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1.  The bHLH Factors Extramacrochaetae and Daughterless Control Cell Cycle in Drosophila Imaginal Discs through the Transcriptional Regulation of the cdc25 Phosphatase string 
PLoS Genetics  2014;10(3):e1004233.
One of the major issues in developmental biology is about having a better understanding of the mechanisms that regulate organ growth. Identifying these mechanisms is essential to understand the development processes that occur both in physiological and pathological conditions, such as cancer. The E protein family of basic helix-loop helix (bHLH) transcription factors, and their inhibitors the Id proteins, regulate cell proliferation in metazoans. This notion is further supported because the activity of these factors is frequently deregulated in cancerous cells. The E protein orthologue Daughterless (Da) and the Id orthologue Extramacrochaetae (Emc) are the only members of these classes of bHLH proteins in Drosophila. Although these factors are involved in controlling proliferation, the mechanism underlying this regulatory activity is poorly understood. Through a genetic analysis, we show that during the development of epithelial cells in the imaginal discs, the G2/M transition, and hence cell proliferation, is controlled by Emc via Da. In eukaryotic cells, the main activator of this transition is the Cdc25 phosphatase, string. Our genetic analyses reveal that the ectopic expression of string in cells with reduced levels of Emc or high levels of Da is sufficient to rescue the proliferative defects seen in these mutant cells. Moreover, we present evidence demonstrating a role of Da as a transcriptional repressor of string. Taken together, these findings define a mechanism through which Emc controls cell proliferation by regulating the activity of Da, which transcriptionally represses string.
Author Summary
Precise control of cell proliferation is critical for normal development and tissue homeostasis. Members of the inhibitor of differentiation (Id) family of helix-loop-helix (HLH) proteins are key regulators that coordinate the balance between cell division and differentiation. These proteins exert this function in part by combining with ubiquitously expressed bHLH transcription factors (E proteins), preventing these transcription factors from forming functional hetero- or homodimeric DNA binding complexes. Deregulation of the activity of Id proteins frequently leads to tumour formation. The Daughterless (Da) and Extramacrochaetae (Emc) proteins are the only members of the E and Id families in Drosophila, yet their role in the control of cell proliferation has not been determined. In this study, we show that the elimination of emc or the ectopic expression of da arrests cells in the G2 phase of the cell cycle. Moreover, we demonstrate that emc controls cell proliferation via Da, which acts as a transcriptional repressor of the Cdc25 phosphatase string. These results provide an important insight into the mechanisms through which Id and E protein interactions control cell cycle progression and therefore how the disruption of the function of Id proteins can induce oncogenic transformation.
PMCID: PMC3961188  PMID: 24651265
2.  Genetic Dissection of Photoreceptor Subtype Specification by the Drosophila melanogaster Zinc Finger Proteins Elbow and No ocelli 
PLoS Genetics  2014;10(3):e1004210.
The elbow/no ocelli (elb/noc) complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. Here we show that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the ‘dorsal rim area’ (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. Our data suggest that Elb/Noc genetically interact with Hth, and we identify two conserved domains crucial for this function. Furthermore, we show that Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. We propose that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease.
Author Summary
The eyes of many animals contain groups of photoreceptor cells with different chromatic sensitivities that can be arranged in complex patterns. It is of great interest to identify the genes and pathways shaping these ‘retinal mosaics’ which include stochastically distributed groups of cells, versus highly localized ones. In many insect eyes, which are composed of large numbers of unit eyes, or ommatidia, specialized photoreceptors are found only in the dorsal periphery, where they face the sky. These ommatidia are responsible for detecting linearly polarized skylight, which serves as an important navigational cue for these animals. Here we describe how two closely related proteins called Elbow and No ocelli interact with the transcription factors Homothorax and Orthodenticle to correctly specify the polarization detectors at the dorsal rim of the retina of Drosophila melanogaster. Interestingly, all four proteins are evolutionarily conserved from worms to humans, and they appear to be involved in similar developmental processes across species. Furthermore, human homologs of Elbow and No ocelli have been identified as promoters of luminal breast cancer. The newly identified role of these two proteins within a regulatory network might therefore enable new approaches in a number of important processes.
PMCID: PMC3953069  PMID: 24625735
3.  Dual mode of embryonic development is highlighted by expression and function of Nasonia pair-rule genes 
eLife  2014;3:e01440.
Embryonic anterior–posterior patterning is well understood in Drosophila, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior.
eLife digest
Networks of genes that work together are widespread in nature. The conservation of individual genes across species and the tendency of their networks to stick together is a sign that they are working efficiently. Furthermore, it is common for existing gene networks to be adapted to perform new tasks, instead of new networks being invented every time a similar but distinct demand arises. One important question is: how can evolution use the same building blocks—such as the genes in a functioning network—in different ways to achieve new outcomes?
The gene network that sets up the ‘body plan’ of insects during development has been well studied, most deeply in the fruit fly, Drosophila. Like all insects, the body of a fruit fly is divided into three main parts—the head, the thorax and the abdomen—and each of these parts is made up of several smaller segments. There is a remarkable diversity of insect body plans in nature, and yet, these seem to arise from the same gene networks in the embryo.
When a Drosophila embryo is growing into a larva, all the different body segments develop at the same time. In most other insects, however, segments of the abdomen emerge later and sequentially during the development process. The ancestors of most insects are also thought to have developed in this way, which is known as ‘short germ embryogenesis’. So how did the so-called ‘long germ embryogenesis’, as observed in Drosophila, evolve from the short germ embryogenesis that is observed in most other insects?
The gene network that controls development includes the ‘pair-rule genes’ that are expressed in a pattern of alternating stripes that wrap around, top to bottom, along most of the length of the embryo. These stripes mark where the edges of each body segment will eventually develop. In fruit flies, this pattern extends along the entire length of the embryo and the stripes all appear at one time. However, in the abdominal region of short germ insects, the pair-rule genes are expressed in waves that pass through the posterior region as it grows, with new segments being added one behind the other.
Now, Rosenberg et al. have attempted to explain how the same genes can be used to direct the segmentation process in such different ways by studying another long germ insect species, the jewel wasp. Analysis of the expression of pair-rule genes in the jewel wasp shows that it uses a mixed strategy to control segmentation. The development of segments at the front of its body is directed in the same way as the fruit fly, with all these segments laid down together. However, the segments at the rear of the body are only patterned later, one after the other, like most other insects.
The work of Rosenberg et al. suggests that the jewel wasp represents an intermediate step between ancestral insects and Drosophila in the evolution of the gene network that patterns the ‘body plan’. Identifying and studying these intermediate forms allows us to understand the ways in which evolution can innovate by building upon what has come before.
PMCID: PMC3941026  PMID: 24599282
Nasonia vitripennis; Tribolium; embryonic patterning; evolution; segmentation; pair-rule genes; D. melanogaster; other
4.  Dying to Entrain: Regulating ipRGC Spacing 
Developmental cell  2013;24(4):338-340.
In a recent issue of Neuron,Chen et al. (2013) show that apoptosis is required to ensure the even distribution of a class of retinal ganglion cells (ipRGCs), which sense luminance both intrinsically and through input from rods and cones. Disrupting apoptosis impairs photoentrainment mediated by rods/cones, but not that mediated by ipRGC-expressed melanopsin.
PMCID: PMC3744582  PMID: 23449468
5.  Temporal Patterning of Neural Progenitors in Drosophila 
Drosophila has recently become a powerful model system to understand the mechanisms of temporal patterning of neural progenitors called neuroblasts (NBs). Two different temporal sequences of transcription factors (TFs) have been found to be sequentially expressed in NBs of two different systems: the Hunchback, Krüppel, Pdm1/Pdm2, Castor, and Grainyhead sequence in the Drosophila ventral nerve cord; and the Homothorax, Klumpfuss, Eyeless, Sloppy-paired, Dichaete, and Tailless sequence that patterns medulla NBs. In addition, the intermediate neural progenitors of type II NB lineages are patterned by a different sequence: Dichaete, Grainyhead, and Eyeless. These three examples suggest that temporal patterning of neural precursors by sequences of TFs is a common theme to generate neural diversity. Cross-regulations, including negative feedback regulation and positive feedforward regulation among the temporal factors, can facilitate the progression of the sequence. However, there are many remaining questions to understand the mechanism of temporal transitions. The temporal sequence progression is intimately linked to the progressive restriction of NB competence, and eventually determines the end of neurogenesis. Temporal identity has to be integrated with spatial identity information, as well as with the Notch-dependent binary fate choices, in order to generate specific neuron fates.
PMCID: PMC3927947  PMID: 23962839
6.  Conservation and Divergence of Regulatory Strategies at Hox Loci and the Origin of Tetrapod Digits 
PLoS Biology  2014;12(1):e1001773.
During development, expression of the Hoxa and Hoxd genes in zebrafish fins and mouse limbs are regulated via a conserved chromatin structure. However, zebrafish lack certain regulatory elements required to produce digits, revealing that radials—the fin's bony elements—are likely not homologous to tetrapod digits.
The evolution of tetrapod limbs from fish fins enabled the conquest of land by vertebrates and thus represents a key step in evolution. Despite the use of comparative gene expression analyses, critical aspects of this transformation remain controversial, in particular the origin of digits. Hoxa and Hoxd genes are essential for the specification of the different limb segments and their functional abrogation leads to large truncations of the appendages. Here we show that the selective transcription of mouse Hoxa genes in proximal and distal limbs is related to a bimodal higher order chromatin structure, similar to that reported for Hoxd genes, thus revealing a generic regulatory strategy implemented by both gene clusters during limb development. We found the same bimodal chromatin architecture in fish embryos, indicating that the regulatory mechanism used to pattern tetrapod limbs may predate the divergence between fish and tetrapods. However, when assessed in mice, both fish regulatory landscapes triggered transcription in proximal rather than distal limb territories, supporting an evolutionary scenario whereby digits arose as tetrapod novelties through genetic retrofitting of preexisting regulatory landscapes. We discuss the possibility to consider regulatory circuitries, rather than expression patterns, as essential parameters to define evolutionary synapomorphies.
Author Summary
Our upper limbs differ from fish fins, notably by their subdivision into arm and hand regions, which are separated by a complex articulation, the wrist. The development of this anatomy is associated with two distinct waves of expression of the Hoxa and Hoxd genes during development. Would such a shared expression pattern be sufficient to infer homology between fish fins and mouse limbs? We investigated this question here, looking at whether the two phases of Hox gene transcription that are observed during tetrapod limb development also occur during zebrafish fin development. We find the answer is “not quite.” For although the mechanisms that regulate the expression of Hoxa and Hoxd are comparable between zebrafish fins and mouse limbs, when the genomic regions that regulate Hox gene expression in fish fins are introduced into transgenic mice, they trigger Hox gene expression in only the proximal limb segment (the segment nearest the body) and not in the presumptive digits. We conclude that although fish have the Hox regulatory toolkit to produce digits, this potential is not utilized as it is in tetrapods, and as a result we propose that fin radials—the bony elements of fins—are not homologous to tetrapod digits.
PMCID: PMC3897358  PMID: 24465181
7.  Temporal patterning of Drosophila medulla neuroblasts controls neural fates 
Nature  2013;498(7455):456-462.
In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains ~40,000 neurons belonging to over 70 different types. We describe how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors--Homothorax, Eyeless, Sloppy-paired, Dichaete and Tailless--are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of either Eyeless, Sloppy-paired or Dichaete blocks further progression of the temporal sequence. We provide evidence that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, our work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts.
PMCID: PMC3701960  PMID: 23783517
8.  The Midline Protein Regulates Axon Guidance by Blocking the Reiteration of Neuroblast Rows within the Drosophila Ventral Nerve Cord 
PLoS Genetics  2013;9(12):e1004050.
Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.
Author Summary
During nervous system development, once formed from neuroblasts, neurons grow axons in the direction of their synaptic partners. Genetic forces guide these axon growth cones towards the target. This is known as axon guidance or pathfinding. There are a number of proteins that regulate axon-pathfinding. The well-known examples are the Slit and its receptor Roundabout, and Netrin and its receptor Frazzled. The Drosophila embryo and the nervous system are divided into segments by segmentation genes. Mutations in segmentation genes affect axon guidance, although how they do so is not well understood. In our work described here, we show that the T-box protein Midline prevents mis-specification of neuroblast rows, in particular, it prevents row 2 from becoming row 5. Thus, in midline mutants, row 2 changes into row 5, ultimately creating a non-permissive barrier that prevents axons from following their defined path. Thus, axons stop and diverge when they reach this barrier. Our results show how mutations in segmentation genes can affect axon guidance and how significant the environment is for axon-pathfinding. Our work is also a cautionary reminder that guidance defects need to be interpreted with care and can arise due to a variety of other defects.
PMCID: PMC3873230  PMID: 24385932
9.  Stochastic spineless expression creates the retinal mosaic for colour vision 
Nature  2006;440(7081):10.1038/nature04615.
Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called ‘pale’ and ‘yellow’, defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. Here we report that the Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless.
PMCID: PMC3826883  PMID: 16525464
10.  The Drosophila eve Insulator Homie Promotes eve Expression and Protects the Adjacent Gene from Repression by Polycomb Spreading 
PLoS Genetics  2013;9(10):e1003883.
Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is “replaced” by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.
Author Summary
Insulators are specialized DNA elements that can separate the genome into functional units. Most of the current thinking about these elements comes from studies done with model transgenes. Studies of insulators within the specialized Hox gene complexes have suggested that model transgenes can reflect the normal functions of these elements in their native context. However, recent genome-wide studies have called this into question. This work analyzes the native function of an insulator that resides between the Drosophila genes eve and TER94, which are expressed in very different patterns. Also, the eve gene is a Polycomb (Pc) domain, a specialized type of chromatin that is found in many places throughout the genome. We show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. Each of these activities are consistent with those seen with model transgenes, and other known insulators can provide these functions in this context. This work provides a novel and convincing example of the normal role of insulators in regulating the eukaryotic genome, as well as providing insights into their mechanisms of action.
PMCID: PMC3814318  PMID: 24204298
11.  Function and Evolution of DNA Methylation in Nasonia vitripennis 
PLoS Genetics  2013;9(10):e1003872.
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.
Author Summary
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.
PMCID: PMC3794928  PMID: 24130511
12.  Multiple Signaling Pathways Coordinate to Induce a Threshold Response in a Chordate Embryo 
PLoS Genetics  2013;9(10):e1003818.
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.
Author Summary
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.
PMCID: PMC3789818  PMID: 24098142
13.  Distinct Regulatory Mechanisms Act to Establish and Maintain Pax3 Expression in the Developing Neural Tube 
PLoS Genetics  2013;9(10):e1003811.
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.
Author Summary
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.
PMCID: PMC3789833  PMID: 24098141
14.  dTULP, the Drosophila melanogaster Homolog of Tubby, Regulates Transient Receptor Potential Channel Localization in Cilia 
PLoS Genetics  2013;9(9):e1003814.
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.
Author Summary
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.
PMCID: PMC3778012  PMID: 24068974
15.  Deterministic or stochastic choices in retinal neuron specification 
Neuron  2012;75(5):739-742.
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.
PMCID: PMC3438524  PMID: 22958814
vertebrate retinogenesis; competence; stochasticity; retinal progenitor; birth order
16.  Divergent Transcriptional Regulatory Logic at the Intersection of Tissue Growth and Developmental Patterning 
PLoS Genetics  2013;9(9):e1003753.
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.
Author Summary
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.
PMCID: PMC3764184  PMID: 24039600
17.  Dynamic Rewiring of the Drosophila Retinal Determination Network Switches Its Function from Selector to Differentiation 
PLoS Genetics  2013;9(8):e1003731.
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.
Author Summary
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.
PMCID: PMC3757064  PMID: 24009524
18.  Sensory Neuron-Derived Eph Regulates Glomerular Arbors and Modulatory Function of a Central Serotonergic Neuron 
PLoS Genetics  2013;9(4):e1003452.
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.
Author Summary
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.
PMCID: PMC3630106  PMID: 23637622
19.  Insulators Target Active Genes to Transcription Factories and Polycomb-Repressed Genes to Polycomb Bodies 
PLoS Genetics  2013;9(4):e1003436.
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.
Author Summary
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.
PMCID: PMC3630138  PMID: 23637616
20.  A Novel Function for the Hox Gene Abd-B in the Male Accessory Gland Regulates the Long-Term Female Post-Mating Response in Drosophila 
PLoS Genetics  2013;9(3):e1003395.
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.
Author Summary
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.
PMCID: PMC3610936  PMID: 23555301
21.  Pax6 Regulates Gene Expression in the Vertebrate Lens through miR-204 
PLoS Genetics  2013;9(3):e1003357.
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.
Author Summary
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.
PMCID: PMC3597499  PMID: 23516376
22.  Age-Dependent Transition from Cell-Level to Population-Level Control in Murine Intestinal Homeostasis Revealed by Coalescence Analysis 
PLoS Genetics  2013;9(2):e1003326.
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.
Author Summary
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.
PMCID: PMC3585040  PMID: 23468655
23.  Power tools for gene expression and clonal analysis in Drosophila 
Nature methods  2011;9(1):47-55.
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.
PMCID: PMC3574576  PMID: 22205518
24.  A Regulatory Pathway, Ecdysone-Transcription Factor Relish-Cathepsin L, Is Involved in Insect Fat Body Dissociation 
PLoS Genetics  2013;9(2):e1003273.
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.
Author Summary
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.
PMCID: PMC3573115  PMID: 23459255

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