[Show abstract][Hide abstract] ABSTRACT: Background:
One of the major challenges in developmental biology is to understand the regulatory events that generate neuronal diversity. During Drosophila embryonic neural lineage development, cellular temporal identity is established in part by a transcription factor (TF) regulatory network that mediates a cascade of cellular identity decisions. Two of the regulators essential to this network are the POU-domain TFs Nubbin and Pdm-2, encoded by adjacent genes collectively known as pdm. The focus of this study is the discovery and characterization of cis-regulatory DNA that governs their expression.
Phylogenetic footprinting analysis of a 125 kb genomic region that spans the pdm locus identified 116 conserved sequence clusters. To determine which of these regions function as cis-regulatory enhancers that regulate the dynamics of pdm gene expression, we tested each for in vivo enhancer activity during embryonic development and postembryonic neurogenesis. Our screen revealed 77 unique enhancers positioned throughout the noncoding region of the pdm locus. Many of these activated neural-specific gene expression during different developmental stages and many drove expression in overlapping patterns. Sequence comparisons of functionally related enhancers that activate overlapping expression patterns revealed that they share conserved elements that can be predictive of enhancer behavior. To facilitate data accessibility, the results of our analysis are catalogued in cisPatterns, an online database of the structure and function of these and other Drosophila enhancers.
These studies reveal a diversity of modular enhancers that most likely regulate pdm gene expression during embryonic and adult development, highlighting a high level of temporal and spatial expression specificity. In addition, we discovered clusters of functionally related enhancers throughout the pdm locus. A subset of these enhancers share conserved elements including sequences that correspond to known TF DNA binding sites. Although comparative analysis of the nubbin and pdm-2 encoding sequences indicate that these two genes most likely arose from a duplication event, we found only partial evidence of sequence duplication between their enhancers, suggesting that after the putative duplication their cis-regulatory DNA diverged at a higher rate than their coding sequences.
[Show abstract][Hide abstract] ABSTRACT: The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.
[Show abstract][Hide abstract] ABSTRACT: Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.
Development 01/2014; 141(2):488. DOI:10.1242/dev.107243 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Analysis of cis-regulatory enhancers has revealed that they consist of clustered blocks of highly conserved sequences. Although most characterized enhancers reside near their target genes, a growing number of studies have shown that enhancers located over 50 kb from their minimal promoter(s) are required for appropriate gene expression and many of these 'long-range' enhancers are found in genomic regions that are devoid of identified exons. To gain insight into the complexity of Drosophila cis-regulatory sequences within exon-poor regions, we have undertaken an evolutionary analysis of 39 of these regions located throughout the genome. This survey revealed that within these genomic expanses, clusters of conserved sequence blocks (CSBs) are positioned once every 1.1 kb, on average, and that a typical cluster contains multiple (5 to 30 or more) CSBs that have been maintained for at least 190 My of evolutionary divergence. As an initial step toward assessing the cis-regulatory activity of conserved clusters within gene-free genomic expanses, we have tested the in-vivo enhancer activity of 19 consecutive CSB clusters located in the middle of a 115 kb gene-poor region on the 3(rd) chromosome. Our studies revealed that each cluster functions independently as a specific spatial/temporal enhancer. In total, the enhancers possess a diversity of regulatory functions, including dynamically activating expression in defined patterns within subsets of cells in discrete regions of the embryo, larvae and/or adult. We also observed that many of the enhancers are multifunctional-that is, they activate expression during multiple developmental stages. By extending these results to the rest of the Drosophila genome, which contains over 70,000 non-coding CSB clusters, we suggest that most function as enhancers.
PLoS ONE 04/2013; 8(4):e60137. DOI:10.1371/journal.pone.0060137 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The primary purpose of this study was to determine if methicillin-resistant Staphylococcus aureus (MRSA) strains could be identified in the milk of dairy cattle in a Paso del Norte region dairy of the United States. Using physiological and PCR-based identification schemes, a total of 40 Staph. aureus strains were isolated from 29 raw milk samples of 133 total samples analyzed. Pulsed-field gel electrophoresis after digestion with the SmaI enzyme revealed that the 40 confirmed strains were represented by 5 pulsed-field types, which each contained 3 or more strains. Of 7 hospital strains isolated from cows undergoing antibiotic therapy, 3 demonstrated resistance to 3 or more antimicrobial classes and displayed similar pulsed-field gel electrophoresis patterns. A secondary purpose of this study was to belucidate the evolutionary relationships of strains isolated in this study to genomically characterized Staph. aureus strains. Therefore, Roche 454 GS (Roche Diagnostics Cor., Dallas, TX) pyrosequencing was used to produce draft genome sequences of an MRSA raw milk isolate (H29) and a methicillin-susceptible Staph. aureus (PB32). Analysis using the BLASTn database (http://blast.ncbi.nlm.nih.gov/) demonstrated that the H29 draft genome was highly homologous to the human MRSA strain JH1, yet the β-lactamase plasmid carried by H29 was different from that carried by JH1. Genomic analysis of H29 also clearly explained the multidrug resistance phenotype of this raw milk isolate. Analysis of the PB32 draft genome (using BLASTn) demonstrated that this raw milk isolate was most related to human MRSA strain 04-02981. Although PB32 is not a MRSA, the PB32 draft genome did reveal the presence of a unique staphylococcal cassette mec (SSCmec) remnant. In addition, the PB32 draft genome revealed the presence of a novel bovine staphylococcal pathogenicity island, SaPIbovPB32. This study demonstrates the presence of clones closely related to human and (or) bovine Staph. aureus strains circulating in a dairy herd.
[Show abstract][Hide abstract] ABSTRACT: Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 39 untranslated region (39UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or .1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The authors thank the subjects and their families who participated in our research and the Division of Intramural Research, National Human Genome Research Institute (NHGRI) and National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), in part, for financial support.
[Show abstract][Hide abstract] ABSTRACT: Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.
PLoS ONE 07/2012; 7(7):e39026. DOI:10.1371/journal.pone.0039026 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the developing CNS, unique functional identities among neurons and glia are, in part, established as a result of successive transitions in gene expression programs within neural precursor cells. One of the temporal-identity windows within Drosophila CNS neural precursor cells or neuroblasts (NBs) is marked by the expression of a zinc-finger transcription factor (TF) gene, castor (cas). Our analysis of cis-regulatory DNA within a cas loss-of-function rescue fragment has identified seven enhancers that independently activate reporter transgene expression in specific sub-patterns of the wild-type embryonic cas gene expression domain. Most of these enhancers also regulate different aspects of cas expression within the larval and adult CNS. Phylogenetic footprinting reveals that each enhancer is made up of clusters of highly conserved DNA sequence blocks that are flanked by less-conserved inter-cluster spacer sequences. Comparative analysis of the conserved DNA also reveals that cas enhancers share different combinations of sequence elements and many of these shared elements contain core DNA-binding recognition motifs for characterized temporal-identity TFs. Intra-species alignments show that two of the sub-pattern enhancers originated from an inverted duplication and that this repeat is unique to the cas locus in all sequenced Drosophila species. Finally we show that three of the enhancers differentially require cas function for their wild-type regulatory behavior. Cas limits the expression of one enhancer while two others require cas function for full expression. These studies represent a starting point for the further analysis of cas gene expression and the TFs that regulate it.
[Show abstract][Hide abstract] ABSTRACT: Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs.
We have generated a Drosophila genome-wide database of conserved DNA consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization.
cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process.
[Show abstract][Hide abstract] ABSTRACT: Evolution and Medicine is a curriculum supplement designed by the National Institutes of Health (NIH) and the Biological Sciences Curriculum Study
(BSCS) for high school students. The supplement is freely available from NIH’s Office of Science Education (OSE) as a part
of the NIH curriculum supplement series. Development of the supplement was a collaborative effort that included input from
a panel of experts in medicine, evolution, education, and educational technology. In total, the curriculum supplement includes
five inquiry-based lessons that are integrated into the BSCS 5E instructional model (based on constructivist learning theory).
The goal was to develop a 2-week curriculum to help students understand major concepts of evolution using the dynamic, modern,
and relevant context of medicine. A diverse group of students and teachers across the US participated in a formative evaluation
of a field test version of the curriculum. High school students made significant learning gains from pretest to posttest,
with a relatively large effect size for student understanding of common ancestry and a relatively small effect size for student
understanding of natural selection. There was no statistically significant difference in achievement gains between white students
and all other racial/ethnic categories. Overall, the evaluation suggests that a curriculum that emphasizes the role of evolution
in medicine, uses a constructivist instructional model, and is grounded in inquiry is relatively well-received by teachers
and students and shows promise for increasing student learning in evolution.
KeywordsEvolution–Natural selection–Common ancestry–Instructional model–Inquiry–High school–Evolution and medicine
Evolution Education and Outreach 12/2011; 4(4):603-612. DOI:10.1007/s12052-011-0361-2
[Show abstract][Hide abstract] ABSTRACT: Many of the key regulators of Drosophila CNS neural identity are expressed in defined temporal orders during neuroblast (NB) lineage development. To begin to understand the structural and functional complexity of enhancers that regulate ordered NB gene expression programs, we have undertaken the mutational analysis of the temporally restricted nerfin-1 NB enhancer. Our previous studies have localized the enhancer to a region just proximal to the nerfin-1 transcription start site. Analysis of this enhancer, using the phylogenetic footprint program EvoPrinter, reveals the presence of multiple sequence blocks that are conserved among drosophilids. cis-Decoder alignments of these conserved sequence blocks (CSBs) has identified shorter elements that are conserved in other Drosophila NB enhancers. Mutagenesis of the enhancer reveals that although each CSB is required for wild-type expression, neither position nor orientation of the CSBs within the enhancer is crucial for enhancer function; removal of less-conserved or non-conserved sequences flanking CSB clusters also does not significantly alter enhancer activity. While all three conserved E-box transcription factor (TF) binding sites (CAGCTG) are required for full function, adding an additional site at different locations within non-conserved sequences interferes with enhancer activity. Of particular note, none of the mutations resulted in ectopic reporter expression outside of the early NB expression window, suggesting that the temporally restricted pattern is defined by transcriptional activators and not by direct DNA binding repressors. Our work also points to an unexpectedly large number of TFs required for optimal enhancer function - mutant TF analysis has identified at least four that are required for full enhancer regulation.
Mechanisms of development 02/2011; 128(3-4):165-77. DOI:10.1016/j.mod.2011.02.001 · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have identified clusters of conserved sequences constituting discrete modular enhancers within the Drosophilanerfin-1 locus. nerfin-1 encodes a Zn-finger transcription factor that directs pioneer interneuron axon guidance. nerfin-1 mRNA is detected in many early delaminating neuroblasts, ganglion mother cells and transiently in nascent neurons. The comparative genomics analysis program EvoPrinter revealed conserved sequence blocks both upstream and downstream of the transcribed region. By using the aligning regions of different drosophilids as the reference DNA, EvoPrinter detects sequence length flexibility between clusters of conserved sequences and thus facilitates differentiation between closely associated modular enhancers. Expression analysis of enhancer-reporter transgenes identified enhancers that drive expression in different regions of the developing embryonic and adult nervous system, including subsets of embryonic CNS neuroblasts, GMCs, neurons and PNS neurons. In summary, EvoPrinter facilitates the discovery and analysis of enhancers that control crucial aspects of nerfin-1 expression.
[Show abstract][Hide abstract] ABSTRACT: Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.
[Show abstract][Hide abstract] ABSTRACT: The presence of highly conserved sequences within cis-regulatory regions can serve as a valuable starting point for elucidating the basis of enhancer function. This study focuses on regulation of gene expression during the early events of Drosophila neural development. We describe the use of EvoPrinter and cis-Decoder, a suite of interrelated phylogenetic footprinting and alignment programs, to characterize highly conserved sequences that are shared among co-regulating enhancers.
Analysis of in vivo characterized enhancers that drive neural precursor gene expression has revealed that they contain clusters of highly conserved sequence blocks (CSBs) made up of shorter shared sequence elements which are present in different combinations and orientations within the different co-regulating enhancers; these elements contain either known consensus transcription factor binding sites or consist of novel sequences that have not been functionally characterized. The CSBs of co-regulated enhancers share a large number of sequence elements, suggesting that a diverse repertoire of transcription factors may interact in a highly combinatorial fashion to coordinately regulate gene expression. We have used information gained from our comparative analysis to discover an enhancer that directs expression of the nervy gene in neural precursor cells of the CNS and PNS.
The combined use EvoPrinter and cis-Decoder has yielded important insights into the combinatorial appearance of fundamental sequence elements required for neural enhancer function. Each of the 30 enhancers examined conformed to a pattern of highly conserved blocks of sequences containing shared constituent elements. These data establish a basis for further analysis and understanding of neural enhancer function.
[Show abstract][Hide abstract] ABSTRACT: Multi-genome comparative analysis has yielded important insights into the molecular details of gene regulation. We have developed EvoPrinter, a web-accessed genomics tool that provides a single uninterrupted view of conserved sequences as they appear in a species of interest. An EvoPrint reveals with near base-pair resolution those sequences that are essential for gene function.
We describe here EvoPrinterHD, a 2nd-generation comparative genomics tool that automatically generates from a single input sequence an enhanced view of sequence conservation between evolutionarily distant species. Currently available for 5 nematode, 3 mosquito, 12 Drosophila, 20 vertebrate, 17 Staphylococcus and 20 enteric bacteria genomes, EvoPrinterHD employs a modified BLAT algorithm [enhanced-BLAT (eBLAT)], which detects up to 75% more conserved bases than identified by the BLAT alignments used in the earlier EvoPrinter program. The new program also identifies conserved sequences within rearranged DNA, highlights repetitive DNA, and detects sequencing gaps. EvoPrinterHD currently holds over 112 billion bp of indexed genomes in memory and has the flexibility of selecting a subset of genomes for analysis. An EvoDifferences profile is also generated to portray conserved sequences that are uniquely lost in any one of the orthologs. Finally, EvoPrinterHD incorporates options that allow for (1) re-initiation of the analysis using a different genome's aligning region as the reference DNA to detect species-specific changes in less-conserved regions, (2) rapid extraction and curation of conserved sequences, and (3) for bacteria, identifies unique or uniquely shared sequences present in subsets of genomes.
EvoPrinterHD is a fast, high-resolution comparative genomics tool that automatically generates an uninterrupted species-centric view of sequence conservation and enables the discovery of conserved sequences within rearranged DNA. When combined with cis-Decoder, a program that discovers sequence elements shared among tissue specific enhancers, EvoPrinterHD facilitates the analysis of conserved sequences that are essential for coordinate gene regulation.
[Show abstract][Hide abstract] ABSTRACT: Acquisition of virulence factors and antibiotic resistance by many clinically important bacteria can be traced to horizontal gene transfer (HGT) between related or evolutionarily distant microflora. Comparative genomic analysis has become an important tool for identifying HGT DNA in emerging pathogens. We have adapted the multi-genome alignment tool EvoPrinter to facilitate discovery of HGT DNA sequences within bacterial genomes and within their mobile genetic elements.
EvoPrinter analysis of 13 different Staphylococcus aureus genomes revealed that one of the human isolates, the hospital epidemic methicillin-resistant MRSA252 strain, uniquely shares multiple putative HGT DNA sequences with different causative agents of bovine mastitis that are not found in the other human S. aureus isolates. MRSA252 shares over 14 different DNA sequence blocks with the bovine mastitis ET3 S. aureus strain RF122, and many of the HGT DNAs encode virulence factors. EvoPrinter analysis of the MRSA252 chromosome also uncovered virulence-factor encoding HGT events with the genome of Listeria monocytogenes and a Staphylococcus saprophyticus associated plasmid. Both bacteria are also causal agents of contagious bovine mastitis.
EvoPrinter analysis reveals that the human MRSA252 strain uniquely shares multiple DNA sequence blocks with different causative agents of bovine mastitis, suggesting that HGT events may be occurring between these pathogens. These findings have important implications with regard to animal husbandry practices that inadvertently enhance the contact of human and livestock bacterial pathogens.
PLoS ONE 02/2008; 3(8):e3074. DOI:10.1371/journal.pone.0003074 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mRNA encoding the Drosophila Zn-finger transcription factor Nerfin-1, required for CNS axon pathfinding events, is subject to post-transcriptional silencing. Although nerfin-1 mRNA is expressed in many neural precursor cells including all early delaminating CNS neuroblasts, the encoded Nerfin-1 protein is detected only in the nuclei of neural precursors that divide just once to generate neurons and then only transiently in nascent neurons. Using a nerfin-1 promoter-controlled reporter transgene, replacement of the nerfin-1 3' UTR with the viral SV-40 3' UTR releases the neuroblast translational block and prolongs reporter protein expression in neurons. Comparative genomics analysis reveals that the nerfin-1 mRNA 3' UTR contains multiple highly conserved sequence blocks that either harbor and/or overlap 21 predicted binding sites for 18 different microRNAs. To determine the functional significance of these microRNA-binding sites and less conserved microRNA target sites, we have studied their ability to block or limit the expression of reporter protein in nerfin-1-expressing cells during embryonic development. Our results indicate that no single microRNA is sufficient to fully inhibit protein expression but rather multiple microRNAs that target different binding sites are required to block ectopic protein expression in neural precursor cells and temporally restrict expression in neurons. Taken together, these results suggest that multiple microRNAs play a cooperative role in the post-transcriptional regulation of nerfin-1 mRNA, and the high degree of microRNA-binding site evolutionary conservation indicates that all members of the Drosophila genus employ a similar strategy to regulate the onset and extinction dynamics of Nerfin-1 expression.