receptor neurons.

Bacteriophage integrase-directed insertion of transgenic constructs into specific genomic loci has been widely used by Drosophila community. The attP40 landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. Here, unexpectedly, we found that homozygous attP40 chromosome disrupts normal glomerular organization of Or47b olfactory receptor neuron (ORN) class in Drosophila. This effect is not likely to be caused by the loss of function of Msp300, where the attP40 docking site is inserted. Moreover, the attP40 background seems to genetically interact with the second chromosome Or47b-GAL4 driver, which results in a similar glomerular defect. Whether the ORN phenotype is caused by the neighbouring genes around Msp300 locus in the presence of attP40-based insertions or a second unknown mutation in the attP40 background remains elusive. Our findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out the attP40 landing site-associated background effects.

Chromatin-based reprogramming of a courtship regulator by concurrent pheromone perception and hormone signaling.

To increase fitness, animals use both internal and external states to coordinate reproductive behaviors. The molecular mechanisms underlying this coordination remain unknown. Here, we focused on pheromone-sensing Drosophila Or47b neurons, which exhibit age- and social experience-dependent increase in pheromone responses and courtship advantage in males. FruitlessM (FruM), a master regulator of male courtship behaviors, drives the effects of social experience and age on Or47b neuron responses and function. We show that simultaneous exposure to social experience and age-specific juvenile hormone (JH) induces chromatin-based reprogramming of fruM expression in Or47b neurons. Group housing and JH signaling increase fruM expression in Or47b neurons and active chromatin marks at fruM promoter. Conversely, social isolation or loss of JH signaling decreases fruM expression and increases repressive marks around fruM promoter. Our results suggest that fruM promoter integrates coincident hormone and pheromone signals driving chromatin-based changes in expression and ultimately neuronal and behavioral plasticity.

Social Context Enhances Hormonal Modulation of Pheromone Detection in Drosophila.

Critical to evolutionary fitness, animals regulate social behaviors by integrating signals from both their external environments and internal states. Here, we find that population density modulates the courtship behavior of male Drosophila melanogaster in an age-dependent manner. In a competitive mating assay, males reared in a social environment have a marked advantage in courting females when pitted against males reared in isolation. Group housing promotes courtship in mature (7-day) but not immature (2-day) males; this behavioral plasticity requires the Or47b pheromone receptor. Using single-sensillum recordings, we find that group housing increases the response of Or47b olfactory receptor neurons (ORNs) only in mature males. The effect of group housing on olfactory response and behavior can be mimicked by chronically exposing single-housed males to an Or47b ligand. At the molecular level, group housing elevates Ca2+ levels in Or47b ORNs, likely leading to CaMKI-mediated activation of the histone-acetyl transferase CBP. This signaling event in turn enhances the efficacy of juvenile hormone, an age-related regulator of reproductive maturation in flies. Furthermore, the male-specific Fruitless isoform (FruM) is required for the sensory plasticity, suggesting that FruM functions as a downstream genomic coincidence detector in Or47b ORNs-integrating reproductive maturity, signaled by juvenile hormone, and population density, signaled by CBP. In all, we identify a neural substrate and activity-dependent mechanism by which social context can directly influence pheromone sensitivity, thereby modulating social behavior according to animals' life-history stage.

Combinations of DIPs and Dprs control organization of olfactory receptor neuron terminals in Drosophila.

In Drosophila, 50 classes of olfactory receptor neurons (ORNs) connect to 50 class-specific and uniquely positioned glomeruli in the antennal lobe. Despite the identification of cell surface receptors regulating axon guidance, how ORN axons sort to form 50 stereotypical glomeruli remains unclear. Here we show that the heterophilic cell adhesion proteins, DIPs and Dprs, are expressed in ORNs during glomerular formation. Many ORN classes express a unique combination of DIPs/dprs, with neurons of the same class expressing interacting partners, suggesting a role in class-specific self-adhesion between ORN axons. Analysis of DIP/Dpr expression revealed that ORNs that target neighboring glomeruli have different combinations, and ORNs with very similar DIP/Dpr combinations can project to distant glomeruli in the antennal lobe. DIP/Dpr profiles are dynamic during development and correlate with sensilla type lineage for some ORN classes. Perturbations of DIP/dpr gene function result in local projection defects of ORN axons and glomerular positioning, without altering correct matching of ORNs with their target neurons. Our results suggest that context-dependent differential adhesion through DIP/Dpr combinations regulate self-adhesion and sort ORN axons into uniquely positioned glomeruli.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Transcriptional profiling of olfactory system development identifies distal antenna as a regulator of subset of neuronal fates.

Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered within distinct sensilla subtypes to decipher their chemical environment. Each sensilla subtype houses 1-4 ORN identities that arise through asymmetric divisions of a single sensory organ precursor (SOP). Despite a number of mutational studies investigating the regulation of ORN development, a majority of the transcriptional programs that lead to the different ORN classes in the developing olfactory system are unknown. Here we use transcriptional profiling across the time series of antennal development to identify novel transcriptional programs governing the differentiation of ORNs. We surveyed four critical developmental stages of the olfactory system: 3rd instar larval (prepatterning), 8 hours after puparium formation (APF, SOP selection), 40 hrs APF (neurogenesis), and adult antennae. We focused on the expression profiles of olfactory receptor genes and transcription factors-the two main classes of genes that regulate the sensory identity of ORNs. We identify distinct clusters of genes that have overlapping temporal expression profiles suggesting they have a key role during olfactory system development. We show that the expression of the transcription factor distal antenna (dan) is highly similar to other prepatterning factors and is required for the expression of a subset of ORs.

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

Morphological variation of carotid artery bifurcation level in digital angiography.

Knowing of the level of carotid artery bifurcation (CB) is important for vascular surgery in the neck, radical neck dissections, carotid sinus baroreceptor stimulation, catheterisations, and aneurysms. The aim of this study was to determine the CB level in relation with the cervical vertebral levels, compare them on the right and the left sides, and investigate the relation of CB level with the length of neck. In this study, 100 conventional carotid angiographies were performed. The CB level was determined in relation with 10 different levels which were the levels of the cervical vertebrae and intervertebral disks, and the relation of CB level with the length of neck was investigated. The right and left CB levels of the patients were also determined, and compared. The highest level of CB was at the level of C2 vertebra, and the lowest level of CB was at the level of C6-C7 intervertebral disk in both male and female. When all patients were taken into consideration, CB level was most frequently seen at the level of C4-C5 (29%) on the right side, and at the level of C4 (26%) on the left side. The CB levels were not symmetrical in 10 female and 23 male. Knowing of the anatomical variations of CB level is important in surgical procedures. The anatomical differences must be taken into consideration since the neighbouring structures of CB change in case of variations. We believe that the results of this study will shed light to planning of all interventional methods concerning common carotid artery and its branches as well as surgery in the neck, and will help to minimise the complications.

An unusual course of the radial artery.

Radial artery variations are of importance for clinicians, whether in angiographic examinations or surgical approaches. The high origin radial artery is the most frequent arterial variation observed in the upper limb, showing an incidence of 14.27% in dissection material and 9.75% in angiographic examination. In the present study an unusual course of the radial artery and its relation with the median nerve has been evaluated. During embryological development the radial artery sprouts from two arterial buds arising from the lateral side of the brachial artery and coalescing with each other. The artery lies in the forearm and is overlapped by the brachioradial muscle. In this particular case the radial artery originated from the medial side of the brachial artery and crossed the median nerve twice in an unusual manner 8 cm below the point at which the deep brachial artery arose and 12 cm above the intercondylar line. These results will enhance anatomical knowledge of the region and reduce complication in surgical approaches.

The incidence and types of sella and sphenopetrous bridges.

The incidence and types of sella and sphenopetrous bridges were investigated in 37 adult male and 43 adult female (a total of 80) dry skulls with removed calvarias. In addition to this, the sellar and parasellar region of ten fixed cadavers (two female and eight male) were carefully dissected, and the individuals were examined for the evidence of sella and sphenopetrous bridges. Sella bridges were seen in 34.17% of the subjects overall. The trace, incomplete and complete types were 11.9%, 3.7% and 17.5%, respectively. On the other hand, sphenopetrous bridges were observed in 15.8% of the male and 4.9% of the female subjects overall. The cadaveric investigation revealed one trace, three incomplete, and one complete sella bridge in three cadavers. In addition to this, a complete sphenopetrous bridge was detected in one of the cadavers. Variations in the cranial base are of importance for surgical approaches in that location.

Supraarticular, supramastoid and suprameatal crests on the outer surface of the temporal bone and the relation between them.

Supraarticular, supramastoid and suprameatal crests on the outer surface of the squamous part of the temporal bone are of clinical importance as they are accepted as landmarks for some surgical approaches relating to the temporomandibular joint, the middle cranial fossa and mastoid air cells, respectively. Because of the surgical importance of the crests, their incidence and the relations between them were studied on a total of 442 (250 male, 192 female) dry skulls. In addition, three-dimensional (3D) surface renderings of the squamous part of the temporal bone were created from routine axial computed tomographic (CT) images in four cadaver specimens to demonstrate the surface anatomy. Suprameatal crests were observed commonly as a trace type on the male dry skulls (51.2%), though no such crest was seen on most of the female skulls (54.4%). Supramastoid crests were commonly observed as a small crest on male dry skulls (46.6%), but as a trace type on female dry skulls (52.05%). Supraarticular crests were commonly seen as a trace type on both male (58.6%) and female (70.6%) dry skulls. In the light of the above data, it can be concluded that the crests on the male dry skulls were stronger than those on the female skulls. The angles between the crests were also examined. It was observed that the angle between the supraarticular and supramastoid crests was larger in male dry skulls compared with the female dry skulls. On the other hand, no gender difference was observed when the angle between the suprameatal and supraarticular crests was examined.

Incidence of os japonicum in Anatolian dry skulls and plain cranium radiographs of modern Anatolian population.

INTRODUCTION: The zygoma may sometimes be divided into two parts by either a horizontal or a vertical suture. Such a bipartite bone has been given the name of os japonicum as it has been mostly observed in Japanese. MATERIAL: In this study 1266 zygomatic bones in 633 Anatolian dry skulls and 1348 zygomatic bones in 674 plain cranium radiographs of adult patients have been examined. RESULTS: Os japonicum was present in 2.2% of female and 1.7% of male subjects. All of the 24 multipartite bones observed in the study were bipartite except one. In addition, of 690 female zygomatic bones examined radiologically 15 (2.2%), and 658 male bones 12 (1.8%) were bipartite or tripartite, a total of 674 plain cranium radiographs. CONCLUSION: The results were compared with those of other populations. As a result evaluation of both dry skulls and plain radiographs as a single sample, our results were again closely similar to Main Island Japan and other East Asia groups.

Various types of suprameatal spines and depressions in the human temporal bone.

Since it covers the lateral wall of the mastoid air system, the suprameatal triangle is of importance to otologic surgeons during mastoidectomy. Because of this clinical importance, topographic anatomy of the suprameatal spine and depression was studied on Anatolian skulls. In all, 363 male and 231 female skulls were studied. The most prevalent type of suprameatal spine resembled a crest and was found in both sexes on the right (77.6%) and left (80%) sides. The absence of a suprameatal depression was significantly higher in females (right 9.1%; left 8.7%) than in males (right 1.7%; left 2.5%). Suprameatal depressions were mostly shallow in female subjects, but were mostly observed in males to be at a medium depth or deep.

The incidence and localization of mastoid foramen and superficial parietomastoid canal and their relations with each other.

Mastoid foramen and some superficial bony canal on the outer surface of the mastoid region are of importance not only as an epigenetic variation but because of the vessels passing through it as well. On the other hand such a canal structure has not been described in classical anatomy textbooks. In this study, the presence, localization and number of both mastoid foramen and superficial bony canal and their relations with each other were studied in 358 male, 228 female, a total of 586 dry Anatolian skulls. The mastoid foramen was absent in 21.5% of the skulls. In its presence it was mostly seen on temporal bone. A superficial bony canal was observed in 45% of the skulls. Some correlations were also observed between the above mentioned structures.

Frequency and classification of bony projections on the lower border of superior orbital fissure.

At the lower border of superior orbital fissure, at the junction of broad medial and narrow lateral portions of the greater wing of sphenoid bone, a bony projection (lateral rectus spine) occurs onto which the lateral rectus muscle and common tendinous ring attach. Such a bony projection has a special clinical significance not only because of its relationship with the contents of superior orbital fissure, but also because it is an attachment point for dura mater. In this study 286 males and 181 females, a total of 467 Anatolian dry skulls in which all the orbits were intact were examined. The bony projections mentioned above were classified according to their shapes. The ones most observed were the tubercle and spine forms which were 30.62% and 23.23% respectively. Of the 467 dry skulls 156 (16.70%) had no bony projection on the lower border of superior orbital fissure.