The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.
Insects use methylation to modulate genome function in a different manner from vertebrates. Here, we quantified the global methylation profile in a parasitic wasp species, Nasonia vitripennis, a model with some advantages over ant and honeybee for functional and genetic analyses of methylation, such as short generation time, inbred lines, and inter-fertile species. Using a highly inbred line permitted us to precisely characterize DNA methylation, which is compared to gene expression variation across developmental stages, and contrasted to other insect species. DNA methylation is almost exclusively on the 5′-most 1 kbp coding exons, and ∼1/3 of protein coding genes are methylated. Methylated genes tend to occur in small clusters in the genome. Unlike many organisms, Nasonia leaves nearly all transposable element genes non-methylated. Methylated genes exhibit more uniform expression across developmental stages for both moderately and highly expressed genes, suggesting that DNA methylation is marking the genes for constitutive expression. Among pairs of differentially methylated duplicated genes, the paralogs that lose DNA methylation after duplication in the Nasonia lineage show lower expression and greater specialization of expression. Finally, by comparative analysis, we show that methylated genes are more conserved at three different time scales during evolution.
In animal development, secreted signaling molecules evoke all-or-none threshold responses of target gene transcription to specify cell fates. In the chordate Ciona intestinalis, the neural markers Otx and Nodal are induced at early embryonic stages by Fgf9/16/20 signaling. Here we show that three additional signaling molecules act negatively to generate a sharp expression boundary for neural genes. EphrinA signaling antagonizes FGF signaling by inhibiting ERK phosphorylation more strongly in epidermal cells than in neural cells, which accentuates differences in the strength of ERK activation. However, even weakly activated ERK activates Otx and Nodal transcription occasionally, probably because of the inherently stochastic nature of signal transduction processes and binding of transcription factors to target sequences. This occasional and undesirable activation of neural genes by weak residual ERK activity is directly repressed by Smad transcription factors activated by Admp and Gdf1/3-like signaling, further sharpening the differential responses of cells to FGF signaling. Thus, these signaling pathways coordinate to evoke a threshold response that delineates a sharp expression boundary.
Graded signals often provide positional information to organize gene expression in animal embryos. In the simplest cases, graded signals are translated into all-or-none threshold responses. However, recent studies have shown that signal transduction processes and binding of transcription factors to target sequences are inherently stochastic. This means that even weak activating signaling might activate target genes stochastically. However, the precise mechanism, by which this stochastic undesirable activation is avoided, is still largely unknown. In the embryo of a simple chordate, Ciona intestinalis, FGF signaling is known to induce neural fate. In the present study, we demonstrate that three additional signaling molecules cooperate to evoke a threshold response for specification of neural fate. First, EphrinA signaling inhibits FGF signaling by attenuating ERK phosphorylation, accentuating differences in the strength of ERK activation. However, even weak ERK activity occasionally turns on the neural genes. This occasional undesirable activation of the neural genes is turned off by Admp and Gdf1/3 signaling through Smad transcription factors. Thus, these two qualitatively different negative regulatory mechanisms evoke an all-or-none threshold response to specify neural fate.
Pattern formation in developing tissues is driven by the interaction of extrinsic signals with intrinsic transcriptional networks that together establish spatially and temporally restricted profiles of gene expression. How this process is orchestrated at the molecular level by genomic cis-regulatory modules is one of the central questions in developmental biology. Here we have addressed this by analysing the regulation of Pax3 expression in the context of the developing spinal cord. Pax3 is induced early during neural development in progenitors of the dorsal spinal cord and is maintained as pattern is subsequently elaborated, resulting in the segregation of the tissue into dorsal and ventral subdivisions. We used a combination of comparative genomics and transgenic assays to define and dissect several functional cis-regulatory modules associated with the Pax3 locus. We provide evidence that the coordinated activity of two modules establishes and refines Pax3 expression during neural tube development. Mutational analyses of the initiating element revealed that in addition to Wnt signaling, Nkx family homeodomain repressors restrict Pax3 transcription to the presumptive dorsal neural tube. Subsequently, a second module mediates direct positive autoregulation and feedback to maintain Pax3 expression. Together, these data indicate a mechanism by which transient external signals are converted into a sustained expression domain by the activities of distinct regulatory elements. This transcriptional logic differs from the cross-repression that is responsible for the spatiotemporal patterns of gene expression in the ventral neural tube, suggesting that a variety of circuits are deployed within the neural tube regulatory network to establish and elaborate pattern formation.
The complex organization of tissues is established precisely and reproducibly during development. In the vertebrate neural tube, as in many other tissues, the interplay between extrinsic morphogens and intrinsic transcription factors produces spatial patterns of gene expression that delineate precursors for specific cell types. One such transcription factor, Pax3, defines the precursors of all sensory neuron subtypes and distinguishes them from precursors fated to give rise to the motor circuits. To gain insight into the molecular mechanisms by which the spinal cord is segregated into these two functional domains, we analysed the genomic regulatory sequences responsible for controlling Pax3 activity. We identified two regions of the genome, the coordinated activity of which establishes and refines Pax3 activity. We showed that the combination of activating signals from secreted Wnt factors together with Nkx family homeodomain repressors restrict Pax3 activity to the presumptive sensory region of the neural tissue. Subsequently, Pax3 acts to directly potentiate its own transcription and this autoregulation sustains Pax3 expression at later developmental stages. Together, our study reveals the way in which intrinsic and extrinsic signals are integrated by cells and converted into a sustained pattern of gene activity in the developing nervous system.
Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.
Tubby is a member of the Tubby-like protein (TULP) family. Tubby mutations in mice (tubby mice) cause late-onset obesity and neurosensory deficits such as retinal degeneration and hearing loss. However, the exact molecular mechanism of Tubby has not been determined. Here we show that Drosophila Tubby homolog, King tubby (dTULP), regulates ciliary localization of transient receptor potential protein (TRP). dTULP-deficient flies showed uncoordinated movement and complete loss of sound-evoked action potentials. dTULP was localized in the cilia of chordotonal neurons of Johnston's organ. Two TRP channels essential for auditory transduction, Inactive and NompC, were mislocalized in the cilia of chordotonal neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and thus provides novel insights into the pathogenic mechanism of tubby mice.
There are two views on vertebrate retinogenesis: a deterministic model dependent on fixed lineages, and a stochastic model in which choices of division modes and cell fates cannot be predicted. In this issue of Neuron, He et al. (2012) address this question in zebra fish using live imaging and mathematical modeling.
vertebrate retinogenesis; competence; stochasticity; retinal progenitor; birth order
The Yorkie/Yap transcriptional coactivator is a well-known regulator of cellular proliferation in both invertebrates and mammals. As a coactivator, Yorkie (Yki) lacks a DNA binding domain and must partner with sequence-specific DNA binding proteins in the nucleus to regulate gene expression; in Drosophila, the developmental regulators Scalloped (Sd) and Homothorax (Hth) are two such partners. To determine the range of target genes regulated by these three transcription factors, we performed genome-wide chromatin immunoprecipitation experiments for each factor in both the wing and eye-antenna imaginal discs. Strong, tissue-specific binding patterns are observed for Sd and Hth, while Yki binding is remarkably similar across both tissues. Binding events common to the eye and wing are also present for Sd and Hth; these are associated with genes regulating cell proliferation and “housekeeping” functions, and account for the majority of Yki binding. In contrast, tissue-specific binding events for Sd and Hth significantly overlap enhancers that are active in the given tissue, are enriched in Sd and Hth DNA binding sites, respectively, and are associated with genes that are consistent with each factor's previously established tissue-specific functions. Tissue-specific binding events are also significantly associated with Polycomb targeted chromatin domains. To provide mechanistic insights into tissue-specific regulation, we identify and characterize eye and wing enhancers of the Yki-targeted bantam microRNA gene and demonstrate that they are dependent on direct binding by Hth and Sd, respectively. Overall these results suggest that both Sd and Hth use distinct strategies – one shared between tissues and associated with Yki, the other tissue-specific, generally Yki-independent and associated with developmental patterning – to regulate distinct gene sets during development.
The Hippo tumor suppressor pathway controls proliferation in a tissue-nonspecific fashion in Drosophila epithelial progenitor tissues via the transcriptional coactivator Yorkie (Yki). However, despite the tissue-nonspecific role that Yki plays in tissue growth, the transcription factors that recruit Yki to DNA, most notably Scalloped (Sd) and Homothorax (Hth), are important regulators of developmental patterning with many tissue-specific functions. Thus, these three transcriptional regulators – Yki, Sd, and Hth – provide a model for exploring the properties of protein-DNA interactions that regulate both tissue-shared and tissue-specific functions. With this goal in mind, we identified the positions in the fly genome that are bound by Yki, Sd, and Hth in the progenitors of the wing and eye-antenna structures of the fly. These data not only provide a global view of the Yki gene regulatory network, they reveal an unusual amount of tissue specificity in the genomic regions targeted by Sd and Hth, but not Yki. The data also reveal that tissue-specific binding is very likely to overlap tissue-specific enhancer regions, provide important clues for how tissue-specific Sd and Hth binding occurs, and support the idea that gene regulatory networks are plastic, with spatial differences in binding significantly impacting network structures.
Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the “retinal determination gene network,” composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development.
Animals develop by using different combinations of simple instructions. The highly conserved retinal determination (RD) network is an ancient set of instructions that evolved when multicellular animals first developed primitive eyes. Evidence suggests that this network is re-used throughout evolution to direct the development of organs that communicate with the brain, providing information about our internal and external world. This includes our eyes, ears, kidneys, and pancreas. An upstream member of the network named eyeless must be activated early to initiate eye development. Eyeless then activates the expression of downstream genes that maintain eyeless expression and define the eye field. Here, we show that eyeless must also be turned off for final steps of eye development. We investigated the mechanism by which eyeless is turned off and we find that feedback regulation by the downstream RD genes changes to repress Eyeless expression during late stages of development. This study shows that tight regulation of eyeless is important for normal development and provides a mechanism for its repression.
Olfactory sensory neurons connect to the antennal lobe of the fly to create the primary units for processing odor cues, the glomeruli. Unique amongst antennal-lobe neurons is an identified wide-field serotonergic neuron, the contralaterally-projecting, serotonin-immunoreactive deutocerebral neuron (CSDn). The CSDn spreads its termini all over the contralateral antennal lobe, suggesting a diffuse neuromodulatory role. A closer examination, however, reveals a restricted pattern of the CSDn arborization in some glomeruli. We show that sensory neuron-derived Eph interacts with Ephrin in the CSDn, to regulate these arborizations. Behavioural analysis of animals with altered Eph-ephrin signaling and with consequent arborization defects suggests that neuromodulation requires local glomerular-specific patterning of the CSDn termini. Our results show the importance of developmental regulation of terminal arborization of even the diffuse modulatory neurons to allow them to route sensory-inputs according to the behavioural contexts.
Serotonin, a major neuromodulatory transmitter, regulates diverse behaviours. Serotonergic dysfunction is implicated in various neuropsychological disorders, such as anxiety and depression, as well as in neurodegenerative disorders. In the central nervous systems, across taxa, serotonergic neurons are often small in number but connect to and act upon multiple brain circuits through their wide-field arborization pattern. We set out to decipher mechanisms by which wide-field serotonergic neurons differentially innervate their target-field to modulate behavior in a context-dependent manner. We took advantage of the sophisticated antennal lobe circuitry, the primary olfactory centre in the adult fruitfly Drosophila melanogaster. Olfactory sensory neurons and projection neurons connect in a partner-specific manner to create glomerular units in the antennal lobe for processing the sense of smell. Our analysis at a single-cell resolution reveals that a wide-field serotonergic neuron connects to all the glomeruli in the antennal lobe but exhibits the glomerular-specific differences in its innervation pattern. Our key finding is that Eph from sensory neurons regulates the glomerular-specific innervation pattern of the central serotonergic neuron, which in turn is essential for modulation of odor-guided behaviours in an odor-specific manner.
Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs), mediate associations among Polycomb Group (PcG) targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.
We have studied the nuclear localization of genes that are regulated by Polycomb mechanisms. The genomes of higher eukaryotes contain hundreds of genes that are regulated by Polycomb mechanisms. Once repressed by Polycomb complexes, they tend to stay repressed; but, when activated, they bind Trithorax protein and tend to maintain the active state epigenetically. Polycomb repression has been reported to make these genes associate in the nucleus to form “Polycomb bodies.” We find that this association is not caused by Polycomb complexes but by insulator elements accompanying the genes. We show that, when these genes are in the active state, the binding of Trithorax targets them to other nuclear regions where transcription occurs, so-called transcription factories. In these nuclear re-positionings the insulator provides the associative power while the state of activity determines whether a gene goes to a Polycomb body or to a transcription factory. The strong effect of Trithorax suggests the possibility that the stable association with a transcription factory it produces may account for the epigenetic memory of the active state.
In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland's other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), we discovered that Abd-B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, we discovered an enhancer from the iab-6 regulatory domain that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. We determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of sex peptide in female mates of iab-6 mutant males. We also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. Our results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR.
Similar to the prostate gland and seminal vesicle in mammals, the Drosophila male accessory gland produces seminal fluid proteins that are critical for individual male reproductive success. In Drosophila, many of these proteins function to induce a suite of long-lasting physiological changes in mated females, like increased egg laying and decreased receptivity to secondary courtships. While investigating the cis-regulatory region of the Hox gene, Abd-B, we found that Abd-B is specifically expressed in a mostly uncharacterized cell-type of the accessory gland, called the secondary cells. Using regulatory mutants of Abd-B, we were able to perturb the function of the secondary cells to show that the secondary cells play a critical role in extending the duration of the post-mating response in females. The induced physiological changes in the female result in a genetic advantage for the genes of the copulating male in populating the next generation. Interestingly, the function of the Abd-B class genes in seminal protein producing tissues seems to be an ancient and conserved function, as the orthologues of Abd-B in mammals, the hox9-13 class of genes, are expressed in the mammalian prostate and seminal vesicle.
During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.
The transcription factor Pax6 is reiteratively employed in space and time for the establishment of progenitor pools and the differentiation of neuronal and non-neuronal lineages of the CNS, pancreas, and eye. Execution of these diverse developmental programs depends on simultaneous activation and repression of gene networks functioning downstream of Pax6. MicroRNAs function as inhibitors of gene expression. Many microRNA genes are transcribed through common promoters of host genes. In this study, using wide-scale analysis of changes in gene expression following Pax6 deletion in the lens, we discover that Pax6 regulates the gene Trpm3 and its hosted microRNA, miR-204. We then show that miR-204 suppresses several target genes in the lens, notably the neuronal gene Sox11. Lastly, by conducting parallel experiments in the medaka fish, we show that Pax6 control of miR-204 and its target genes is evolutionarily conserved between mammals and fish, stressing the biological importance of this pathway. Pax6 regulation of miR-204 explains part of the complex, divergent inhibitory activity of Pax6 in ocular progenitor cells, which is required to establish and maintain the identity and function of ocular tissues.
In multi-cellular organisms, tissue homeostasis is maintained by an exquisite balance between stem cell proliferation and differentiation. This equilibrium can be achieved either at the single cell level (a.k.a. cell asymmetry), where stem cells follow strict asymmetric divisions, or the population level (a.k.a. population asymmetry), where gains and losses in individual stem cell lineages are randomly distributed, but the net effect is homeostasis. In the mature mouse intestinal crypt, previous evidence has revealed a pattern of population asymmetry through predominantly symmetric divisions of stem cells. In this work, using population genetic theory together with previously published crypt single-cell data obtained at different mouse life stages, we reveal a strikingly dynamic pattern of stem cell homeostatic control. We find that single-cell asymmetric divisions are gradually replaced by stochastic population-level asymmetry as the mouse matures to adulthood. This lifelong process has important developmental and evolutionary implications in understanding how adult tissues maintain their homeostasis integrating the trade-off between intrinsic and extrinsic regulations.
In multi-cellular organisms, there is a static equilibrium maintaining cells of various forms. This homeostasis is achieved by an exquisite balance between stem cell proliferation and differentiation. Understanding how different species and organ types maintain this dynamic equilibrium has been an interesting question for both evolutionary and developmental biologists. Using population genetic theory together with previously published single-cell sequencing data collected from mouse intestinal crypts at two points in development, we have revealed a dynamic picture of stem cell renewal in intestinal crypts. We found that intestinal equilibrium is maintained at the single-cell level through predominantly asymmetric stem cell divisions at early life stages, but progressively switches to a population level homeostasis with only symmetric divisions as the mouse matures to adulthood. This dynamic process, likely to be conserved across species, has important developmental and evolutionary implications in understanding how adult tissues maintain their homeostasis integrating lifelong trade-offs between intrinsic and extrinsic factors.
The development of two-component expression systems in Drosophila melanogaster, one of the most powerful genetic models, has allowed the precise manipulation of gene function in specific cell populations. These expression systems, in combination with site-specific recombination approaches, have also led to the development of new methods for clonal lineage analysis. We present a hands-on user guide to the techniques and approaches that have greatly increased resolution of genetic analysis in the fly, with a special focus on their application for lineage analysis. Our intention is to provide guidance and suggestions regarding which genetic tools are most suitable for addressing different developmental questions.
Insect fat body is the organ for intermediary metabolism, comparable to vertebrate liver and adipose tissue. Larval fat body is disintegrated to individual fat body cells and then adult fat body is remodeled at the pupal stage. However, little is known about the dissociation mechanism. We find that the moth Helicoverpa armigera cathepsin L (Har-CL) is expressed heavily in the fat body and is released from fat body cells into the extracellular matrix. The inhibitor and RNAi experiments demonstrate that Har-CL functions in the fat body dissociation in H. armigera. Further, a nuclear protein is identified to be transcription factor Har-Relish, which was found in insect immune response and specifically binds to the promoter of Har-CL gene to regulate its activity. Har-Relish also responds to the steroid hormone ecdysone. Thus, the dissociation of the larval fat body is involved in the hormone (ecdysone)-transcription factor (Relish)-target gene (cathepsin L) regulatory pathway.
Insect fat body is the intermediary metabolism organ and the main source of hemolymph components, and it is crucial for insect development and metamorphosis. However, molecular mechanism for the fat body remodeling is almost unknown other than in Drosophila melanogaster. A pupal diapause species the cotton bollworm, Helicoverpa armigera (Har), is a useful model to study individual or tissue remodeling, because larval fat body will remain integral in diapause-type pupae for months, whereas the dissociation of larval fat body will start on day 0 after pupation in nondiapause-type ones. Here, we find that H. armigera cathepsin L (Har-CL) is released from fat body cells into the extracellular matrix for tissue dissociation. A nuclear protein is identified to be transcription factor Har-Relish, which regulates the promoter activity of Har-CL gene. Har-Relish also responds to the steroid hormone ecdysone. Thus, a new regulatory mechanism, ecdysone-Relish-cathepsin L signaling pathway, is involved in the larval fat body dissociation.
Soluble circulating proteins play an important role in the regulation of mating behavior in Drosophila melanogaster. However, how these factors signal through the blood–brain barrier (bbb) to interact with the sex-specific brain circuits that control courtship is unknown. Here we show that male identity of the blood–brain barrier is necessary and that male-specific factors in the bbb are physiologically required for normal male courtship behavior. Feminization of the bbb of adult males significantly reduces male courtship. We show that the bbb–specific G-protein coupled receptor moody and bbb–specific Go signaling in adult males are necessary for normal courtship. These data identify sex-specific factors and signaling processes in the bbb as important regulators of male mating behavior.
Complex behaviors such as mating behavior are controlled by the brain. Ensembles of brain cells work in networks to ensure proper behavior at the right time. While the state of these cells plays an important role in whether and how the behavior is displayed, information from outside the brain is also required. Often, this information is provided by hormones that are present in the circulating fluid (such as the blood). However, the brain is protected by a layer of very tight cells, the so-called blood–brain barrier, that keeps unwanted molecules out. So how then do hormones and other regulatory factors “talk” to the brain? We are studying this question by examining the mating behavior of males of a model organism, the fruit fly Drosophila melanogaster. We have found that the blood–brain barrier cells themselves contain male-specific molecules that play an important role. When they are absent, courtship behavior is compromised. We have also identified how outside factors talk to the brain: by using a cellular signaling protein and a particular signaling pathway. Together they are well suited to pass on outside information to the brain network that regulates mating behavior.
Transport of newly synthesized Rhodopsin upon light stimulation in adult Drosophila photoreceptors is mediated by a Crag/Rab11-dependent vesicular trafficking process.
Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.
Animals sense light through receptors called Rhodopsins. These proteins are typically localized to stacked membranes in photoreceptors. In flies, upon light exposure, Rhodopsin undergoes conformational changes and becomes active as metarhodopsin. Metarhodopsin then initiates a signaling cascade that activates the photoreceptor cell. To deactivate the light response, metarhodopsin is converted back into Rhodopsin by absorption of another photon of light. Under certain conditions, metarhodopsin cannot be converted back to Rhodopsin, and it is then endocytosed and degraded. Rhodopsin then needs to be synthesized and delivered back to the membrane stacks. Here, we show that the Calmodulin-binding protein Crag is required for the delivery of newly made Rhodopsin to the membrane stacks. Loss of Crag leads to the accumulation of Rhodopsin in the cytosol, followed by shrinkage of membrane stack volume, and, eventually, photoreceptor cell degeneration. We also show that Crag activates a target protein, Rab11, which mediates the vesicular transport of Rhodospin to the membrane. Finally, we document that the human homolog of Crag, DENND4A, is able to rescue the loss of Crag in flies, suggesting that DENND4A functions in a similar process in vertebrates.
Colour vision is found in many invertebrate and vertebrate species. It is the ability to discriminate objects based on the wavelength of emitted light independent of intensity. As it requires the comparison of at least two photoreceptor types with different spectral sensitivities, this process is often mediated by a mosaic made of several photoreceptor types. In this review, we summarize the current knowledge about the formation of retinal mosaics and the regulation of photopigment (opsin) expression in the fly, mouse and human retina. Despite distinct evolutionary origins, as well as major differences in morphology and phototransduction machineries, there are significant similarities in the stepwise cell-fate decisions that lead from progenitor cells to terminally differentiated photoreceptors that express a particular opsin. Common themes include i) the use of binary transcriptional switches that distinguish classes of photoreceptors, ii) the use of gradients of signaling molecules for regional specializations, iii) stochastic choices that pattern the retina and iv) the use of permissive factors with multiple roles in different photoreceptor types.
photoreceptor; opsin; colour vision; retinal mosaic; transcription factors
The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.
Neurons generally differentiate and never divide again. One barrier to understanding the mechanisms has been the paucity of genetic mutations that result in neuronal cell cycles. Here we show that mutation in three genes lead to cell cycle re-entry by a particular class of developing photoreceptor neurons in the fly retina. Strikingly, these neurons do not complete cell division but only divide their nuclei. The binucleated neurons then typically retain one nucleus in its normal location in the cell body, while transporting the other into the growing axon like other axonal material. Our findings identify Cyclin A regulation as crucial to maintaining cell cycle exit by at least some neurons and identify a neuron-specific defect in cell division as a further barrier to neuron proliferation. Because defects in transporting axonal material have been implicated in the origin of multiple neurodegenerative diseases, our findings also suggest a possible connection between defective cell cycle regulation and neuronal cell death.
A high-resolution neuronal lineage analysis in the Drosophila antennal lobe reveals the complexity of lineage development and Notch signaling in cell fate specification.
Binary cell fate decisions allow the production of distinct sister neurons from an intermediate precursor. Neurons are further diversified based on the birth order of intermediate precursors. Here we examined the interplay between binary cell fate and birth-order-dependent temporal fate in the Drosophila lateral antennal lobe (lAL) neuronal lineage. Single-cell mapping of the lAL lineage by twin-spot mosaic analysis with repressible cell markers (ts-MARCM) revealed that projection neurons (PNs) and local interneurons (LNs) are made in pairs through binary fate decisions. Forty-five types of PNs innervating distinct brain regions arise in a stereotyped sequence; however, the PNs with similar morphologies are not necessarily born in a contiguous window. The LNs are morphologically less diverse than the PNs, and the sequential morphogenetic changes in the two pairs occur independently. Sanpodo-dependent Notch activity promotes and patterns the LN fates. By contrast, Notch diversifies PN temporal fates in a Sanpodo-dispensable manner. These pleiotropic Notch actions underlie the differential temporal fate specification of twin neurons produced by common precursors within a lineage, possibly by modulating postmitotic neurons' responses to Notch-independent transcriptional cascades.
The Drosophila brain develops from a limited number of neural stem cells that produce a series of ganglion mother cells (GMCs) that divide once to produce a pair of neurons in a defined order, termed a neuronal lineage. Here, we provide a detailed lineage map for the neurons derived from the Drosophila lateral antennal lobe (lAL) neuroblast. The lAL lineage consists of two distinct hemilineages, generated through differential Notch signaling in the two GMC daughters, to produce one projection neuron (PN) paired with a local interneuron (LN). Both hemilineages yield distinct cell types in the same sequence, although the temporal identity (birth-order-dependent fate) changes are regulated independently between projection neurons and local interneurons, such that a series of analogous local interneurons may co-derive with different projection neurons and vice versa. We also find that Notch signaling can transform a class of nonantennal lobe projection neurons into antennal lobe projection neurons. These findings suggest that Notch signaling not only modulates temporal fate but itself plays a role in the distinction of antennal lobe versus nonantennal lobe neurons.
Patterning the Drosophila retina for color vision relies on post-mitotic specification of photoreceptor subtypes. R8 photoreceptors express one of two light-sensing Rhodopsins, Rh5 or Rh6. This fate decision involves a bistable feedback loop between Melted, a PH-domain protein, and Warts, a kinase in the Hippo growth pathway. Here, we show a subset of the Hippo pathway—Merlin(Mer), Kibra(Kib), and Lethal(2)giant larvae(Lgl), but not Expanded or Fat--is required for Warts expression and activity in R8 to specify Rh6 fate. Melted represses warts transcription to disrupt Hippo pathway activity and specify Rh5 fate. R8 Hippo signaling therefore exhibits ON-or-OFF regulation, promoting mutually exclusive fates. Furthermore, Mer and Lgl are continuously required to maintain R8 neuronal subtypes. These results reveal a role for Mer, Kib, and Lgl in neuronal specification and maintenance, and show that the Hippo pathway is re-implemented for sensory neuron fate by combining canonical and non-canonical regulatory steps.
binary cell fate; color vision; photoreceptor; rhodopsin; Hippo pathway; neural development; neural maintenance; Merlin; Warts; Kibra; Lgl
The homeodomain and adjacent CVC domain in the visual system homeobox (VSX) proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele.
Problems with the early development of the mammalian retina can cause congenital eye defects such as microphthalmia, in which the eye is dramatically smaller and functionally compromised. Severe microphthalmia is associated with mutations in the retinal-expressed visual system homeobox 2 (Vsx2) gene, but how Vsx2 controls retinal development, and ultimately eye formation, has remained unclear. We assessed the impact of two missense mutations, discovered in humans, on Vsx2 function and eye development in mice. One mutation altered a highly conserved residue of the homeodomain, and the other altered a highly conserved residue in the CVC domain, a region of unresolved function. Both mutations impacted the DNA binding properties of the protein, although to differing extents. Likewise, both mutations caused microphthalmia and disruptions in retinal development, also to differing extents and by distinct mechanisms. Our data suggest that Vsx2 acts as a gatekeeper of the retinal gene expression program by preventing the activation of interfering or competing gene expression programs. We propose that the evolutionary stable association between the VSX-class homeodomain and CVC domain set the stage for Vsx2 or its archetype to assume a gatekeeper function for retinal development and ultimately eye organogenesis.
Understanding how novel complex traits originate involves investigating the time of origin of the trait, as well as the origin of its underlying gene regulatory network in a broad comparative phylogenetic framework. The eyespot of nymphalid butterflies has served as an example of a novel complex trait, as multiple genes are expressed during eyespot development. Yet the origins of eyespots remain unknown. Using a dataset of more than 400 images of butterflies with a known phylogeny and gene expression data for five eyespot-associated genes from over twenty species, we tested origin hypotheses for both eyespots and eyespot-associated genes. We show that eyespots evolved once within the family Nymphalidae, approximately 90 million years ago, concurrent with expression of at least three genes associated with early eyespot development. We also show multiple losses of expression of most genes from this early three-gene cluster, without corresponding losses of eyespots. We propose that complex traits, such as eyespots, may have originated via co-option of a large pre-existing complex gene regulatory network that was subsequently streamlined of genes not required to fulfill its novel developmental function.
Butterfly eyespots play an essential role in natural and sexual selection, yet the evolutionary origins of eyespots and of their underlying gene regulatory network remain unknown. By scoring phenotypes and wing expression of five genes in 399 and 21 nymphalid species, respectively, we tested when eyespots and expression of their associated genes evolved. We found that the origin of eyespots was concurrent with the origin of the gene expression patterns, approximately 90 million years ago. Following this event, many genes expressed in eyespot development were lost in some lineages without a corresponding loss of eyespots, indicating substantial evolution in the cluster of genes associated with eyespots. This finding suggests that complex traits such as butterfly eyespots may initially evolve by re-deploying pre-existing gene regulatory networks, which are subsequently trimmed of genes that are unnecessary in the novel context.
How complex networks of activators and repressors lead to exquisitely specific cell type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In the R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict, and combinatorial activation to induce cell type-specific expression. Further, Dve levels are finely tuned to yield cell type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell fate specification.
Sensory systems with high discriminatory power employ neurons that express only one of several alternative sensory receptor proteins. This exclusive receptor gene expression restricts the sensitivity spectrum of neurons and is coordinated with the choice of their synaptic targets1-3. However, little is known about how it is maintained throughout the life of a neuron. Here we show that the green-light sensing receptor Rhodopsin 6 (Rh6) acts to exclude an alternative blue-sensitive Rhodopsin 5 (Rh5) from a subset of Drosophila R8 photoreceptor neurons4. Loss of Rh6 leads to a gradual expansion of Rh5 expression into all R8 photoreceptors of the aging adult retina. The Rh6 feedback signal results in repression of the rh5 promoter and can be mimicked by other Drosophila Rhodopsins; it is partially dependent on activation of Rhodopsin by light, and relies on Gαq activity, but not on the subsequent steps of the phototransduction cascade5. Our observations reveal a thus far unappreciated spectral plasticity of R8 photoreceptors, and identify Rhodopsin feedback as an exclusion mechanism.