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Cell Reports, Volume 2, Issue 4, 1002-1013, 11 October 2012
A Resource for Manipulating Gene Expression and Analyzing cis-Regulatory Modules in the Drosophila CNS
Laurina Manning1, Ellie S. Heckscher1, Maria D. Purice1, Jourdain Roberts1, Alysha L. Bennett1, Jason R. Kroll1, Jill L. Pollard1, Marie E. Strader1, Josh R. Lupton1, Anna V. Dyukareva1, Phuong Nam Doan1, David M. Bauer1, Allison N. Wilbur1, Stephanie Tanner1, Jimmy J. Kelly1, Sen-Lin Lai1, Khoa D. Tran1, Minoree Kohwi1, Todd R. Laverty2, Joseph C. Pearson3, Stephen T. Crews3, Gerald M. Rubin2 and Chris Q. Doe1, ,
1 Institutes of Neuroscience and Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA
2 Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
3 Program in Molecular Biology and Biophysics, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Graphical Abstract
► Five thousand Drosophila genomic fragments were analyzed for embryonic central nervous system expression ► More than 1,500 lines are expressed in small subsets of neurons ► The lines provide markers and functional access for >95% of central nervous system neurons
- Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data sets are available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizing interneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.
All model organisms can benefit from tools that allow targeted gene expression. In mouse, collections of Cre lines allow floxed or flox-stop transgenes to be expressed or inactivated, respectively (reviewed in Branda and Dymecki, 2004). In Drosophila and zebrafish, the yeast GAL4/UAS sytem has become a standard method for generating cell-type-specific gene expression (reviewed in Baier and Scott, 2009; Scott, 2009; Scott et al., 2007; Venken and Bellen, 2012). Drosophila is a powerful model system for studying cell biology, development, and neuroscience, primarily because of its facile genetics. Over the past two decades, genetic screens have identified key regulators of highly conserved cellular processes (e.g., cell-cycle progression, cell death, and cell migration), developmental events (e.g., Hox function, cellular memory, and cell-cell signaling pathways), and neurobiological processes (e.g., axon pathfinding, learning and memory, and behavior).
We are interested in characterizing the generation of cellular diversity within the embryonic central nervous system (CNS) and linking neuronal development with function and behavior. The CNS starts to develop at stage 8 with the formation of specialized ventral midline cells, and at stage 9 the bilateral neuroectoderm generates an array of ∼30 neural progenitors, called neuroblasts (NBs) (Doe, 2008; Skeath and Thor, 2003). All ventral midline progenitors and NBs are stereotyped and individually identifiable (Broadus et al., 1995; Wheeler et al., 2006). Each NB divides asymmetrically to generate smaller ganglion mother cells (GMCs) and a self-renewed NB (reviewed in Skeath and Thor, 2003). Each GMC either divides once to generate a pair of sibling neurons (Pearson and Doe, 2003), divides more than once to generate a small number of glia (Akiyama-Oda et al., 1999), or directly differentiates into a neuron (Baumgardt et al., 2009). By stage 16, NB divisions have largely ceased and each segment of the CNS typically contains ∼22 midline cells (Wheeler et al., 2006) and ∼335 bilateral cells (∼35 motoneurons, ∼35 glia, and ∼260 interneurons or unknown cell types; Beckervordersandforth et al., 2008; Landgraf et al., 1999; E.S.H. and C.Q.D., unpublished data).
Embryonic motoneurons are relatively well characterized. Most can be identified by the presence of nuclear phosphorylated Mothers against Dpp (pMad) protein or Zfh1 protein (Layden et al., 2006), and subsets can be identified that express the even-skipped (eve) gene, encoding a transcription factor that promotes axon targeting to dorsal muscle groups (Fujioka et al., 2003; Landgraf et al., 1999), or hb9 (Flybase: exex), which encodes a transcription factor that promotes axon targeting to ventral muscles (Broihier and Skeath, 2002; Odden et al., 2002). GAL4 lines that are expressed in subsets of the Eve+ neurons (Fujioka et al., 1999) and the entire population of Hb9+ neurons (Broihier and Skeath, 2002) are available. Similarly, glial cells can be identified based on position, molecular markers, and GAL4 line expression (Beckervordersandforth et al., 2008). Despite the work of many investigators over the past few decades, very little is known about the majority of interneurons within the CNS. There are few molecular markers for interneuronal subsets and even fewer GAL4 lines with well-characterized expression in subsets of interneurons. This is a barrier to understanding how interneurons develop and how they participate in neural circuits regulating larval behavior.
A powerful tool for characterizing neuronal morphology and function in Drosophila is the GAL4/UAS system (Brand and Perrimon, 1993). GAL4 transgenes with distinct patterns have been made by random insertion of constructs containing a basal promoter and the GAL4 coding sequence, a variation of the enhancer-trapping method (Bellen et al., 1989; Bier et al., 1989; O'Kane and Gehring, 1987) that has been widely used to identify cell-type-specific enhancers and their associated genes. The growing collection of Drosophila GAL4 transgenes is useful for marking neuronal cell types or expressing upstream activating sequence RNA interference (UAS-RNAi) constructs to assay gene function (see above), and in the Mosaic Analysis with Repressible Cell Marker (MARCM) system to generate positively-marked homozygous mutant neuronal clones (Lee and Luo, 2001). These are powerful methods, but they all have limitations. First, most GAL4 lines have been generated by random insertion of a basal GAL4 transgene randomly within the genome (enhancer traps), which makes it difficult to generate additional lines with the same pattern (e.g., LexA versions of the line) and impossible to perform bioinformatic comparisons of the DNA sequences that confer different GAL4 expression patterns. Second, relatively few GAL4 lines are expressed in small subsets of the CNS, which makes it impossible to characterize the morphology and function of the vast majority of neurons.
Here we describe the embryonic CNS expression of 5,000 GAL4 lines made by inserting molecularly defined cis-regulatory DNA into a single attP genomic location using the PhiC31 integrase system (Bischof et al., 2007; Groth et al., 2004). This will facilitate the production of non-GAL4 drivers that can be used in combination with GAL4 lines. It will also make it possible to use bioinformatics to look for cis-regulatory elements that are shared by GAL4 lines with coexpression in one or more neurons. We describe the embryonic CNS expression at stages 9–11 (when neurogenesis is maximal) and stage 16 (when neuronal diversity is maximal). We comprehensively annotate expression patterns in the CNS midline cells, NBs, and neurons at both stages. We note expression in other embryonic tissues, such as the lateral body wall (e.g., muscle, sensory neurons, and trachea) and viscera. Companion papers report on the expression of the same collection of GAL4 lines in the adult brain (see the accompanying paper by Jenett et al., 2012, in this issue of Cell Reports) and imaginal discs (see the accompanying paper by Jory et al., 2012, in this issue of Cell Reports), and all three data sets are publicly available online (http://www.janelia.org/gal4-gen1). The embryonic data derived from this collection of GAL4 lines will be valuable for determining the morphology and function of all neurons in the embryonic CNS, and especially for shedding light on the function of interneurons, which have only begun to be defined although they comprise the majority of all neurons in the CNS.
Results and Discussion
Overview of Embryonic Patterns and Database Features
We stained >5,500 enhancer-gal4 lines generated at Janelia Farm Research Campus (JFRC) to determine their pattern of expression during embryogenesis (Table S1. Annotated Embryonic Expression Patterns of the GAL4 Lines, Related to Figures 2 and 3), and acquired interpretable images for 5,000 lines representing 796 genes. The method used to generate the lines was described previously (Pfeiffer et al., 2008). Each line contains an average of 3 kb cis-regulatory DNA from intergenic and intronic regions of genes known or suspected to be expressed in the adult brain, and each transgene was integrated into the same attP2 site on the second chromosome. We chose embryos at two stages for detailed analysis: germband elongated (GBE; embryonic stages 9–12) and germband shortened (stages 15–17; most embryos were at stage 16). We crossed each line to a line containing UAS-GFP:NLS (green fluorescent protein [GFP] with a nuclear localization signal) and/or cd8:GFP (membrane-tethered GFP), and stained for GFP to determine the expression pattern of each line. We costained each line for the Even-skipped (Eve) transcription factor, which detects a subset of neurons and muscle precursors and anal pad (Frasch et al., 1987); these cells can be used as well-characterized landmarks to help annotate the GAL4 patterns. Our goal was to comprehensively annotate the gene expression patterns in NBs, GMCs, neurons, and glia, including the specialized neurons and glia at the ventral midline. We noted expression outside the CNS but did not attempt to annotate nonneural tissue patterns.
Our data are included in a publicly available database hosted by JFRC (http://www.janelia.org/gal4-gen1; see Figures 1A–1C for screenshots describing how to search the database for embryonic patterns). Additional details regarding the website construction and database functions are described in an accompanying paper (Jenett et al., 2012). The database integrates expression data from embryos (this work) as well as from the adult brain (Jenett et al., 2012) and larval imaginal discs (Jory et al., 2012). Our embryonic data sets include a maximum intensity projection of the most relevant portion of the CNS staining pattern, as well as at least one movie that steps through a z-stack of confocal images to illustrate all aspects of the pattern. One can search the database using selected keywords (see Table 1 for GBE categories, Table 2 for stage 16 categories, and Table 3 for midline categories).
Table 1 |
Expression Pattern | Number of Lines (%) | Number of Genes (%) | ||
Total | 5,000 (100%) | 796 (100%) | ||
Head | 3,806 (76%) | 736 (92%) | ||
NBs, subset | 662 (13%) | 313 (39%) | ||
NBs, most or all | 99 (2%) | 69 (9%) | ||
GMCs/neurons, subset | 1,411 (28%) | 480 (60%) | ||
GMCs/neurons, most or all | 46 (1%) | 36 (5%) | ||
Stripe or column pattern | 224 (4%) | 140 (18%) | ||
Other: body wall | 2,176 (44%) | 577 (72%) | ||
Other: gut/internal | 613 (12%) | 336 (42%) | ||
Scattered | 756 (15%) | 377 (47%) | ||
Poor stain | 385 (8%) | 226 (28%) | ||
Table 2 |
Expression Pattern | Number of Lines (%) | Number of Genes (%) | ||
Total | 5,000 (100%) | 796 (100%) | ||
Head | 3,712 (74%) | 731 (92%) | ||
Neurons, small subset | 1,862 (37%) | 553 (69%) | ||
Neurons, most or all | 822 (16%) | 381 (48%) | ||
Eve medial cells | 661 (13%) | 309 (39%) | ||
Eve lateral cells | 785 (16%) | 366 (46%) | ||
Other: body wall | 2,631 (53%) | 635 (80%) | ||
Other: gut/internal | 1,997 (40%) | 619 (78%) | ||
Scattered | 1,423 (28%) | 520 (65%) | ||
Poor stain | 764 (15%) | 341 (43%) | ||
No expression | 28 (1%) | 27 (3%) | ||
Table 3 |
Expression Pattern | Number of Lines (%) | Number of Genes (%) | ||
Total | 5,000 (100%) | 796 (100%) | ||
Midline expression | 1,285 (26%) | 471 (59%) | ||
HS total | 438 (9%) | 253 (32%) | ||
LS total | 847 (17%) | 387 (49%) | ||
GBE midline expression | 389 (8%) | 244 (31%) | ||
GBE HS all | 24 (1%) | 16 (2%) | ||
GBE HS subset | 113 (2%) | 91 (11%) | ||
St16 midline expression | 1,261 (25%) | 468 (59%) | ||
St16 HS | 435 (9%) | 252 (32%) | ||
St16 HS all | 26 (1%) | 18 (2%) | ||
St16 HS midline glia | 109 (2%) | 83 (10%) | ||
St16 HS midline glia (all) | 83 (2%) | 69 (9%) | ||
St16 HS VUMs | 89 (2%) | 66 (8%) | ||
St16 HS MNB progeny | 26 (1%) | 23 (3%) | ||
St16 HS neuron subset | 331 (7%) | 205 (26%) | ||
St16 HS neuron subset, VUM-MNB | 223 (4%) | 156 (20%) | ||
St16 LS | 826 (17%) | 383 (48%) | ||
Dorsal median cells | 24 (1%) | 20 (3%) | ||
Channel glia | 15 (<1%) | 14 (2%) | ||
We found that nearly all of the 5,000 lines imaged had expression in both young GBE embryos (4,289; 86%) and older stage 16 embryos (4,672; 93%; Figure 1D). This is an underestimate of the GBE expression because we did not image this stage in 410 lines (see Experimental Procedures ); thus, most of the lines contained active cis-regulatory modules (CRMs or enhancers). The most common pattern was head expression (GBE, 76%; stage 16, 74%; Figure 1D), possibly because the genes expressed in the adult brain were preferentially used to generate the GAL4 lines (Jenett et al., 2012). It should be noted, however, that head expression was observed in 59% of the 500 lacZ enhancer trap lines (Bellen et al., 1989), so the head may utilize a large number of CRMs. The next most common patterns were CNS expression (GBE, 33%; stage 16, 57%) and body wall expression (GBE, 44%; stage 16, 53%; Figure 1D); this may be due to the complexity of cell types in these tissues. We saw relatively fewer lines expressed in the midline and viscera, and in a scattered pattern, but for each of these we observed nearly double the number of expressing lines observed in older embryos (Figure 1D), perhaps due to the increasing complexity of these tissues as development proceeds. We observed one expression pattern common to most of the 5,000 lines assayed: a pattern of four neuronal clusters in the stage 16 subesophageal CNS (Figure S1). This gene expression pattern is presumably derived from a DNA sequence shared by all GAL4 constructs (see Discussion ).
GBE Patterns—Embryonic Stages 9–12
At this stage of neurogenesis, the neuroectoderm covers the ventral surface of the embryo, and just internal to these cells is the NB array. Progressively more internal are GMCs and newly born neurons (reviewed in Skeath and Thor, 2003). We used the Eve protein as a fiduciary marker (Figure 2A). Eve is detected in the first-born GMC from NB1-1 and NB4-2, the first five GMCs from NB7-1, and a segment-specific number of GMCs from NB3-3 (five in T1-T2, six in T3, and ten in A1-A7). Each of these GMCs divides to generate an Eve+ neuron and an Eve− sibling (Skeath and Doe, 1998). In addition, Eve is detected in pericardial cells at the dorsal surface of the embryo and the anal pad (Frasch et al., 1987; Figure 2A).
We found 4,320 lines expressed at GBE (Table 1; Figure 1D). We were interested in pan-NB lines, with the rationale that the associated genes may show NB-specific gene expression. We identified NBs based on their relatively large size and position just internal to the ventral neuroectoderm. Despite the lack of a definitive NB marker such as Deadpan or Miranda (Doe, 2008), we feel confident that size and position are sufficient criteria to identify NBs. Approximately 2% (99) of the GBE+ lines were detected in most or all NBs (Figure 2B). We were also interested in identifying GAL4 lines that label subsets of NBs, particularly single NBs that could be used for lineage analysis. More than 15% (662) of the GBE+ lines were detected in a subset of NBs, with some being in just a few NBs per hemisegment (Figures 2C and 2D). Additional experiments will be necessary to map these GAL4+ NBs onto the NB map (Broadus et al., 1995), and this may be best achieved using new semiautomated methods for creating gene expression atlases (Long et al., 2009; Peng et al., 2010; Qu et al., 2011). The lines expressed in subsets of NBs should be useful as NB markers to study the specification of unique NB identity. They could also be used to drive yeast Flippase to excise an FRT-stop-FRT cassette (del Valle Rodríguez et al., 2012) to allow permanent tracing of the NB and its progeny beyond embryonic stages, which would help investigators align embryonic NB identity with larval NB identity (Broadus et al., 1995; Truman and Bate, 1988) and determine the contribution of specific NBs to the adult CNS.
We detected 46 lines expressed in most or all GMCs/neurons (Figure 2E) and 1,411 lines expressed in a subset of GMCs/neurons (Figures 2F–2H). We did not try to distinguish GMCs from neurons, because these populations are intermingled at this stage and have the same cell size. Some of the expression patterns were regionally restricted, e.g., in stripes at different anterior/posterior positions in a segment, in medial or lateral columns along the length of the CNS, or specific to a segment (thoracic only or abdominal only). These are annotated in the database and can be retrieved by checking the stripe/column box (quantified in Table 1). The GAL4 lines with regional expression (stripe or column) may be used to identify flanking genes that are involved in spatial patterning of the CNS, or as markers to study the effect of earlier patterning genes on the specification of regional neuronal identity. They may also be useful for designing split GAL4 transgenes (Luan et al., 2006; Pfeiffer et al., 2010) to restrict GAL4 expression to a very small subset of cells (e.g., using a columnar enhancer to drive the GAL4 DNA-binding domain and the stripe enhancer to drive the GAL4 VP16 transactivation domain). These experiments are straightforward because the enhancer and attP integration sites are defined, and thus new split GAL4 transgenes should have highly predictable expression patterns.
A large fraction of lines with GAL4 patterns showed expression in the head (3,806/4,320; 88%). We did not try to map the identity of these cells to NBs, neurons, glia, or nonneuronal cell types of the head, due to the complexity of the pattern (Figures 2I–2L). A bias toward head expression may reflect the fact that the most cis-regulatory DNA was selected based on its proximity to genes expressed in the adult brain.
We found many lines expressed in nonneural tissues (Figures 2M–2P), but did not comprehensively annotate these patterns. We note that 2,176 lines were expressed in the body wall, which could include trachea, muscles, sensory neurons, histoblasts, or other cell types (e.g., Figures 2M–2O). We also observed expression of 613 lines in the viscera, termed "internal/gut" in the database (Figure 2P). A total of 756 lines showed scattered expression (data not shown; retrieve from the database by checking the "scattered" box). This could reflect stochastic expression of the line within a reproducible pattern of cells, expression in hemocytes that have variable positions due to their migratory nature, or unknown cell types that have a variable pattern.
Stage 16 CNS Patterns
At stage 16, the ventral nerve cord is composed of ∼335 bilateral cells, including ∼35 motoneurons and ∼35 glia (Beckervordersandforth et al., 2008; Landgraf et al., 1999; E.S.H. and C.Q.D., unpublished data). Most of the remaining 260 cells are presumed to be interneurons, but in fact are mostly uncharacterized. A major goal of this study was to identify GAL4 lines that are expressed in subsets of these presumed interneurons to allow further morphological and functional characterization. We detected 4,672 lines expressed at stage 16 (Figure 1D). At stage 16, the CNS shows a segmentally reiterated group of medial Eve+ neurons consisting of the RP2 motoneuron, the aCC/pCC motoneuron/interneuron siblings, and the U1–U5 motoneurons (Skeath and Doe, 1998). In addition, there is a cluster of Eve-lateral (EL) interneurons (Figure 3A). We identified 1,862 lines (37%) that are expressed in small subsets of neurons per hemisegment (<30 neurons per hemisegment, or ∼10% of total cells; see Figures 3B–3E for examples). The more-specific of these lines will be especially useful for characterizing neuronal morphology and function. In contrast, we identified just 822 lines (16%) expressed in most or all neurons in the CNS; some are specific to the CNS and others have additional expression outside the CNS (examples shown in Figures 3F and 3G). Interestingly, previous enhancer-trap experiments showed a similar or lower percentage of lines with all-CNS expression (7/49, 14%; O'Kane and Gehring, 1987) and 42/3768 (1%; Bier et al., 1989), even though each insertion presumably queries a broader cis-regulatory region. It is somewhat surprising how few lines are expressed in a pan-neuronal pattern. This suggests that there may be relatively few CRMs devoted to pan-neuronal expression, and that genes that are widely expressed may utilize multiple, dispersed CRMs to achieve pan-neuronal expression.
In the future, the GAL4 lines expressed in <10 neurons per hemisegment can be used for many experiments. We are currently characterizing about 100 of these lines in more detail, with the goal of mapping each of the GAL4+ neurons into a CNS atlas of gene expression that would allow us to register each GAL4+ neuron to known neurons or each other (E.S. Heckscher et al., unpublished data). The lines can be used to express axon or dendrite markers to determine their morphology, possibly using GFP reconstitution across synaptic partners (Feinberg et al., 2008) to identify candidate synaptic partners. The lines can be used to express Ca2+ sensors to identify neurons with rhythmic activity that matches the periodicity of larval body wall contractions during locomotion. They can be used to express neuronal silencers to screen for behavioral defects, and they can be used to express channelrhodopsin to allow the neurons to be light activated. The pan-neuronal lines, particularly those without additional expression, will also be quite useful. Surprisingly few GAL4 lines are known to be pan-neuronal without additional expression. The most commonly used pan-neuronal GAL4 line is Elav-GAL4, but this line is expressed in NBs that give rise to glia (Berger et al., 2007), and thus is not appropriate for driving neuron-specific expression. We have also observed lines expressed in subsets or all embryonic glia, such as enhancers near the glial cells missing gene (data not shown); some examples of glial patterns are shown in Figures 3H–3J.
Previously, other investigators and members of our laboratory characterized the genetic regulatory network that specifies the Eve+ RP2 motoneuron (McDonald and Doe, 1997; McDonald et al., 2003), and similar work has been done on the Eve+ U1-U5 neurons (Grosskortenhaus et al., 2006; Isshiki et al., 2001; Kohwi et al., 2011; Tran and Doe, 2008; Tran et al., 2010) and EL neurons (Tsuji et al., 2008). Identification of GAL4 lines expressed by Eve+ neurons would be useful for providing tools to manipulate gene expression in these neurons, and the flanking genes may be important for Eve+ neuron development or function. Thus, we searched for lines coexpressed by medial or lateral Eve+ neurons. We found 661 lines expressed in the medial Eve+ neurons, and 785 lines expressed in the lateral Eve+ neurons (ELs; Table 2; some examples of each are shown in Figures 3K–3O). In some cases many other cells express the line, which is not very useful, but in other cases the lines are fairly specifically expressed in the Eve+ neurons (Figures 3M and 3O). These lines should help define the genetic regulatory network that can be used to generate a specific neuron from an identified precursor, because the Eve+ neurons are among the best-characterized neurons in the CNS.
We also detected 3,712 lines expressed in the head (Figuress 1D, 3P, and 3Q), although this number may be artificially high due to the "four clusters of neurons" general background pattern in nearly all lines (Figure S1). Many lines were also expressed in nonneural tissues such as the gut (e.g., Figures 1D and 3U–3W; quantified in Table 2) and body wall (which includes somatic body wall muscles, trachea, sensory neurons, and undefined cell types; Figures 1D, 3X, and 3Y), and in scattered cells that may include hemocytes (e.g., Figures 3R–3T).
CNS Midline Patterns
The ventral nerve cord contains a specialized set of neurons and glia that lie along the midline. By virtue of their appearance as a stripe down the midline of the embryo (Figure 4), it is relatively easy to identify expression of genes or transgenic lines that are midline expressed. Consequently, the midline cells are an attractive system for studying gene regulation, and this provides one rationale for our annotation of the JFRC GAL4 line collection for embryonic midline expression patterns.
There are two developmentally distinct groups of midline cells (Kearney et al., 2004). The cells that are generally considered midline cells are derived from single-minded+ mesectodermal cells and consist of the midline glia, which ensheath the axon commissures, and a diverse set of neurons. The second group is referred to as midline accessory cells. They reside at the midline but are either mesodermal cells that reside internal to the midline (dorsal-median cells) or are glia that arise from lateral neuroglioblasts and migrate to the midline (channel glia and medial-most cell body glia [MM-CBG]). The ∼22 mesectodermal-derived mature midline cells/segment at stage 16 are diverse and consist of (1) approximately three midline glia; (2) two MP1 peptidergic neurosecretory cells; (3) H-cell, a dopaminergic interneuron; (4) H-cell sib, a glutamatergic interneuron; (5) three glutamatergic and octopaminergic motoneuron ventral unpaired median interneurons (mVUMs); (6) three GABAergic interneuron VUMs (iVUMs); and (7) the median NB (MNB) and its approximately eight interneuronal progeny (Wheeler et al., 2006). A previous large-scale in situ hybridization analysis documented midline expression of 286 genes (Kearney et al., 2004); these data are publically available from the Drosophila CNS Midline Gene Expression Database (MidExDB; http://midline.bio.unc.edu/MDB_Home.aspx; Wheeler et al., 2009). A confocal microscopic analysis of ∼100 genes yielded detailed maps of midline cell expression at each embryonic stage, so that each midline cell type can be identified at each stage of development (Wheeler et al., 2006, Wheeler et al., 2008).
Analysis of horizontal views of GBE and stage 16 GAL4 transgenic embryos using anti-Eve staining was particularly useful for identifying lines with midline GFP expression, since medial Eve+ neurons, including aCC, pCC, and RP2 cells, bracket midline-expressing cells (Figures 4A–4J). At the GBE stage, midline cells are just acquiring their fates, and cannot easily be distinguished except by staining with cell-type-specific markers. Consequently, we designate GBE midline-expressing enhancers as expressed in either all midline cells or a subset of midline cells. In contrast, at stage 16 it is often possible to make judgments as to the identity of the midline cell type based on the position along the apical-basal axis and morphology (Wheeler et al., 2006, Wheeler et al., 2008). In this manner, the midline glia, VUM neurons, and progeny of the MNB can often be determined. In contrast, without appropriate markers, it is not possible to identify the other midline neurons. In annotating midline-expressed lines, we chose to liberally assign midline GFP+ cells to a specific midline cell type.
In our analysis, we analyzed 5,000 lines for midline expression corresponding to 796 genes (Table 3; Table S2. Annotated Midline Expression Patterns of the GAL4 Lines, Related to Figure 4). We identified 1,285 lines that had midline expression at either the GBE stage (389 lines) or at stage 16 (1,261 lines). Overall, 26% of all lines examined had midline expression, corresponding to 59% of the genes analyzed. We subdivided the 1,285 lines with midline expression into two classes: high-specificity (HS) lines and low-specificity (LS) lines. The LS lines (847 lines, 66% of total) had midline expression but usually also had expression in a large number of neurons in the lateral CNS. In contrast, the HS lines (438 lines, 34% of total) generally showed strong midline expression and were not generally expressed in large numbers of CNS cells. Nevertheless, many HS lines had expression in other embryonic and CNS cell types. The HS lines corresponded to 253 genes, and 111 of these genes had multiple HS lines with an average of 2.7 lines per gene. Generally, when a gene had multiple lines with the same midline pattern, they were overlapping (and thus likely contained the same CRM). In contrast, when a gene had multiple lines with distinct midline expression patterns, they were in nonoverlapping DNA segments and thus must represent different CRMs. Only rarely were lines with identical patterns of expression in the same gene found in nonoverlapping DNA segments (sometimes referred to as shadow enhancers; Barolo, 2012).
Many of the HS genes are known to be expressed in midline cells. For example, MidExDB currently contains expression data for 286 genes expressed in CNS midline cells. Of the 253 HS genes, 56 are listed in MidExDB. Another 20 HS genes are expressed in midline cells, based on published accounts. Consequently, although a significant fraction of the HS gene enhancers reside within known midline-expressed genes, new midline-expressed genes may be identified based on our discovery of new midline enhancers. However, there are examples of midline enhancers that reside within well-studied genes that do not have corresponding strong midline expression. For example, the similar (Drosophila Hypoxia Inducible Factor ortholog) bHLH-PAS gene, which is ubiquitously expressed (Nambu et al., 1996), has two intronic overlapping midline-expressed lines (R14D11 and R14E09). Consequently, the role of these enhancers in vivo remains unknown. Many of the midline enhancers reside in 5′-flanking regions, 3′-flanking regions, and introns, often within relatively close distance to a midline-expressed gene. However, there are examples of midline enhancers that may act as relatively long distances. For example, the overlapping lines R25F10 and R27A10 reside ∼15 kb upstream of the charlatan gene, which is broadly expressed in the CNS but does not have prominent midline expression (Escudero et al., 2005; Yamasaki et al., 2011). However, the midline-expressed hibris gene is located 74 kb upstream of charlatan (Artero et al., 2001; Dworak et al., 2001), and the enhancer, which is ∼58 kb downstream of hibris, may control hibris midline expression.
The HS midline-expressed lines include a variety of midline patterns that are listed in Table 3, Table S2. Annotated Midline Expression Patterns of the GAL4 Lines, Related to Figure 4, and Figure 4. At the GBE stage, 24 lines were expressed in all midline primordium cells (Figure 4A) and 113 were expressed in subsets (Figures 4B–4E). The subsets included large subsets (Figure 4B) and subsets with as few as a single cell per segment (Figure 4C). In some cases, the subsets of midline primordium cells can be tentatively assigned to midline glia or neural precursors based on the identity of the stage 16 midline cell types. For example, R30C01 (netrin-B) is expressed only in midline glia at stage 16 (Figure 4G) and is found in a subset of midline cells (likely midline glia) in GBE embryos (Figure 4D).
At stage 16, the midline cells can in many cases be distinguished as to midline cell type. In 26 HS lines, expression was present in all midline cells (Figure 4F); these were generally the same lines with GBE all-midline cell expression. Stage 16 expression in all midline cells may reflect perdurance from expression at the earlier midline primordium stages, because few genes, if any, are prominently expressed in all midline cells at stage 16, or this expression could be due to an incomplete CRM lacking a late repression element. Many HS lines were expressed in subsets of midline cells (Figures 4G–4J); in some cases expression was present in a single midline cell type, and in other cases expression was present in multiple midline cell types. We categorized HS lines expressed in specific cell types as midline glia, VUMs, MNB progeny, and small subsets of neurons. The midline glia can generally be identified based on their characteristic dorsal position, number, and elongated nuclear morphology. We annotated 83 HS lines with midline glia expression, excluding lines expressed in all midline cells. In 27 of these 83 lines, the only midline cell type is midline glia (Figure 4G). The VUM neurons can also be identified based on their characteristic ventral position and relatively large nuclei (Figure 4H). In the small number of cases in which we assign a VUM subset to either iVUMs or mVUMs, this is based on the existing knowledge that the corresponding gene is expressed in either iVUMs or mVUMs. For example, Gad1 is expressed in the GABAergic iVUMs (Wheeler et al., 2006), making it likely that line R58G12 whose fragment resides in an intron of Gad1 is expressed in iVUMs (Figure 4H). Eighty-nine HS lines are expressed in the VUM neurons, many in VUM subsets, likely either iVUMs or mVUMs. The MNB progeny tend to form a cluster of small neuronal nuclei (Figure 4I), and were the predominant midline cell type in 26 HS lines. The neural subset category consists of 223 HS lines that express GFP in one or a small number of neuronal nuclei, but could not be assigned to a specific midline neuron without staining with cell-type-specific markers. In those rare cases in which a GFP+ neuron is assigned to a specific type (e.g., H-cell; R29A07), this is because the corresponding gene is known to be expressed in that cell type (pale; Wheeler et al., 2006). The last class of annotated midline lines (which includes HS and LS) corresponds to the midline accessory cells: the dorsal median cells (24 lines), channel glia (15 lines), and MM-CBG (14 lines). The large number of GAL4 lines that correspond to each midline cell type have the potential to be useful for functional studies of midline cell types, as well analysis of enhancer function and the regulation of midline gene expression.
Spatial and Temporal Changes in Gene Expression during Development
We identified >4,000 GAL4 lines with expression in early embryos (GBE) or in late embryos (stage 16), which allowed us to determine whether there are different CRMs for each tissue at each stage of development, or whether individual CRMs give persistent tissue-specific expression over time. We found that the vast majority of GAL4 lines have some embryonic expression in both early and late embryos (85%, with only 3% early only and 12% late only; Figure 5A). Within the CNS there is more stage-specific expression (6% early specific, 45% late specific, and 49% at both stages; Figure 5A). The increased number of lines specifically expressed in the stage 16 CNS is likely due to the greater complexity of cell types present in the CNS at this stage. Similar results were observed for the midline, body wall, and viscera patterns (Figure 5A), reflecting the increased cellular complexity in these tissues. Most tissues had a relatively small number of early-embryo-specific patterns (6% CNS, 2% midline, and 5% viscera; Figure 5A). This suggests that once a CRM is active in young embryos, it often may remain active as the embryo develops. Although we cannot rule out the possibility that the perdurance of GAL4 or the GFP reporter proteins contributes to the observed persistent expression from young to old embryos, we note that some tissues do in fact show a much higher percentage of early-embryo-specific expression (19% body wall and 22% scattered; Figure 5A), which shows that GAL4 and GFP can be turned over during early embryogenesis.
We were also interested in whether the GAL4 lines showed a bias toward any cell type or subset of the CNS. We chose to focus on the Eve+ neurons and the midline neurons, which were the most comprehensively annotated cell types. The Eve+ neurons can be divided into a medial group that is primarily motoneurons (seven motoneurons and one interneuron) and a lateral group that is all interneurons. Interestingly, we found no significant bias in gene expression between these two groups: the lines were nearly equally divided among medial-specific, lateral-specific, and both (Figure 5B). This shows that the CRMs in this study appear equally likely to be expressed in the medial and lateral regions of the CNS, and within motoneurons and interneurons—with the caveat that Eve represents only a subset of motoneuron and interneurons in the CNS. Analysis of midline and lateral CNS expression gave a different result: 58% of the lines were expressed in the lateral CNS only, 37% were expressed in both the midline and lateral CNS, and only 5% were expressed in the midline only (Figure 5B). This suggests that the lateral CNS has more gene regulatory complexity than the midline, a result that is consistent with the greater number of unique cell types in the lateral CNS (Bossing et al., 1996; Schmid et al., 1999; Schmidt et al., 1997; Wheeler et al., 2009).
Conclusions and Future Directions
We have characterized the embryonic expression of 5,000 GAL4 lines, and provided the images and cis-regulatory DNA sequence information in a public website. All GAL4 lines were made with molecularly defined cis-regulatory DNA inserted into a single attP site in the genome, and thus the observed patterns can be replicated with the use of other gene products. For example, we have already used the R9D11 cis-regulatory DNA to make a direct fusion to red fluorescent protein, to be used as a marker in combination with other GAL4/UAS transgenes (O. Bayraktar and C.Q.D., unpublished data). Other applications could use cis-regulatory DNA to drive LexA or FLPase, which could then be used in combination with another GAL4/UAS transgene or to express split GAL4 components. The collection of GAL4 lines will allow investigators to target many different NB lineages or neuronal subsets using misexpression or UAS-RNAi screens. As mentioned above, perhaps the most powerful use of these lines will be in neural circuit analysis: lines with GAL4 expression in fewer than ten neurons per hemisegment may be further refined by using smaller fragments of cis-regulatory DNA or a split GAL4 methodology, allowing single neurons to be targeted. Combining GAL4 lines with highly restricted expression patterns with permanent-labeling transgenes will be a powerful method for linking embryonic neurons to their adult morphology and function. Lastly, it should be possible to perform bioinformatic analysis on the cis-regulatory DNA from lines with overlapping expression patterns to identify motifs that may drive cell-specific gene expression, as has been done for larval imaginal disc CRMs (Jory et al., 2012).
Experimental Procedures
Generation of Fragment Enhancer-GAL4 Lines
Details regarding the design and manufacture of the enhancer-GAL4 constructs, transformant fly stocks, and all related protocols were previously described (Pfeiffer et al., 2008).
Drosophila Genetics
Males from each GAL4-expressing line were crossed to virgin females of y,w; UAS-GFP::lacZ.nls (Bloomington Indiana stock #6452), which produces nuclear localized GFP. In a few cases, the male GAL4 line was crossed to virgin females of y,w; UAS-mCD8::GFP, w+ (Bloomington Indiana stock #5137) that produces membrane-bound GFP.
Antibody Staining and Image Analysis
We performed 24 hr embryo collections at room temperature (22.5°C) on apple agar caps with yeast paste. Standard methods were used to fix the embryos (Odden et al., 2002), which were stored in 100% ethanol at −20°C until antibody staining was performed. Embryos were incubated in primary antibody for 1.5 hr at room temperature (22.5°C), rinsed in phosphate-buffered saline (PBS) with 0.1% Triton X-100, 3% bovine serum albumin, and 10 mM glycine (PTBG) for 60 min. They were then incubated in secondary antibody for 1.5 hr at room temperature (22.5°C), rinsed twice for 10 min in PTBG, followed by a single 15 min rinse in 10% glycerol in PBS on a rocker. Embryos were dehydrated by passing through a 25%, 50%, 90% glycerol series and stored overnight in mounting media (90% glycerol with 2% N-propyl gallate). We used the following primary and secondary antibodies: chicken anti-GFP (1:1000; Aves, Tigard OR), mouse anti-Eve 2B10 concentrate with a working concentration of 4 μg/ml, preincubated on fixed wild-type embryos prior to use (Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development [NICHD] and maintained by the Department of Biology, University of Iowa [Iowa City, IA]), donkey anti-chicken DyLight 488, and donkey anti-mouse DyLight 549 (1:400; Jackson Immunochemicals, West Grove PA). Reduction of the rinsing time dramatically increased the signal of Eve antibody. Embryos were imaged on a Bio-Rad Radiance 2100MP or a Zeiss 700 confocal microscope. We did not determine the gender of the imaged embryos. ImageJ was used to produce QuickTime movies and TIFF projections.
Data Collection
We collected z-stack images of the stage 16 ventral nerve cord in the T2-A3 segment region. For each line, our goal was to customize the region imaged to the extent of the staining. We also imaged GBE embryos (stages 9–12), typically imaging the entire embryo. We did not image GBE stages for 410 lines due to the lack of appropriately staged embryos or poor staining quality. Most of the image stacks were collected at 20× + 1.5 zoom at 1.0 μm z-steps, with the number of z-steps depending on the extent of the staining. Eve protein was a marker for the location and general dimensions of the CNS, and allowed accurate orientation and staging of the embryos.
We thank R. Mann for comments on the manuscript, Tallon Lamoreaux for assistance with fly stocks, and Janet Hanawalt for administrative support. This work was supported by an NRSA postdoctoral award to J.C.P. (NICHD), NIH grants R01 NS64264 (NINDS) and R37 RD25251 (NICHD) to S.T.C., and NIH grant HD27056 and support from the Howard Hughes Medical Institute to C.Q.D.
Supplemental Information
Web Resources
The URLs for data presented herein are as follows:
FlyLight Image Database, http://ww.janelia.org/gal4-gen1
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Publication Information
Received: August 15, 2012
Revised: September 11, 2012
Accepted: September 17, 2012
Published online: October 11, 2012


text:
Results 6 to 10 of 26
Thread: twin girls!
April 23rd, 2013 08:37 AM #6Little Bean arriving September 2014
Zoe | Noemí Esther
Lucas Emmanuel | Levi Alexander
Adele | Adira | Arabella | Aurora | Eliana | Elizabeth | Milena | Susannah | Zara
April 23rd, 2013 08:42 AM #8Senior Member
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- Jul 2012
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- London, England
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April 23rd, 2013 08:47 AM #10Junior Member
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Ok I'm confused, are you having twins or not? Is this baby a time traveler?
My Cupcake: Daisha
Family Pets: Azula and Batgirl
Sherlock, Doctor Who, Jane Austen, Merlin, Luther, (I was born in the wrong country...)
Ladies: Alice, Daisha, Lila/Lilia, Danielle, Zella, Freya, Romana, Victoria, Madge, Persephone, Elisabeth, River, Sarah-Jane, Amelia-Rose, Maeve, Daphne
Gents: Dashiell, Daniel, Zane, Matthew, David, Rhys, Oliver, Henry, Theodore, Felix, Bowen, Derek, Apollo, Baker, Zephyr, Rylan, Morgan, Callen, Robert, Rupert
April 23rd, 2013 10:09 AM #12
Yeah, you mentioned earlier you already knew you were carrying one daughter-- which means you had a Level One sono which examined the baby closely and diagnosed its gender. It is simply not possible to have a long, detailed ultrasound and miss a second baby.
Blade, MD
XY: Antoine Raphael (3.1.2012)
XX: Cassia Viviane Noor (11.30.2013)
April 23rd, 2013 10:19 AM #14Senior Member
- Join Date
- Nov 2009
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Wow! that would be a huge surprise, and highly rare!! How many weeks were you when your first sonogram was done???
~ "How can there be too many children? That is like saying there are too many flowers."~ Mother Teresa

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Captain Underpants #02: Captain Underpants and the Attack of the Talking Toiletsby Dav Pilkey
Synopses & ReviewsPublisher Comments:The second book in Dav Pilkey's mega-bestselling Captain Underpants series. Now with super-cool foil covers! Shiny! Fun! George and Harold are usually responsible kids...whenever anything bad happens, George and Harold are usually responsible! And it looks like they're up to their old tricks again! First, they ruined the school's science fair with a series of silly pranks. Then, they accidentally created an army of evil, vicious, talking toilets, intent on taking over the world. Who will stop the carnivorous commodes?!!!? This looks like a job for Captain Underpants! Review:Harold and George run into trouble when they use the school brain's science project to print copies of their latest Captain Underpants adventure — they accidentally unleash an invasion of Talking Toilets! The boys have to hypnotize Principal Krupp again so that, as Captain Underpants, he can battle the menace. But will it be enough?
Review:"The combination of total departure from reality, smirky insider humor, and crucial taboos as plot points will draw middle-graders like magnets....A tome for the ages this ain't, but it'll make kids laugh until soda comes out of their noses." Bulletin for the Center of Children's Books
Review:"[A] worthy sequel to The Adventures of Captain Underpants; Pilkey maintains the original's satiric, self-referential formula....Bart Simpson could learn a few things from the subversively hilarious Harold and George." Publishers Weekly
Review:"The fun is in the reading, which is full of puns, rhymes, and nonsense along with enough revenge and wish fulfillment for every downtrodden fun-seeking kid who never wanted to read a book." School Library Journal
Synopsis:George and Harold are up to their usual tricks. Last time they hypnotized Principal Krupp into believing he was Captain Underpants. Now, they bring to life the most disgusting monster — the terrible Turbo Toilet 2000. Illustrations.
Synopsis:Now with super-cool foil covers! Shiny! Fun! About the AuthorDav Pilkey has written and illustrated numerous popular, award-winning books for children, including the Captain Underpants and Dumb Bunnies series; DOG BREATH, winner of the California Young Reader Medal; and THE PAPERBOY, a Caldecott Honor Book. He lives with his wife in the Pacific Northwest. Visit him online at www.pilkey.com. What Our Readers Are SayingBe the first to add a comment for a chance to win!Product Details
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Monday, November 26, 2007
Monday Morning Update
Hey everyone! I don't really have a lot today. I did post my thoughts on Torii Hunter signing with the Los Angeles
Angels of Anaheim on Friday, so be sure to check that out. Here are just a couple of quick bullet points.
I do have a couple of postings ready for the next couple of days, so be sure to
come back!
Bob Nightengale is
right up there, in my opinion, with Ken Rosenthal in terms of having great
inside baseball information. However, he has an
incredible article about the last couple of weeks in the life of Torii Hunter.
It is interesting to read the thoughts of some of the people directly involved
in his negotiations with other teams and some of the people that tried to
convince Torii to play for their team.
Of course, everyone has an opinion on the Torii
Hunter signing. I hate to pick a couple out, but I would just encourage you to
go to the Twins blogs on the left side of this screen for plenty of opinion on
the subject.
Pat Neshek is down in
Florida preparing for the 2008 season. He has also been able to write on his blog a
bit more. First, he had another Game Used Auction. But he also recently signed
1,000 certified cards for Topps that will be inserted into 2008 packs (unsure
which set). It was incredibly interesting to read how it all worked and get his
thoughts on it. It's so fun to read this kind of stuff from someone who truly
cares about fans and collectors!
Over at John Sickels site, there is an interesting diary on
potential trade partners for Johan Santana.
At www.mlbtraderumors.com, there is an interesting
on the rumored Matt Garza for Delmon Young
discussions. The Comments are very interesting.
Thanks to Blogonoscopy, but this is hilarious!
The Vikings
Defense had quite a day against the NY Giants. I was surprised by how dominant
the team was all-around, even the offense. Tavaris
Jackson was good. Chester Taylor was at least solid. The defense returned three
interceptions for touchdowns which is amazing. They are 5-6 and the schedule is
pretty weak the next three weeks (although I have to believe that the Lions are
better than they have played lately!). Could this team really still be in
playoff contention? Probably not, but it would be cool!
Devon Hester. He's
Kurt Warner – you're
in the end zone, you don't have all day to find a
AJ Feeley may have made himself a little money!
That is it for today. If you are hearing any Twins rumors, please be sure to
let me know. Send me an e-mail on such things, or if you have ideas for future
blog postings. Have a great week!
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Why We Fight
This article needs additional citations for verification. (November 2012) |
Why We Fight is a series of seven documentary films commissioned by the United States government during World War II whose purpose was to show American soldiers the reason for U.S. involvement in the war. Later on they were also shown to the general U.S. public to persuade them to support American involvement in the war.
Most of the films were directed by Frank Capra, who was daunted yet also impressed and challenged by Leni Riefenstahl's propaganda film Triumph of the Will and worked in direct response to it. The series faced a tough challenge: convincing a recently non-interventionist nation of the need to become involved in the war and ally with the Soviets, among other things. In many of the films, Capra and other directors spliced in Axis powers propaganda footage going back twenty years, and re-contextualized it so it promoted the cause of the Allies.
Why We Fight was edited primarily by William Hornbeck, although some parts were re-enacted "under War Department supervision" if there was no relevant footage available. The animated portions of the films were produced by the Disney studios – with the animated maps following a convention of depicting Axis-occupied territory in black.
Purpose of films as war information[edit]
Director Frank Capra enlisted shortly after the Japanese attack on Pearl Harbor. He was assigned to work directly under Chief of Staff George C. Marshall, the most senior officer in command of the Army, who would later create the Marshall Plan and be awarded a Nobel Peace Prize. Marshall felt that Signal Corps was incapable of producing "sensitive and objective troop information films." One colonel explained the importance of these future films to Capra:
You were the answer to the General's prayer. . . You see, Frank, this idea about films to explain "Why" the boys are in uniform is General Marshall's own baby, and he wants the nursery right next to his Chief of Staff's office.[1]
During his first meeting with General Marshall, Capra was given his mission:
Now, Capra, I want to nail down with you a plan to make a series of documented, factual-information films—the first in our history—that will explain to our boys in the Army why we are fighting, and the principles for which we are fighting... You have an opportunity to contribute enormously to your country and the cause of freedom. Are you aware of that, sir?[2]
Countering enemy propaganda films[edit]
Shortly after his meeting with General Marshall, Capra viewed Leni Riefenstahl's "terrifying motion picture," Triumph of the Will. Capra describes the film as "the ominous prelude of Hitler's holocaust of hate. Satan couldn't have devised a more blood-chilling super-spectacle. . . . Though panoplied with all the pomp and mystical trappings of a Wagnerian opera, its message was as blunt and brutal as a lead pipe: We, the Herrenvolk, are the new invincible gods!"[3]
According to Capra, Triumph of the Will "fired no gun, dropped no bombs. But as a psychological weapon aimed at destroying the will to resist, it was just as lethal." Capra at this point had no assistants or facilities, and he began to see his assignment as overwhelming:
I sat alone and pondered. How could I mount a counterattack against Triumph of the Will; keep alive our will to resist the master race? I was alone; no studio, no equipment, no personnel.[3]
Creating a powerful film idea[edit]
Capra made his primary focus the creation of "one basic, powerful idea" that would spread and evolve into other related ideas. Capra considered one important idea that had always been in his thoughts:
I thought of the Bible. There was one sentence in it that always gave me goose pimples: "Ye shall know the truth, and the truth shall make you free."[3]
As a result, his goal became to "let the enemy prove to our soldiers the enormity of his cause—and the justness of ours." He would compile enemy speeches, films, newsreels, newspaper articles, with a list of the enemy's hostile actions. He presented his ideas to other officers now assigned to help him:
I told them of my hunch: Use the enemy's own films to expose their enslaving ends. Let our boys hear the Nazis and the Japs shout their own claims of master-race crud—and our fighting men will know why they are in uniform.[3]
Weeks later, after major efforts and disappointments, Capra located hard-to-reach archives within government facilities, and by avoiding normal channels was able to gain access:
Peterson and I walked away on air. We had found the great cache of enemy films—and it was ours![3]
Description of films[edit]
- Prelude to War (1942) (51:35) (Academy award as Documentary Feature) – this examines the difference between democratic and fascist states, and covers the Japanese conquest of Manchuria and the Italian conquest of Ethiopia.[4] Capra describes it as "presenting a general picture of two worlds; the slave and the free, and the rise of totalitarian militarism from Japan's conquest of Manchuria to Mussolini's conquest of Ethiopia."[3]
- The Nazis Strike (1943) (40:20) – covers Nazi geopolitics and the conquest of Austria, Czechoslovakia and Poland.[5] Capra's description: "Hitler rises. Imposes Nazi dictatorship on Germany. Goose-steps into Rhineland and Austria. Threatens war unless given Czechoslovakia. Appeasers oblige. Hitler invades Poland. Curtain rises on the tragedy of the century—World War II."[3]
- Divide and Conquer (1943) (56:00) [6] – about the campaign in Benelux and the Fall of France. Capra's description: "Hitler occupies Denmark and Norway, outflanks Maginot Line, drives British Army into North Sea, forces surrender of France."[3]
- The Battle of Britain (1943) (51:30) [7] – depicts Britain's victory against the Luftwaffe. Capra's synopsis: "Showing the gallant and victorious defense of Britain by Royal Air Force, at a time when shattered but unbeaten British were only people fighting Nazis."[3]
- The Battle of Russia (1943) (76:07) Part I [8] and Part II [9] – shows a history of Russian defense and Russia's battle against Germany. Capra's synopsis: "History of Russia; people, size, resources, wars. Death struggle against Nazi armies at gates of Moscow and Leningrad. At Stalingrad, Nazis are put through meat grinder."[3]
- The Battle of China (1944) (62:16) [10] – shows Japanese aggression such as the Nanking Massacre and Chinese efforts such as the construction of the Burma Road and the Battle of Changsha. Capra's synopsis: "Japan's warlords commit total effort to conquest of China. Once conquered, Japan would use China's manpower for the conquest of all Asia."[3]
- War Comes to America (1945) (64:20) [11] – shows how the pattern of Axis aggression turned the American people against isolationism. Capra's synopsis: "Dealt with who, what, where, why, and how we came to be the U.S.A.—the oldest major democratic republic still living under its original constitution. But the heart of the film dealt with the depth and variety of emotions with which Americans reacted to the traumatic events in Europe and Asia. How our convictions slowly changed from total non-involvement to total commitment as we realized that loss of freedom anywhere increased the danger to our own freedom. This last film of the series was, and still is, one of the most graphic visual histories of the United States ever made."[3]
Production details[edit]
The films were directed by Academy Award-winning director Frank Capra and narrated by Academy Award winning actor Walter Huston. This narration, though factual for the most part, is replete with rhetoric describing implacably warlike German and Japanese armed forces. Conversely, it lionizes the courage and sacrifice of the British, Soviets, and Chinese.
At the end of each film, the quotation from Army Chief of Staff George Marshall that "...the victory of the democracies can only be complete with the utter defeat of the war machines of Germany and Japan," is shown on screen, followed by a ringing Liberty Bell over which is superimposed a large letter "V" zooming into the screen, accompanied by patriotic or military music on the soundtrack.
Why We Fight also contains many scenes from Triumph of the Will when talking about the Nazis.
Made from 1942 to 1945, the seven films range from 40 to 76 minutes in length, and all are available on DVD or online at no cost, since they have always been public domain films produced by the U.S. government.
Film as medium of choice to present war information[edit]
After World War I the methods used to gain support from troops and civilians needed to change. Giving speeches to both soldier recruits and the American public was no longer effective. Film became the medium of choice to inform American soldiers and recruits about why fighting was necessary.[12] As Kathleen German states, "this was the first massive attempt to influence opinion in the U.S. military" through film.[13] Film was also chosen because it combined the senses of sight and hearing, giving it an advantage over radio or print.[14] Capra, who had no experience in documentary films, was chosen because "of his commitment to American ideals" and because of the popularity of some of his earlier feature films. He was thought "to understand the heart and soul of American audiences."[12] Once the documentary series was completed, it was said to contain the "Capra touch."[15]
The series' appeal was furthered by how the film was edited. "Throughout his career Capra depended upon his skill as an editor to achieve the contrast of the individual and the group, critical in the success of his Hollywood movies."[16] Capra thought it would be most effective to use the enemy's original film and propaganda in the series in order to expose the enemies with their own images. By taking pieces of the enemy material to edit together and placing his own narration over the results, Capra gave meaning and purpose to the war with added narrative.[17] This "parallel editing"[18] created an "us vs. them" image by re-framing and showing clips out of their original order and context.
By such careful editing, the films compared and contrasted the forces of evil with America and its traditional values. Capra highlighted the differences between America and the enemy and showed how the enemy would attack these values if "we" did not fight.[19] This worked to create a battle not only between Allies and the Axis Powers but between good and evil. Capra treated it as a matter of showing the enormity of the Axis cause and the justness of the Allied.[20] In order to justify the Western Allies' alliance with the Soviet Union, the series omitted many facts, which could have cast doubts on the "good guy" status of the Soviets, such as the Nazi-Soviet alliance, Soviet invasion of Poland; Soviet occupation of the Baltic States, Winter War and others.[21] Virtually in line with Soviet propaganda, the series was not only screened but widely acclaimed in the Soviet Union.[22] To exonerate the Soviets, the series casts even less important Allies, like the Poles, in the bad light, even repeating Nazi propaganda claims such as the Tuchola Forest myth or false claims about the Polish Air Force being destroyed on the ground.[21][22]
The Why We Fight series became a heavily used means of presenting information about Axis powers for the American government during World War II. General Surles, the director of the Department of War's Bureau of Public Relations, had hoped that the series would be effective enough to allow similar kinds of army films to be shown to the general public.[23] Surles saw this goal realized when Prelude to War, the most successful of the seven films, was shown to President Franklin D. Roosevelt. Roosevelt saw this film as so important that he ordered it to be distributed in civilian arenas for public viewing.[17] However, some objections were raised against the Why We Fight series because it was so persuasive. Lowell Mellett, the coordinator of government films and aide to Roosevelt, saw the films as dangerous. He was most concerned with the effect the series would have after the war was over and the "hysteria" the films would create in their wake.[23] At least 54 million Americans had seen the series by the end of the war, and studies were done to gauge the impact of the films. However, the results were inconclusive, and therefore the effectiveness of the series is still in question.[24]
Post war[edit]
Prelude to War and The Battle of China refer several times to the Tanaka Memorial, portraying it as "Japan's Mein Kampf" to raise American morale for a protracted war against Japan. The authenticity of this document remains a topic of historical debate. Even though its authenticity has been called into question by some today, the Tanaka Memorial was widely accepted as authentic in the 1930s and 40s because Japan's actions corresponded so closely to these plans.
In 2000 the United States Library of Congress deemed the films "culturally significant" and selected them for preservation in the National Film Registry. Created by the U.S. Army Pictorial Services, the films are in the public domain; all of them are available for download at the Internet Archive.
The Battle of Russia, part 1
The Battle of Russia, part 2
See also[edit]
- Propaganda in the United States
- War film
- List of films in the public domain in the United States
- Frank Capra subsection "Why We Fight series" for additional details
- ^ Capra 1971, p. 322.
- ^ Capra 1971, p. 326.
- ^ a b c d e f g h i j k l Capra, Frank. The Name Above the Title: An Autobiography. New York: The Macmillan Company, (1971) pp. 325–343 ISBN 0-306-80771-8
- ^ "Why We Fight: Prelude to War : Frank Capra". Archive.org. 1942. Retrieved 2014-03-16.
- ^ "Why We Fight: The Nazi Strike : Frank Capra". Archive.org. 1943. Retrieved 2014-03-16.
- ^ "Why We Fight: Divide and Conquer : Frank Capra". Archive.org. 1943. Retrieved 2014-03-16.
- ^ "Why We Fight: The Battle of Britain : Frank Capra". Archive.org. 1943. Retrieved 2014-03-16.
- ^ "Why We Fight: The Battle of Russia Part I : Frank Capra". Archive.org. 1943. Retrieved 2014-03-16.
- ^ "Why We Fight: The Battle of Russia Part II : Frank Capra". Archive.org. 1943. Retrieved 2014-03-16.
- ^ "Why We Fight: The Battle of China : Frank Capra". Archive.org. 1944. Retrieved 2014-03-16.
- ^ "Why We Fight: War Comes to America : Frank Capra". Archive.org. 1945. Retrieved 2014-03-16.
- ^ a b Combs and Combs, 1994, p. 69.
- ^ German, 1990, p. 237.
- ^ German, 1990, p. 238.
- ^ Koppes and Black, 1987, p. 76.
- ^ German, 1990, p. 240.
- ^ a b Combs and Combs, 1994, p. 70.
- ^ German, 1990, p. 241.
- ^ German, 1990.
- ^ Richard Overy, Why the Allies Won, p295 ISBN 0-393-03925-0
- ^ a b Mieczysław B. Biskupski (January 2010). Hollywood's war with Poland, 1939–1945. University Press of Kentucky. pp. 148–150. ISBN 978-0-8131-2559-6. Retrieved 4 March 2011.
- ^ a b Mieczysław B. Biskupski (January 2010). Hollywood's war with Poland, 1939–1945. University Press of Kentucky. pp. 151–152. ISBN 978-0-8131-2559-6. Retrieved 4 March 2011.
- ^ a b Koppes and Black, 1987, p. 122.
- ^ Rollins, 1996, p. 81.
- Combs, James, and Sara Combs. Film Propaganda and American Politics: An Analysis and Filmography. New York: Garland Publishing, Inc., 1994.
- German, Kathleen. "Frank Capra's Why We Fight Series and the American Audience." Western Journal of Speech Communication 54. (1990): 237–248.
- Koppes, Clayton, and Gregory Black. Hollywood Goes to War: How Politics, Profits, and Propaganda Shaped World War II Movies. New York: The Free Press, 1987.
- Rollins, Peter. "Frank Copra's Why We Fight Film Series and Our American Dream." Journal of American Culture. 19. (4): 81, 6.
- Shindler, Colin. Hollywood Goes to War: Films and American Society 1939–1952. Boston: Routledge and Kegan Paul, 1979.
External links[edit]
Wikimedia Commons has media related to Why We Fight. |
- Why we fight: Prelude to War at the Internet Movie Database
- Why we fight: Prelude to War is available for free download at the Internet Archive [more]
- Why we fight: The Nazis Strike at the Internet Movie Database
- Why we fight: The Nazis Strike is available for free download at the Internet Archive [more]
- Why we fight: Divide and Conquer at the Internet Movie Database
- Why we fight: Divide and Conquer is available for free download at the Internet Archive [more]
- Why we fight: The Battle of Britain at the Internet Movie Database
- Why we fight: The Battle of Britain is available for free download at the Internet Archive [more]
- Why we fight: The Battle of Russia at the Internet Movie Database
- Why we fight: The Battle of Russia (Part 1) is available for free download at the Internet Archive [more]
- Why we fight: The Battle of Russia (Part 2) is available for free download at the Internet Archive [more]
- Why we fight: The Battle of China at the Internet Movie Database
- Why we fight: The Battle of China is available for free download at the Internet Archive [more]
- Why we fight: War Comes to America at the Internet Movie Database
- Why we fight: War Comes to America is available for free download at the Internet Archive [more]
- USAAF First Motion Picture Unit at Magic Lantern
- American World War II propaganda films
- United States National Film Registry films
- Documentary films about World War II
- World War II films made in wartime
- American documentary films
- Black-and-white films
- 1940s war films
- 1940s documentary films
- Film series
- Films directed by Frank Capra
- Why We Fight
- Public domain films


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