Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. This study investigates the iab-8 ncRNA from the Drosophila Bithorax Complex and shows that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, this study found that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote (Castro Alvarez, 2021).

Several noncoding RNAs (ncRNAs) have been identified from the Hox clusters of different species; a few of these have been shown to play key roles in gene regulation. One of these ncRNAs is the 92 Kb, spliced and polyadenylated transcript called the iab-8 ncRNA. Located within the Drosophila Bithorax Complex (BX-C), the iab-8 ncRNA originates from a promoter located about 4.5Kb downstream of the Abd-B transcription unit and continues until within about 1 Kb of the abd-A promoter. In situ hybridization experiments show that it is transcribed specifically in the very posterior epidermis of the embryo from the cellular blastoderm stage. From later embryonic stages, its expression becomes limited to parasegments (PS) 13 and 14 of the CNS. Loss of the iab-8 ncRNA has been shown to result in both male and female sterility, likely due to problems in the innervation of muscles important for reproduction. Much of its function has been attributed to a microRNA located between its sixth and seventh exons, called miR-iab-8 (miRNA). miR-iab-8 targets multiple transcripts including the Ubx and abd-A homeotic genes and their cofactors hth and exd. Indeed female sterility has been directly linked to ectopic hth, Ubx and abd-A in the CNS (Castro Alvarez, 2021).

In the embryonic CNS, abd-A expression is normally limited to PS7-12. Studies have shown that the restriction of abd-A expression from PS13 in the CNS is dependent upon expression of the iab-8 ncRNA. Although, the miR-iab-8 miRNA plays a part in the repression of abd-A in PS13, a deletion of the miRNA template sequence only results in a mild derepression of abd-A in PS13. On the contrary, mutations preventing the production of the iab-8 ncRNA cause a complete de-repression of abd-A, such that the abd-A expression pattern in PS13 mimics that of PS12), suggesting the existence of a second repression mechanism. This study explored the mechanism by which the iab-8 ncRNA represses abd-A. Using deletions spanning different regions of the iab-8 transcript, this study failed to identify specific parts of the transcript that can account for the additional repression of abd-A by the iab-8 ncRNA. Furthermore, it was found that the iab-8 transcript can repress an exogenous reporter gene placed downstream of its sequence. Based on these findings, it is concluded that it is the act of transcription that is necessary for repression, rather than the sequence transcribed (a phenomenon called transcriptional interference). Examination of the iab-8 transcript in the CNS, shows that there is a 3' extension made specifically within the CNS. This elongated transcript seems to be essential for abd-A down-regulation and requires the neuronal-specific, RNA-binding protein ELAV (or its paralogue FNE) for its creation. Overall, this work suggests that ELAV mediates a 3' extension of the iab-8 ncRNA that, in turn, allows it to specifically repress abd-A expression in the posterior CNS through transcriptional interference (Castro Alvarez, 2021).

Previous papers reported that the main transcript of the iab-8 ncRNA terminates ~1 kb upstream of the abd-A transcription unit. This finding was based on 3'RACE experiments performed on RNA isolated from relatively early embryos (6-12 hours). This study shows that, in later embryos, where the iab-8 ncRNA is restricted to the CNS, the iab-8 transcript extends well into the abd-A transcription unit. In every condition tested, it was seen that abd-A is repressed when there is an extended transcript. However, outside of the sequences required for normal transcription and RNA processing, the iab-8 transcript, itself, does not seem to require any specific sequences to mediate this repression. Based on these findings, and the cis nature of this repression, it is concluded that the act of transcribing the extended iab-8 ncRNA is what represses abd-A expression in PS13 of the CNS. This type of inhibition is called transcriptional interference (Castro Alvarez, 2021).

In transcriptional interference the transcription of one gene spreading over the coding or regulatory sequences of another gene is able to downregulate the target gene's expression. The mechanisms mediating transcriptional interference seem to depend on the relative position of both promoters. In the case of the iab-8 ncRNA and the repression of abd-A, there exist tandem promoters, where the genes are transcribed in the same direction and the upstream transcript transcribes over portions of the downstream gene (promoter, enhancers, transcription unit). Studies performed in single cell organisms (yeast and bacteria) suggest that there are two main mechanisms mediating transcriptional interference of tandem promoters. The first is called the 'sitting duck' mechanism, where an initiating RNA polymerase or activating transcription factors are knocked off of the target gene by the passing polymerase. The potential second mechanism to mediate transcriptional interference is called the 'occlusion' mechanism, where activating transcription factors (or RNA polymerase itself) for the downstream gene are prevented from binding to their binding sites by the passing RNA polymerase or by the modified chromatin structure following the passage of an elongating polymerase. Thus far, it is not possible to distinguish between these two mechanisms in this system. However, both mechanisms have been shown to be dependent upon the strength of the silencing transcript's promoter relative to the target transcript promoter. The stronger the promoter activity from the upstream gene, the stronger the repression of the downstream gene. In the case of transcriptional interference by the iab-8 ncRNA, it is believed that its level of transcription is approximately equivalent to that of abd-A. Indeed, using an abd-A intronic probe to compare levels (a probe not subject to possible stabilization of the exonic probes of the abd-A coding mRNA), a similar level is seen of transcription from both the iab-8 (PS13 and 14) and abd-A promoters (PS7 through PS12). Given the slower nature of transcription initiation vs transcriptional elongation, this high level of transcription might favor downstream gene repression (Castro Alvarez, 2021).

From work on mammalian cells, it has long been known that the final exon of coding genes often promotes termination by the recruitment of the termination machinery to the poly(A) site [26]. Although in recent years, ELAV has been studied as a protein whose function lies in extending the 3'UTRs of neuron-specific genes by altering the selection of poly-A signals, RT-PCR results suggest that, here, ELAV may play a role in the alternative splicing of the final exons of the iab-8 transcript. This function in alternative splicing is consistent with the role described for ELAV as an RNA binding protein involved in the alternative splicing of the neuronal isoforms of the Nrg and fne gene products. In fact, ELAV family members have been shown to be particularly important for splicing into a terminal exon. Thus, ELAV might extend the iab-8 ncRNA by suppressing the ability of the iab-8 transcript to splice into its normal terminal exon. This would then prevent the transcribing RNA polymerase from terminating, causing it to continue transcribing until it finds a new terminal exon. Published ChIP-seq experiments (where nascent transcripts were cross-linked to the genomic DNA along with proteins) on ELAV from early and later embryos support this interpretation. According to these results, there is additional ELAV binding at the junction between intron 7 and exon 8 of the iab-8 transcript in later embryos when iab-8 is expressed only in the CNS (Castro Alvarez, 2021).

Interestingly, among the spliced fusions between iab-8 and abd-A, this study found one isoform that contains the abd-A ATG sequence. This would seem counter-productive, if the function of transcriptional interference is to prevent abd-A expression. Although it is not possible to judge the amount of this specific transcript based on the current experiments, previous results have suggested that exons one and two of the iab-8 ncRNA act as translational repressors. Indeed, the male specific abdominal (MSA) RNA, which is identical to the iab-8 ncRNA except that the first two exons of iab-8 are replaced by an alternative first exon, actually codes for a peptide whose coding sequence lies in the shared last exon. A GFP fusion to this peptide and other reporters placed in the iab-8 sequence have shown that these proteins are never expressed in the CNS, but can be expressed in the male accessory gland, where MSA is expressed. Thus, even if this form is produced in a significant quantity, it seems that the embryos have further buffered themselves against ectopic abd-A, by repressing its translation (Castro Alvarez, 2021).

Lastly, it is of note that even in the elav, fne, miR-iab-8 triple mutant, the derepression of abd-A, while strong, may not be complete. There are a few cells, that still seem to repress abd-A in the posterior CNS. At the moment, this result cannot be explained. It is believed that some of this change may be due to fact that elav, fne, miR-iab-8 mutant nerve cords are very much abnormal and may have certain cellular defects. It was noticed, for example that these nerve cords were more difficult to dissect as they were to the extremely fragile relative to wild type. However, it is also possible that there are additional factors that allow transcriptional readthrough in these embryos or perform a repressive function on abd-A by another mechanism. Interestingly, RT-qPCR results still seem to detect a low level of transcriptional readthrough even in elav, fne double mutants, hinting that some transcriptional interference might occur even in the absence of these factors. One possible candidate to mediate this transcriptional readthrough is the rbp9 gene, the third elav homologue in Drosophila. Like elav and fne, rbp9 that is expressed in neurons and has been shown to be capable of promoting 3'RNA extensions when ectopically expressed in cultured cells (Castro Alvarez, 2021).

As a mechanism of transcriptional repression, transcriptional interference has mostly been found in organisms with compact genomes like yeast and bacteria. Because most of the multicellular eukaryotes studied in the lab have much larger genomes, containing a large proportion of 'non-essential' DNA, transcriptional interference has often been disregarded as a common mechanism for gene repression. However, due to co-regulation and/or gene duplication events, eukaryotic genes may be more compact at certain locations than generally assumed. This is very evident in the HOX gene clusters where there are numerous examples of tightly packed or overlapping transcription units. With all of these examples of overlapping transcription units and possible transcriptional interference, it is interesting to ask if this association could relate to an ancient gene regulatory mechanism. Within the Hox genes there is a known phenomenon called posterior dominance. According to the principle of posterior dominance, the more posterior Hox gene expressed in a segment generally plays the dominant role in patterning the segment. In Drosophila, this is often seen by down-regulation of the more-anterior gene. It is interesting to note that in the most studied Hox clusters, the Hox genes are organized on the chromosome in a way in which each Hox gene is located directly 3' to the next more-posterior segment specifying Hox gene. If it is considered that the Hox clusters are thought to have arisen from successive gene duplication events and after such duplication events, the two genes should have equal regulatory potential, then how could the upstream gene consistently take on a more dominant role? Transcriptional interference provides a possible explanation for this. According to this model, the upstream gene might have a slight advantage over the downstream gene due to transcriptional interference. This advantage, although potentially weak in many cases, could then be intensified and fixed through evolving cross-regulatory interactions. In the case being studied, the finding that ectopic abd-A in the posterior CNS leads to female sterility would help to drive such interactions (Castro Alvarez, 2021).

Although this phenomenon has been studied in a HOX cluster, other situations might exist where genes are located in similar tight configurations that induce transcriptional interference. An interesting bioinformatic analysis of nested genes in Drosophila suggests that transcriptional interference might be a natural consequence of tight, tandem gene arrangement. There is a significantly lower number of nested genes transcribed from the same strand in the Drosophila genome. Furthermore, nested genes in the same orientation contained fewer or no introns. Examining the expression data of the tandem, nested genes showed that these genes were often downregulated in tissues where the upstream gene was expressed, leading to s suggestion that the genetic arrangement of the genes might lead to transcriptional interference through mechanisms like unnatural splicing. This is very similar situation to what was found in the Hox complex and may hint that transcriptional interference exists at other loci displaying a similar arrangement of genes. Examining the mechanism that mediates transcriptional interference at model loci like iab-8 may help to define the conditions necessary for transcriptional interference to occur and potential lead to the identification other loci regulated in similar fashion (Castro Alvarez, 2021).

Distinct elements confer the blocking and bypass functions of the Bithorax Fab-8 boundary

Boundaries in the Drosophila bithorax complex (BX-C) enable the regulatory domains that drive parasegment specific expression of the three Hox genes to function autonomously. The four regulatory domains (iab-5, iab-6, iab-7 and iab-8) that control the expression of the Abdominal-B (Abd-B) gene are located downstream of the transcription unit and are delimited by the Mcp, Fab-6, Fab-7 and Fab-8 boundaries. These boundaries function to block crosstalk between neighboring regulatory domains. In addition, three of the boundaries (Fab-6, Fab-7 and Fab-8) must also have bypass activity so that regulatory domains distal to the boundaries can contact the Abd-B promoter. In these studies a functional dissection was undertaken of the Fab-8 boundary using a boundary replacement strategy. The studies indicate that the Fab-8 boundary has two separable sub-elements. The distal sub-element blocks crosstalk, but can not support bypass. The proximal sub-element has only minimal blocking activity but is able to mediate bypass. A large multiprotein complex, the LBC, binds to sequences in the proximal sub-element and contributes to its bypass activity. The same LBC complex has been implicated in the bypass activity of the Fab-7 boundary (Kyrchanova, 2019).

The lncRNA male-specific abdominal plays a critical role in Drosophila accessory gland development and male fertility

Although thousands of long non-coding RNAs (lncRNA) have been identified in the genomes of higher eukaryotes, the precise function of most of them is still unclear. This study shows that a >65 kb, male-specific, lncRNA, called male-specific abdominal (msa) is required for the development of the secondary cells of the Drosophila male accessory gland (AG). msa is transcribed from within the Drosophila bithorax complex and shares much of its sequence with another lncRNA, the iab-8 lncRNA, which is involved in the development of the central nervous system (CNS). Both lncRNAs perform much of their functions via a shared miRNA embedded within their sequences. Loss of msa, or of the miRNA it contains, causes defects in secondary cell morphology and reduces male fertility. Although both lncRNAs express the same miRNA, the phenotype in the secondary cells and the CNS seem to reflect misregulation of different targets in the two tissues (Maeda, 2018).

Previous, work showed that the iab-6^cocuD1 mutation removes a secondary cell specific enhancer for Abd-B and that this mutation causes the loss of Abd-B expression in the secondary cells. Furthermore, this mutation causes both a cytological phenotype in these cells and a reduction in the long-term post-mating response (LTR) in the mates of these mutant males. Sequence analysis of a lncRNA discovered by large-scale RNA-seq, called male-specific abdominal (msa), indicate that the ~1.1 kb iab-6^cocuD1 deletion also removes the promoter and first exon of the msa lncRNA. By genetic and molecular analyses, this study showed that the expression of the msa lncRNA allows the mir-iab-8 miRNA to be produced in the secondary cells and that this miRNA is responsible for some of the male’s ability to induce an LTR in his mate. Based on the data presented in this study, and from previous work, it is clear that both Abd-B and the miRNA play overlapping, but not completely-redundant roles in secondary cell development; reduction of either Abd-B or the mir-iab-8 miRNA alone show weaker phenotypes than the removal of both elements together (Maeda, 2018).

That there is some non-redundancy between the effects of the ABD-B transcription factor and the mir-iab-8 miRNA in secondary cells can also be seen with the LTR-mediating proteins CG1652 and CG1656. In both iab-6^cocuD1 and iab-6^cocuD1/miR iab-8 mutants, shifts were observed in the gel-migration of these proteins relative to wildtype. However, both proteins migrate slightly differently in extracts of iab-6^cocuD1/miR iab-8 vs. extracts of iab-6^cocuD1 homozygotes, indicating differences between these genotypes. Previous work linked the subtle shifts in the migration pattern of CG1656 in iab-6cocu mutants to changes in N-linked glycosylation. PGNase F treatment of extracts from iab-6^cocuD1 and iab-6^cocuD1/miR iab-8 mutants does not remove the subtle difference in SDS-PAGE migration, thus further highlighting the differences between the two genotypes and likely, the role of Abd-B vs the miRNA (Maeda, 2018).

Earlier work has shown that mir-iab-8 is important for male and female fertility through its role in the developing CNS. In the early posterior CNS, mir-iab-8 is important for the repression of specific homeotic genes and homeotic gene cofactors (abd-A, Ubx, exd and hth). Failure to repress these targets in the posterior CNS leads to male and female sterility, as males lack the ability to curl their abdomens for mating and females lack the ability to lay eggs. These phenotypes seem to be due to CNS defects that prevent proper innervation of particular abdominal muscles. The results presented in this study suggest that mir-iab-8 has at least some different primary targets in the secondary cells that are also needed for fertility. It has not yet been possible to determine a primary targets for mir-iab-8 in the secondary cells, though a number of genes have been examined that are upregulated in iab-6^cocu mutants and contain predicted miRNA binding sites. Given that many miRNA loss-of-function phenotypes are thought to be caused by mildly affecting the expression level of many target genes, lack of success in finding a primary target could indicate that removing miR-iab8 causes defects in the secondary cells through the mild overexpression of a network of targets (Maeda, 2018).

It is now known that the iab-8 miRNA plays a dual role in male fertility. One role is in the CNS and is accomplished through the regulation of the hox genes and their cofactors. This study describes a second role for mir-iab-8; in the secondary cells of the male AG, it plays an important fertility function that seems to be through the regulation of a different set of genes. Given the high conservation of mir-iab-8 in arthropod hox complexes and the conservation of its target sites in the homeotic genes and their effectors, it is believed the homeotic function of the miRNA is likely its ancestral role. In this context, it is intriguing to speculate on the genesis of the msa transcript. In both flies and humans, male gonadal tissues have the highest levels of ncRNA expression. This has been suggested to reflect the high concentration of transcription factors in this tissue regulating cryptic promoters in intergenic regions. If the ncRNA provided advantages in fertility it could be selected; perhaps such a scenario led to selection for secondary cell expression of msa (Maeda, 2018).

The msa promoter seems to be tied to an Abd-B enhancer. Interestingly, Abd-B class hox genes have evolutionarily conserved functions in the male reproductive tissues. For example, egl-5, the C. elegans Abd-B ortholog is expressed in the male worm seminal fluid producing organs and is sufficient to induce markers associated with the male-specific, seminal fluid-producing cell fates in hermaphrodites. In mammals, Abd-B class hox genes have been shown to be expressed in the male seminal fluid creating organs like the prostate and the seminal vesicle and have been shown to be critical for the production of secreted gene products. Based on these conservations, it may be that the accessory gland function of msa/mir-iab-8 arose from the co-opting of the Abd-B secondary cell enhancer (iab-6^cocuD1) by a neighboring, potentially cryptic, promoter. As the creation of the msa transcript would not disturb hox gene regulation in the secondary cell (since its normal targets do not seem to be expressed in these cells), its appearance could have been tolerated, adding increased genetic flexibility for selection. One can imagine that transcripts of secondary cell-expressed genes whose repression was beneficial to male fertility might then have acquired/retained regulation by miR-iab8. Based on this, it would be particularly interesting to look for species that do not express the msa lncRNA and to examine predicted accessory gland targets for changes in mir-iab8 binding sites (Maeda, 2018).

MicroRNA-encoded behavior in Drosophila

The relationship between microRNA regulation and the specification of behavior is only beginning to be explored. This study finds that mutation of a single microRNA locus (miR-iab4/8) in Drosophila larvae affects the animal's capacity to correct its orientation if turned upside-down (self-righting). One of the microRNA targets involved in this behavior is the Hox gene Ultrabithorax whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants while thermogenetic manipulation of SRN activity results in changes in self-righting behavior. These data thus reveal a microRNA-encoded behavior and suggests that other microRNAs might also be involved in behavioral control in Drosophila and other species (Picao-Osorio, 2015).

The regulation of RNA expression and function is emerging as a hub for gene expression control across a variety of cellular and physiological contexts, including neural development and specification. Small RNAs such as microRNAs (miRNAs) have been shown to affect neural differentiation, but their roles in the control of behavior are only beginning to be explored (Picao-Osorio, 2015).

Previous work has focused on the mechanisms and impact of RNA regulation on the expression and neural function of the Drosophila Hox genes. These genes encode a family of evolutionarily conserved transcription factors that control specific programs of neural differentiation along the body axis, offering an opportunity to investigate how RNA regulation relates to the formation of complex tissues such as the nervous system (Picao-Osorio, 2015).

This study used the Hox gene system to investigate the roles played by a single miRNA locus (miR-iab4/iab8) on the specification of the nervous system during early Drosophila development. This miRNA locus controls the embryonic expression of posterior Hox genes. Given that no detectable differences were found in the morphological layout of the main components of the nervous system in late Drosophila embryos of wild type and miR-iab4/iab8-null mutants [herein ΔmiR], this study analyzed early larval behavior as a stratagem to probe the functional integrity of the late embryonic nervous system (Picao-Osorio, 2015).

Most behaviors in early larva were unaffected by the miRNA mutation, except self-righting (SR) behavior: miRNA mutant larvae were unable to return to their normal orientation at the same speed as their wild-type counterparts (Picao-Osorio, 2015).

By means of selective target overexpression followed by SR phenotype analyses, this study identified the Drosophila Hox gene Ultrabithorax (Ubx) as a miRNA target implicated in the genetic control of SR behavior. Overexpression of Ubx within its expression domain did not affect any larval behavior tested except SR, which is in agreement with the effects observed in miRNA mutants. Analysis of Ubx 3' untranslated region (3'UTR) fluorescent reporter constructs expressed in the Drosophila central nervous system (CNS) indicates that the interaction between miR-iab4/iab8 and Ubx is direct, which is in line with prior observations in other cellular contexts (Picao-Osorio, 2015).

To identify the cellular basis for SR control, Ubx was systematically overexpressed within subpopulations of neurons. Increased levels of Ubx within the pattern of Cha(7.4kb)-Gal4, which largely targets cholinergic sensory and interneurons, phenocopied the miRNA SR anomalies. Further overexpression analysis identified two metameric neurons as the minimal node required for the SR behavior [self-righting node (SRN)] (Picao-Osorio, 2015).

Several lines of evidence confirm the role of miRNA-dependent Ubx regulation within the SRN as a determinant of SR. First, both Ubx and miRNA transcripts (miR-iab4) derived from the miR-iab4/iab8 locus were detected within the SRN. Second, in the context of miRNA mutation, Ubx protein expression is increased within the SRN. Third, reduction of Ubx (Ubx RNAi) specifically enforced within SRN cells is able to ameliorate or even rescue the SR phenotype observed in miRNA mutants (Picao-Osorio, 2015).

Two plausible scenarios arise to explain the effects of miR-iab4/iab8 in regard to SR behavior. One is that miRNA input is required for the late embryonic development of the neural networks underlying SR, arguing for a 'developmental' role of the miRNA; another is that miRNA repression affects normal physiological/behavioral functions largely without disrupting neural development in line with a 'behavioral' role. Two independent experiments support that the primary roles of miR-iab4/8 are behavioral. First, anatomical analysis of SRN cells in wild type (wt), ΔmiR, and R54503>Ubx [SRN-driver line] show no significant differences in total numbers of SRN cells or in SRN cell body size; furthermore, analysis of wt, ΔmiR, and R54503>Ubx show indistinguishable SRN-projection patterns. Second, Gal-80ts-mediated conditional expression experiments show that SRN-specific Ubx overexpression after embryogenesis is sufficient to trigger the SR behavior (Picao-Osorio, 2015).

These results suggest that miRNA-dependent Hox regulation within the SRN must somehow modify the normal physiology of SRN cells so that when the miRNA is mutated, these neurons perform functions different from those in wild-type animals. To test this hypothesis, genetically encoded calcium sensors [GCaMP6] specifically expressed in SRN cells were used, and spontaneous profiles of neural activity were tracked down. SRN cells in miRNA mutants produce activity traces that are significantly different from those observed in wild-type SRN cells. Quantification of maximal amplitude and proportion of active cells in each genotype also reveal significant differences in SRN function across the genotypes, but no change in cell viability is observed. Neural activity differences across genotypes are significant within regions of expression of miR-iab4, suggesting that this miRNA (and not miR-iab8) might be the main contributor to SR control. Analysis of mutations that selectively affect miR-iab4 or miR-iab8 strongly suggests that miR-iab4 is the key regulator of SR (Picao-Osorio, 2015).

To demonstrate that the changes in SRN neural activity were causal to SR behavior, SRN cells were artificially activated or inhibited this was shown to trigger the aberrant SR phenotype. This suggested that activation of SRN cells in larvae placed 'right side up' might be sufficient to 'evoke' actions reminiscent of a self-righting response. An optogenetic system was developed in which SRN cells were activated by means of R54F03-driven channelrhodopsin 2 (ChR2) in trans-retinal fed larvae. Under blue light stimulation, larvae performed an atypical bending movement, frequently adopting a 'lunette' position. Neither parental line R54F03-Gal4 nor UAS-Ch2R showed similar reactions to stimulation, confirming the specificity of this effect (Picao-Osorio, 2015).

To study the links between SRN neurons and the SR movement, SRN projections were labeled with myr-GFP and SRN cells were discovered to innervate two of the lateral transverse (LT) muscles and can be colabeled antibodies against Fasciclin 2 (Fas2), demonstrating these to be motorneurons. LT muscles are innervated by Bar-H1+ motorneurons, so Bar-H1-Gal4 was used as a second driver to demonstrate that appropriate Ubx levels in these cells are required for normal SR behavior, establishing the SRN cells as the LT-MNs (Picao-Osorio, 2015).

This study has therefore shown that miRNA-dependent Hox gene repression within a distinct group of motorneurons (SRN/LT-MNs) is required for the control of a specific locomotor behavior in the early Drosophila larva. The finding that Hox gene posttranscriptional regulation is involved in SR control suggests that other RNA-based regulatory processes affecting Hox gene expression might also impinge on specific neural outputs; this possibility is currently being investigated, with special regard to the roles of the Hox genes in the specification of neural lineages with axial-specific architectures, and the roles of other miRNAs on behavior are being systematically tested (Picao-Osorio, 2015).

That no obvious neuro-anatomical changes in miRNA mutant embryos could be detected suggests that these are either very subtle or that the role of miRNA regulation may be primarily behavioral, in the sense of affecting the performance of a correctly wired neural system, rather than developmental, contributing to the development of the network. Given that miR-iab4/iab8 is involved in adult ovary innervation, it seems that miRNAs -- much like ordinary protein-coding genes -- can be involved in several distinct roles within the organism (Picao-Osorio, 2015).

The results of this study contribute to the understanding of how complex innate behaviors are represented in the genetic program. The data lead to a proposal that other miRNAs might also be involved in the control of behavior in Drosophila and other species (Picao-Osorio, 2015).

Hox gene regulation in the central nervous system of Drosophila

Hox genes specify the structures that form along the anteroposterior (AP) axis of bilateria. Within the genome, they often form clusters where, remarkably enough, their position within the clusters reflects the relative positions of the structures they specify along the AP axis. This correspondence between genomic organization and gene expression pattern has been conserved through evolution and provides a unique opportunity to study how chromosomal context affects gene regulation. In Drosophila, a general rule, often called “posterior dominance,” states that Hox genes specifying more posterior structures repress the expression of more anterior Hox genes. This rule explains the apparent spatial complementarity of Hox gene expression patterns in Drosophila. This paper reviews a noticeable exception to this rule where the more-posteriorly expressed Abd-B Hox gene fails to repress the more-anterior abd-A gene in cells of the central nervous system (CNS). While Abd-B is required to repress ectopic expression of abd-A in the posterior epidermis, abd-A repression in the posterior CNS is accomplished by a different mechanism that involves a large 92 kb long non-coding RNA (lncRNA) encoded by the intergenic region separating abd-A and Abd-B (the iab8ncRNA). Dissection of this lncRNA revealed that abd-A is repressed by the lncRNA using two redundant mechanisms. The first mechanism is mediated by a microRNA (mir-iab-8) encoded by intronic sequence within the large iab8-ncRNA. Meanwhile, the second mechanism seems to involve transcriptional interference by the long iab-8 ncRNA on the abd-A promoter. Recent work demonstrating CNS-specific regulation of genes by ncRNAs in Drosophila, seem to highlight a potential role for the iab-8-ncRNA in the evolution of the Drosophila Hox complexes (Gummalla, 2014).

MicroRNAs in the Drosophila bithorax complex

The iab-4 noncoding RNA from the Drosophila bithorax complex is the substrate for a microRNA (miRNA). Gene conversion was used to delete the hairpin precursor of this miRNA; flies homozygous for this deletion are sterile. Surprisingly, this mutation complements with rearrangement breakpoint mutations that disrupt the iab-4 RNA but fails to complement with breaks mapping in the iab-5 through iab-7 regulatory regions. These breaks disrupt the iab-8 RNA, transcribed from the opposite strand. This iab-8 RNA also encodes a miRNA, detected on Northern blots, derived from the hairpin complementary to the iab-4 precursor hairpin. Ultrabithorax is a target of both miRNAs, although its repression is subtle in both cases (Bender, 2008).

A large number of microRNA (miRNA) clones prepared from Drosophila RNA have been characterized at a variety of developmental stages. Two of these clones matched sequences from the BX-C, mapping to the 3' end of a ncRNA discovered by Cumberledge (1990). This ncRNA was called the 'iab-4 RNA,' because it was thought to come from the iab-4 segmental domain of the BX-C, and the miRNAs were named miR-iab-4 5p (five prime) and miR-iab-4 3p (three prime). More recent mapping of segmental domains (Bender, 2000) has shown that the RNA actually lies in the iab-3 domain (regulating parasegment 8), and indeed, the ncRNA is expressed from parasegment 8 through parasegment 12 (Cumberledge, 1990). However, the iab-4 nomenclature is maintained in this study to avoid confusion with the designations in other studies. Two cDNA clones for the iab-4 RNA were described by Cumberledge (1990), with alternate 3' poly(A) sites separated by 304 base pairs (bp). The two miRNAs come from this region between these two poly(A) sites; both are presumably derived from a 70-base hairpin RNA precursor predicted from the sequence (Bender, 2008).

A recent study (Ronshaugen, 2005) suggested that the miR-iab-4 5p miRNA might be responsible for repression of Ubx in the abdominal segments where the miRNA is expressed. The conclusion was based on experiments in which miR-iab-4 5p was expressed at high levels in tissues, including the wing and haltere discs, where miR-iab-4 5p is not normally found. However, the pattern of UBX expression in PS8, where the miRNA is expressed, is very similar to the UBX pattern in PS7, which lacks the miRNA. The obvious repression of Ubx in both of these parasegments is clearly dependent on abd-A; any effect of miR-iab-4 5p must be subtle or redundant. Moreover, misexpression of miRNAs in other systems have been shown to give misleading effects. The function of miR-iab-4 5p can best be examined by mutating or deleting the miRNA (Bender, 2008).

Prior studies characterized a large number of P element insertions in the BX-C, including one called HCJ200, which maps only ~200 bp proximal to the miRNAs. This provided the opportunity to mutate the miRNAs by P-element-mediated gene conversion. Loss of the miRNAs derived from the iab-4 ncRNA causes no apparent morphological or behavioral phenotype, but the analysis revealed a functional miRNA derived from the opposite strand (Bender, 2008).

A 3.7-kb conversion donor fragment was constructed with a mutated version of the miRNA precursor sequence. The precursor sequence is symmetrically cut by the BstZ17I restriction endonuclease; this permitted the replacement of most of the precursor sequence with a double-stranded oligonucleotide-containing sites for the HindIII and I-SceI endonucleases. A plasmid with the donor sequence and a plasmid to supply P-element transposase were both injected into embryos with the HCJ200 (rosy⁺) P insertion. Offspring from the injected individuals were screened for loss of the HCJ200 P element (i.e., rosy^-), and progeny from these exceptional flies were screened by PCR for a change in the size of the genomic DNA at the site of the insertion. One of 86 fertile injected animals gave the expected convertants. The conversion events were verified by sequencing the PCR product, and by whole-genome Southern blots. Genomic DNA was cut with HindIII, which cuts the oligonucleotide introduced in the conversion but does not cut the wild-type sequence within the 3.7-kb donor fragment. The sizes of the HindIII fragments on the Southern blot confirmed that the donor sequence was at the expected position in the BX-C and not at another genomic location (Bender, 2008).

Flies homozygous for the conversion chromosome (henceforth called 'δ') appeared normal. In particular, no evidence of segmental transformation was seen in mounted adult abdominal cuticles of either sex. However, both sexes were sterile. Females had ovaries with eggs of normal size, but only very rare individuals ever laid an egg, even after mating with wild-type males, and these rare eggs never hatched. Males had morphologically normal testes containing motile sperm. In single-fly tests for mating behavior, δ homozygous females mated with wild-type males as readily as their heterozygous siblings. The δ homozygous males showed normal courtship behavior toward wild-type females, except that they never completed copulation. The mutant males mounted the females, but they did not bend their abdomens quite far enough to mate. Thus, the sterility in both sexes appeared to be behavioral, due to a defect in the nerves or muscles required to lay eggs or to curl the abdomen (Bender, 2008).

The δ mutation was tested for complementation with rearrangement mutations in the BX-C, including several that should disrupt the iab-4 RNA transcript upstream of the position of the miRNA precursor. Surprisingly, breaks disrupting the iab-4 transcription unit complemented with δ -- i.e., trans-heterozygotes were fertile. Thus, the iab-4 RNA is not the precursor for any miRNA that is responsible for fertility. In contrast, rearrangements distal to the position of the miRNAs failed to complement, even with breaks >50 kb distal. Assuming that noncomplementing rearrangements are upstream in the precursor, one can deduce that the precursor is transcribed distal to proximal on the chromosome, and that the miRNA responsible for fertility comes from the opposite strand to those detected by Aravin (2003). Similarly, one would predict that the precursor transcript for the fertility miRNA spans at least the iab-7 through the iab-3 segmental domains (Bender, 2008).

Several studies have detected such a transcript, which is usually called the iab-8 ncRNA. It has been detected and mapped solely by in situ hybridization to RNA in embryos, although the complementation analysis now corroborates the in situ mapping, at least for the iab-4 through iab-7 region. Its start site is near Abd-B; it was detected by probes 4 kb proximal to the 3' end of the Abd-B transcripts. A potential promoter has been defined for the iab-8 transcript, which lies ~5 kb downstream from Abd-B, although the evidence did not preclude a start site further upstream. The iab-8 RNA has been detected upstream of the Abd-B class A RNA start site. However, hybridization to the Abd-B class B RNA in the ninth abdominal segment (PS14) could have been mistaken for the iab-8 RNA pattern. Moreover, the iab-8^S10 breakpoint, just proximal to Abd-B, does complement the sterility phenotype of δ, and so the promoter for the iab-8 fertility function should be to the left of that break (Bender, 2008).

At the 3' end, the iab-8 RNA extends through abd-A. The iab-8 RNA in situ pattern was detected by a probe 5.5 kb proximal to the 3' end of the abd-A poly(A) site. Thus the transcription unit spans at least 120 kb. The iab-8 RNA has not been detected by probes in the bxd regulatory region, further proximal to abd-A (Bender, 2008).

The iab-8 transcript initiates at the cellular blastoderm stage, as do most of the other embryonic ncRNAs. However, it should take ~45 min to transcribe to the position of the miRNA precursor hairpin, assuming a transcription speed of ~1.3 kb/min. This would account for the developmental delay in the appearance of the RNA signal. The iab-8 RNA is located in the eighth abdominal segment and in more posterior segmental rudiments. In late embryos, the transcript persists in the posterior end of the ventral nerve chord (Bender, 2008).

Embryos homozygous for the δ mutation showed no apparent changes in the patterns of ABD-A and ABD-B, but there were subtle differences in the UBX pattern. UBX is expressed strongly and comprehensively in the cells of parasegment 6 (PS6, primarily the first abdominal segment). In the second abdominal segment (PS7), ABD-A appears and turns off UBX, especially in the more anterior cells of the parasegment. In the more posterior segments, the UBX staining pattern weakens progressively, and the ABD-A pattern becomes somewhat stronger. However, in δ embryos, the UBX pattern is nearly constant from PS7 through PS12. Thus, the progressive posterior decline in wild-type embryos appears not to be caused by ABD-A or ABD-B but rather by miR-iab-4 5p, whose expression shows a progressive posterior increase in PS8-12 (Bender, 2008).

In the eighth abdominal segment (PS13) of a wild-type embryo, UBX is almost completely absent in both the epidermis and the CNS. Homozygotes for δ show a partial derepression of UBX in the CNS in PS13. The derepression is similar in pattern and intensity to that seen in AbdB^-/+ heterozygotes (data not shown). The repression of Ubx could be indirect; miR-iab-8 could be a positive regulator of Abd-B (and miR-iab-4 a positive regulator of abd-A). But all known targets of miRNAs are negatively regulated, and so it seems more likely that both miRNAs directly regulate Ubx (Bender, 2008).

It is possible that the effects of these two miRNAs are masked by functional redundancy with abd-A (for miR-iab-4) and Abd-B (for miR-iab-8). Embryos lacking ABD-A (but retaining miR-iab-4) show a dramatic derepression of UBX in the second through seventh abdominal segments. There does appear to be a slight decline in UBX levels in the more posterior segments, which could be attributed to miR-iab-4 repression. A complete analysis would include the UBX expression in embryos lacking both abd-A and miR-iab-4, but that will require construction of an abd-A, δ double mutant chromosome. In any case, miR-iab-4 repression of Ubx is subtle, even in the absence of ABD-A. Similarly, in an Abd-B homozygous mutant embryo (retaining miR-iab-8), UBX expression in the eighth abdominal segment closely resembles that in the seventh. Again, a δ, Abd-B double mutant chromosome would be useful for comparison, but again it is clear that the repression of Ubx by miR-iab-8 is still subtle in the absence of ABD-B. There is no reason to expect that Ubx is the only target of these miRNAs; perhaps other target genes will be discovered which the miRNAs repress more dramatically (Bender, 2008).

MiR-iab-8 is the first example of a functional product of a ncRNA in the BX-C. There are no other predicted miRNA precursor sequences in the iab-8 RNA or elsewhere in the BX-C (the Antennapedia complex includes miR-10), but there are many other ncRNAs. The possibility that they also include functional products now seems more likely (Bender, 2008).

Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci

Many microRNA loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. Antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4 /iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. miR-iab-8-5p, like miR-iab-4-5p, is also relevant to Hox gene regulation. Ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A via extensive arrays of conserved target sites, and can induce a dramatic homeotic transformation of halteres into wings. The antisense miRNA principle is generalizable: it has been shown that several other loci in both invertebrates and vertebrates are endogenously processed on their antisense strands into mature miRNAs with distinct seeds. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes (Tyler, 2008).

These studies of BX-C miRNAs reveal two principal insights into miRNA regulatory biology. First, a new Hox cluster miRNA, mir-iab-8, was identified. Using gain-of-function assays it was shown that it can strongly inhibit Ubx and abd-A and generate homeotic phenotypic transformations. Indeed, the Hox-regulatory properties of mir-iab-8 are far more potent than those of mir-iab-4 (Ronshaugen, 2005), and correlate directly with the properties of its target sites in their 3' UTRs. Curiously, both BX-C miRNAs obey organizational and functional rules previously defined for the protein-encoding members of the BX-C. These regulatory RNAs exhibit colinearity, in that transcription of pri-mir-iab-8 initiates more distally on the chromosome and is expressed more posteriorly in the embryo relative to pri-mir-iab-4. They also exhibit posterior prevalence, in that both sense and antisense iab-4 miRNAs directly repress multiple homeotic genes located more anteriorly in the Hox cluster. In fact, the next most-anterior Hox gene Antp is a third likely endogenous iab-miRNA target that contains highly conserved target sites with t1A features for miR-iab-8-5p. In contrast, Abd-B contains no conserved sites for either iab-4 or iab-8 miRNAs in its long (>2 kb) 3' UTR. Therefore, BX-C miRNAs and homeobox genes are governed by the same regulatory logic (Tyler, 2008).

It is worth recalling that saturation mutagenesis screens of the BX-C revealed only three loci that are required for viability and exhibit homeotic defects, corresponding to the homeobox genes Ubx, abd-A, and Abd-B. In contrast, pioneering studies by Lewis (1978) considered rearrangements and dominant alleles suggested the existence of at least eight homeotic 'factors' in this region of the genome. Although many of these are now recognized as cis-regulatory elements that regulate Hox gene transcription, this work with BX-C miRNAs reveals two bona fide Hox regulators that are capable of inducing severe dominant homeotic transformations. The endogenous requirement for iab-4 /iab-8 miRNAs appears to be subtle, possibly due to compensatory transcriptionally based regulatory mechanisms. Nevertheless, loss of function analysis corroborates that these miRNAs are required for normal expression of Hox targets in the nervous system and for normal development. These data emphasize that loss-of-function and gain-of-function genetics are complementary approaches to uncover important regulatory molecules (Tyler, 2008).

Antisense transcription and processing were uncovered in this study as a mechanism to generate new functional miRNAs. Bioinformatic analysis suggests that a large fraction of miRNA loci are theoretically competent to produce antisense miRNAs. Extant cloning efforts suggest that few miRNA loci actively produce large quantities of antisense miRNAs. Nevertheless, the sequencing effort reported in this study has revealed additional instances of putative antisense miRNAs. Although none of these was cloned more than twice, genetics demonstrates that rare miRNAs (e.g., lsy-6, expressed by a handful of neurons in a whole animal) can be critical components of regulatory networks and can have potent biological activities. Therefore, assessment of the biological relevance of the other antisense miRNA candidates awaits further study (Tyler, 2008).

In the case of the iab-4 locus, the regulatory diversity afforded by sense and antisense transcription of a single miRNA hairpin is manifested by altering the seed regions of their respective miRNA products and by deploying the sense and antisense pri-miRNA transcripts in distinct spatial domains. It might in fact be deleterious for a given locus to be simultaneously transcribed on both strands -- either because of transcriptional interference from colliding polymerase complexes, or because of the possibility to inadvertently generate dsRNA. Further analysis is needed to test the notion that it is favorable for sense/antisense miRNA pairs not to be expressed in the same cells. Overall, though, as animal genomes are quite extensively transcribed, and many miRNA genes adopt extensive hairpins on both strands, the potential for endogenous antisense processing of miRNA hairpins is theoretically quite broad. It is proposed that antisense transcription of other miRNA loci might generate novel small RNAs whose potentially beneficial regulatory activities are available for selection and stabilization by natural selection. This identification of several confident examples of antisense miRNAs, whose processing and/or targets have been conserved among diverse species, provides compelling support for this hypothesis (Tyler, 2008).

Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods

In Drosophila melanogaster, the iab-4/iab-8 locus encodes bi-directionally transcribed microRNAs that regulate the function of flanking Hox transcription factors. This study showed that bi-directional transcription, temporal and spatial expression patterns and Hox regulatory function of the iab-4/iab-8 locus are conserved between fly and the beetle Tribolium castaneum. Computational predictions suggest iab-4 and iab-8 microRNAs can target common sites, and cell-culture assays confirm that iab-4 and iab-8 function overlaps on Hox target sites in both fly and beetle. However, w key differences were observed in the way Hox genes are targeted. For instance, abd-A transcripts are targeted only by iab-8 in Drosophila, whereas both iab-4 and iab-8 bind to Tribolium abd-A. This evolutionary and functional characterization of a bi-directionally transcribed microRNA establishes the iab-4/iab-8 system as a model for understanding how multiple products from sense and antisense microRNAs target common sites (Hui, 2013).

The iab-4 miRNA locus has some unusual properties: the locus is transcribed in both directions, producing two primary miRNA transcripts and two hairpin precursors. Each precursor is processed to produce two mature miRNAs, one from each arm of each precursor hairpin. Only a handful of other miRNAs have been shown by deep sequencing data to be transcribed in both directions; currently, the miRBase database has only 27 animal examples. This study shows that bi-directional transcription of the iab-4/8 locus and production of miRNAs from both transcripts is conserved in insects. However, the relative abundance of the four mature miRNAs varies significantly between fly and beetle (Hui, 2013).

The four mature miRNAs produced from the iab-4 locus are extremely similar. Indeed, they are all seed-shifted variants of each other. This state is possible only because the mature sequences are partially palindromic. Thus, sense and antisense sequences are highly similar, as are partially complementary mature sequences from opposite arms of the same hairpin. As the mature sequences are closely related, the predicted targets of the four mature products overlap significantly. Previous work suggests that this is an unusual situation: the targets of alternate miRNAs derived from the 5'- and 3'-arms of almost all miRNAs are largely different. It was shown that iab-4 -5p and iab-8-5p have more common targets that expected by chance. This functional overlap of antisense products may have facilitated the maintenance of the bi-directionality in the iab-4/iab-8 locus. Indeed, the same pattern was observed in mir-307, the other miRNA locus that produces mature miRNAs from both genomic strands (Hui, 2013).

This analysis of the repression of engineered perfect target sites clearly shows significant cross-targeting for three of the four mature miRNAs. Furthermore, it was shown that the Hox gene Ubx/Utx is a conserved target of both iab-4 and iab-8 miRNAs in both Drosophila and Tribolium. However, between fly and beetle, differences were found in Hox gene targets of iab-4/8 miRNAs and differences in the sites that mediate those targets. For example, abd-A is regulated only by iab-8 miRNAs in Drosophila, whereas both iab-4 and iab-8 miRNAs target abd-A transcripts in Tribolium. There are, therefore, both conserved and variable aspects of the targeting properties of the four mature miRNAs produced from the iab-4/8 locus in insects. The conservation of the Hox genes Ubx and abd-A as targets of the iab-4/8 miRNAs further establishes the ancient connection between the miRNAs of the Hox complex and their role in modulating the function of the Hox genes themselves. No other intergenic miRNA has been linked by genomic position to its function, yet all Hox complex miRNAs (iab-4, mir-196 and mir-10) have been found to modulate Hox gene function (Hui, 2013).

The iab-4/8 locus provides for fundamental insight into the mechanisms of evolution and the function of sense/antisense miRNA pairs. The production of functional products from both strands of the same locus may impose an evolutionary trade-off, driven on one hand by sequence conservation because of structural constraints, and on the other hand by constraints imposed by target specificity. It is proposed that the deep conservation can be explained in part by the common targeting properties of the multiple mature products generated from these two transcripts. Given the functional similarity of the miRNA products of iab-4 and iab-8, the antisense transcription of the locus can be considered analogous to the acquisition of an enhancer by the sense transcript to drive expression and miRNA production in the additional domain. Furthermore, the palindromic nature of the iab-4/iab-8 mature sequences determines that the novel antisense miRNA will share targets with the pre-existing sense miRNA. It is suggestd that this explains the apparent contradiction between extreme conservation of mature miRNA sequences on both arms, yet significant plasticity between organisms as to which arm is the dominant product. However, the evolution of target sites in abd-A demonstrates that functional target sites can be differentially regulated between even closely related species (Hui, 2013).

Search PubMed for articles about Drosophila lncRNA:iab8

Bender, W. (2008). MicroRNAs in the Drosophila bithorax complex. Genes Dev. 22: 14-19. PubMed citation: 18172161

Castro Alvarez, J. J., Revel, M., Carrasco, J., Cleard, F., Pauli, D., Hilgers, V., Karch, F. and Maeda, R. K. (2021). Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference. PLoS Genet 17(11): e1009843. PubMed ID: 34780465

Gummalla, M., Galetti, S., Maeda, R. K. and Karch, F. (2014). Hox gene regulation in the central nervous system of Drosophila. Front Cell Neurosci 8: 96. PubMed ID: 24795565

Hui, J. H., Marco, A., Hunt, S., Melling, J., Griffiths-Jones, S. and Ronshaugen, M. (2013). Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods. Nucleic Acids Res 41: 3352-3361. PubMed ID: 23335784

Kyrchanova, O., Wolle, D., Sabirov, M., Kurbidaeva, A., Aoki, T., Maksimenko, O., Kyrchanova, M., Georgiev, P. and Schedl, P. (2019). Distinct elements confer the blocking and bypass functions of the Bithorax Fab-8 boundary. Genetics. PubMed ID: 31551239

Maeda, R. K., Sitnik, J. L., Frei, Y., Prince, E., Gligorov, D., Wolfner, M. F. and Karch, F. (2018). The lncRNA male-specific abdominal plays a critical role in Drosophila accessory gland development and male fertility. PLoS Genet 14(7): e1007519. PubMed ID: 30011265

Picao-Osorio, J.,Johnston, J., Landgraf, M., Berni, J. and Alonso, C.R. (2015). MicroRNA-encoded behavior in Drosophila. Science 350(6262): 815-20. PubMed ID: 26494171

Tyler, D. M., et al. (2008). Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci. Genes Dev. 22(1): 26-36. PubMed citation: 18172163

date revised: 25 August 2022

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