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The developmental expression dynamics of Drosophila melanogastertranscription factors
- Boris Adryan1, 2Email author and
- Sarah A Teichmann1Email author
DOI: 10.1186/gb-2010-11-4-r40
© Adryan and Teichmann; licensee BioMed Central Ltd. 2010
Received: 23 November 2009
Accepted: 12 April 2010
Published: 12 April 2010
Site-specific transcription factors (TFs) are coordinators of developmental and physiological gene expression programs. Their binding to cis-regulatory modules of target genes mediates the precise cell- and context-specific activation and repression of genes. The expression of TFs should therefore reflect the core expression program of each cell.
We studied the expression dynamics of about 750 TFs using the available genomics resources in Drosophila melanogaster. We find that 95% of these TFs are expressed at some point during embryonic development, with a peak roughly between 10 and 12 hours after egg laying, the core stages of organogenesis. We address the differential utilization of DNA-binding domains in different developmental programs systematically in a spatio-temporal context, and show that the zinc finger class of TFs is predominantly early expressed, while Homeobox TFs exhibit later expression in embryogenesis.
Previous work, dissecting cis-regulatory modules during Drosophila development, suggests that TFs are deployed in groups acting in a cooperative manner. In contrast, we find that there is rapid exchange of co-expressed partners amongst the fly TFs, at rates similar to the genome-wide dynamics of co-expression clusters. This suggests there may also be a high level of combinatorial complexity of TFs at cis-regulatory modules.
In the post-genomic era, transcriptional regulatory relationships are often displayed in the form of a gene regulatory network (GRN), in which the nodes represent genes and the edges are the regulatory interaction of 'activation' or 'repression'. Developmental geneticists turned to 'network biology' decades ago, because the complex relationships between genes can often best be summarized in a wiring diagram. A good example is the early patterning of the Drosophila embryo, a relatively well understood process involving the concerted action of about ten or so transcription factors (TFs). The anterior-posterior axis of the fly embryo is initially defined by a maternally deployed protein gradient of the Bicoid (Bcd) TF. Regionalized gap gene expression domains establish a genetic circuitry that defines pair rule and segment polarity gene expression (see [1] for review). This GRN model allows one to make inferences about the behavior of the network upon perturbation (for example, in a logical analysis [2] or in an experimental approach [3]). These works clearly show the added value of a systems approach. Note that signal transduction cascades are not disregarded in these models; they provide the connections between otherwise separate GRNs. However, within specific developmental lineages, the body plans of animals primarily underlie the concerted action of TFs within GRNs (see [4, 5] for review).
GRNs are hierarchical structures that can be dissected into functional modules and network motifs (see [6] for review). Single input motifs in which a target gene receives regulatory input from only one TF are commonly seen in unicellular organisms [7]. Most genes involved in metazoan development, however, require more complex modes of regulation: multiple TFs contribute to the expression state of the target gene. At the heart of this regulation lie the cis-regulatory modules, genomic clusters of specific binding sites for TFs (see [8] for review). The availability of TFs to bind to those cis-regulatory modules is fine-tuned and plays an important role in the precise regulation of the genes. One of the most intriguing examples for the modulation of developmental expression is the 'stripe 2' enhancer of the gene even skipped (eve) in Drosophila. This pair rule gene is expressed in seven stripes along the anterior-posterior axis of the early embryo. Individual stripes or combinations of stripes are under the control of individual enhancers. The possibly best-characterized enhancer drives the expression of the second stripe [9, 10]. This enhancer contains activating binding sites for the TFs Bcd and Hunchback (Hb), and repressing binding sites for Giant (Gt) and Krüppel (Kr). At one, and only one position within the fly embryo (a domain of only a few nuclei in width), the gradients of the maternal TFs and the regionalized gap genes establish an environment under which the 'stripe 2' enhancer can drive expression of eve.
The microenvironment under which gene expression through a cis-regulatory module is facilitated is thus primarily, but not exclusively, influenced by the qualitative and quantitative presence of binding TFs [11]. The structures of the factors themselves provide additional means to modulate network function [12]. Some TF classes such as the zinc finger TFs recognize their target sequences as monomers, while other classes often require binding as homo- or heterodimers, at foremost the developmentally important helix-loop-helix (HLH) TFs [13]. This has direct implications for the minimal TF configuration necessary to drive transcription. Together, the availability of a TF in a given biological context as well as the combination of the TFs modulate the biological outcome.
For the life cycle of unicellular organisms such as yeast, a large proportion of the TFs and their regulatory interconnections have been identified [14]. Even the dynamics of the entire GRN under varying physiological conditions has been elucidated [15, 16]. Metazoan development is clearly more complex, and although there has been progress in the description of some developmental GRNs at a systems level in model organisms [17–19] and many individual studies, we lack fundamental information about the number, structure and functions of their regulatory networks. For most organisms we even lack reliable details about the TFs contributing to the development of tissues and organ systems. And for those organisms and tissues where we have this information, we at most know a few of their interconnections.
As the first step in the analysis of GRNs, Bolouri and Davidson [20] propose a strategy to obtain 'a parts list' and then 'map how these parts fit together'. We have recently undertaken an investigation to create such a parts list and have defined the repertoire of site-specific TFs in Drosophila melanogaster, work that resulted in the FlyTF database [21]. Experimental studies have determined the genome-wide binding of some of these TFs (see, for example, [22]). A comprehensive screen aiming to determine the binding of the majority of factors is underway as part of the modENCODE projects [23]. We have started a computational analysis of currently available genomics data in Drosophila in order to shed light on developmental TFs and their role in GRNs.
Here, we present our results to integrate the repertoire of D. melanogaster TFs with information about their expression. We determined that approximately 95% of the roughly 750 TFs are utilized in a developmental context. For those TFs where spatio-temporal gene expression information is available, we investigated which tissues showed the highest degree of TF involvement. Interestingly, we found that the embryonic nervous system does not only express more genes than other body parts, but also expresses a high percentage of TFs compared to other body parts. There is considerable overlap between the TF expression repertoires of tissues, such that only a small fraction of TFs exhibits real tissue-specificity. Interestingly, tissue-specificity is not strictly conserved between embryonic and adult tissues, though we do observe nervous system-specific expression of TFs conserved between the two stages. For the first time in a metazoan organism, we show the degree of TF exchange between developmental co-expression clusters, which we interpret as an approximation of their mode of collaboration in different tissues.
Results and discussion
DNA-binding domain families in the Drosophilatranscription factor repertoire
Our previous work using computational predictions and a large-scale literature curation identified 753 site-specific TFs in D. melanogaster [21]. Most can uniquely be mapped to 731 genes (97% of the repertoire), whereas 22 are only assigned to a transcription-related or DNA-binding 'activity' that has not been assigned to any gene. An example for the latter group is the Glue enhancer binding factor-I (FBgn0013970) [24].
Fifty different types of DNA-binding domains (DBDs) are encoded in the fly genome. Our previous work established that only 14 types of DBDs are present in more than 5 TFs (Additional file 1). The zinc finger DBD of the C2H2 type is the most abundant domain and can be found in 249 TFs, which is also the case in most other eukaryotic genomes [25, 26]. The Homeobox is the second most common DBD with 99 counts. Other frequently used DBDs are the helix-loop-helix (55 counts), zf-C4 type zinc finger (22 counts), the BESS (20 counts) and the Forkhead domains (19 counts), and 19 TFs with various bZIP-like leucine zipper DBDs. In contrast, there are single copy DBDs in well-established TFs that occur only once or twice in the Drosophila genome - for example, the SRF (Mef2, Blistered), the Stat (Stat92E) and the Prox1 (Prospero) domains.
A compilation of all raw data used in this study is available as 'mini-website' in Additional file 2.
Almost all transcription factors are expressed in embryonic development
Assuming that gene expression implies the activity of the encoded protein, we first assessed TF activity in development and physiology. Independent of the experimental strategy used for data acquisition, the proportion of TFs active at some stage during embryonic development is high. Active transcription [27] overlapping with at least part of the gene can be observed for 95% of TFs (694 of 731 TFs on the array). In the developmental time-course experiment published by Hooper et al. [28], 98% of TFs (667 of the 679 TFs present on the array) show either average or high expression during embryogenesis. This is in keeping with the results obtained by in situ hybridization [29, 30], by which 94% of TFs (351 of 373 TFs in the database) are detected in the embryo.
We were also interested in the utilization of TFs in adult tissues as their gene expression profiles should exhibit the footprint of both morphological and physiological homeostasis. About 94% of the site-specific TFs (687 of 724 TFs present on the array) are expressed in one or more adult tissues in the FlyAtlas [31].
Four broad classes of TF expression in embryonic development
We were especially intrigued by the large number of TFs that are maternally contributed (those of classes a and d): 58.7% (219 of 373) of the TFs expressed during stages 1 to 3 in the BDGP database. This clearly shows that the maternal contribution is not restricted just to the dozen or so TFs that participate in axis determination, but may be involved in developmental processes beyond.
In the microarray experiments, we interpreted gene expression based on embryo material younger than 2 h after egg lay (AEL) as maternal contribution. In the time-course published by Hooper et al. [28], 48.2% (327 of 679) of TFs show high expression at 1 to 2 h AEL. Although by 2 h AEL a number of genes are expressed from the zygotic genome, this compares to 47.1% (337 of 715) of TFs in the 0 to 30 minute AEL data point of [32], and significant early expression is further supported by the 58.7% (429 of 731) of expressed TFs in the 0 to 2 h AEL data point in the active transcription map. Although the thresholds for the microarray experiments are somewhat arbitrarily chosen, there is general agreement and consistency between the different approaches of gene expression profiling.
We restricted our further analysis to the BDGP dataset as each gene is manually curated and spatially resolved [29, 30]. A significant proportion, roughly one-third, of the maternally contributed factors is expressed in neuronal structures at later developmental stages (please refer to Additional file 3 for details.)
In many other publications, individual duplicate TFs are dissected in terms of their conservation or divergence in expression patterns [13, 33–37]. Here, we survey global trends of expression for large families of distantly related TFs that share a DBD. We observe a tendency for members of DBD TF families to have broadly similar timing of expression during embryogenesis (Figure 1b; Additional file 4). For instance, the number of TFs in the Homeobox and HLH families that are expressed during embryogenesis increases (Figure 1b). In contrast, the zf-C2H2 family includes 82 TFs expressed in the last stage, but also 85 members that are present in the earliest stage. Notably, almost half of these early TFs belong to the subfamily of zf-C2H2 TFs that also contain a zinc finger-associated domain (zf-AD), while many fewer of the late TFs have this additional domain.
Viewed as a percentage of all TFs present at each stage, the zinc finger family is strongly represented at the earliest stage (Additional file 4). Of the 46 exclusively early TFs in the BDGP database, half utilize a zinc finger DBD (15% zinc finger TFs, and 35% zinc finger with zf-AD TFs). In contrast, the number of HLH TFs (0% to 15%) and Homeobox TFs (4% to 27%) starts low and then increases significantly throughout embryogenesis (χ2 test, P < 0.001). The trends observed for the in situ hybridization-based approach are confirmed on a genome-wide level in the microarray studies (Additional file 4).
The microarray data cover a longer time span of embryogenesis than the BDGP and so extend our view: they show that the percentage of factors with a zinc finger out of all expressed TFs decreases continuously until the very end of embryogenesis, while the percentage of Homeobox TFs remains high. At the same time, beyond the temporal coverage of the BDGP database, the bZIP class of TFs increases its share of all expressed TFs between 16 and 24 h AEL in the Hooper et al. dataset. This is consistent with their biological function, as more than a quarter of the bZIP TFs have annotations connected to late embryonic or larval development, with an over-representation of Gene Ontology (GO) term 'instar larval or pupal development' reflecting this (P < 0.002). Microarray data further support the continuous expression of about half of the zf-C4 hormone receptor TFs, an important finding considering that, for example, an essential role for ecdysone signaling in embryogenesis has only been established relatively recently [38].
The functional subdivision of TFs and their expression behavior suggests a model in which many maternally contributed TFs show continued expression, and additional TF classes are subsequently enhanced or switched on to control tissue-specific expression programs. The role of the group of maternal TFs with a zf-AD whose expression diminishes later in development remains enigmatic, as only few of them seem to have an essential function or are evolutionarily conserved ([39] and references therein). The expression behavior of the other TF families makes sense in the light of the following observations: many developmental regulators that commit cells to specific lineages are members of the HLH TF family - for example, Twist is the master regulator of mesoderm development [40], Single minded of central nervous system midline development [41], and Trachealess induces the tracheal cell fate [42]. Homeodomain TFs, like the zinc finger TFs, contribute to axis determination (for example, Bicoid [43]) and segmentation (for example, Ultrabithorax [44]), but after specific lineages have been specified also facilitate differentiation processes (for example, Drifter [45]).
Other TF classes with little or no maternal contribution, whose number of expressed TFs increases during embryogenesis, are the aforementioned bZIP family, and the Forkhead, Ets, T-box, and GATA TF families. This expression behavior does not imply that individual TF family members are not important for early development. In fact the Sloppy paired TFs (with a Forkhead DBD) play a role in segmentation [46], Ets98B is important for pole cell migration [47] and Brachyenteron (with a T-box DBD) is involved in gastrulation and gut formation [48]. However, it is only in later stages that more members of these families are expressed during the formation of organ systems, as shown in Figure 1b. Examples of this are the Forkhead TFs Forkhead (hindgut and malpighian tubules [48], and central nervous system [49]), Binou (visceral mesoderm) [50], Jumeaux (neuroblasts) [51], and fd3F (sensory neurons) [52].
TF expression peaks in gastrulation and the onset of organogenesis
Though in absolute terms the number of TFs expressed peaks at about 12 h AEL, the proportion of TFs relative to non-TFs expressed in embryogenesis peaks around 4 to 8 h AEL (Figure 2b). During this time, up to 10% of the genes expressed in the embryo are TFs, which is twice the percentage of TF genes amongst all genes in the fly genome [21]. Both the temporal gene expression array and the active transcription map confirm this trend, with small differences in the timing and intensity of the TF expression peak. In agreement with this, the expression classes 'II' and 'II:a' (showing transient expression) identified by Hooper et al. [28] fall into the 6 to 8 h AEL time frame, and these two groups have the largest proportions of TFs ('II', 10%; 'II:a', 13.5%) compared to their other classes. This peak in the proportion of TFs expressed coincides with gastrulation and the onset of organogenesis.
In all three datasets, the proportion of TFs expressed relative to non-TFs rapidly rises after the onset of zygotic expression, and continuously falls after stage 10 (BDGP database) or 6 to 8 h AEL (microarray experiments). This means that despite large numbers of TFs being expressed in embryonic tissues at later stages of development, the regulatory complexity in terms of the TF:non-TF expression ratio is greatest during the period of gastrulation and the onset of organogenesis.
Transcription factor expression along the lineage of embryonic tissues
The maternally contributed transcripts exhibit the lowest fraction of TFs relative to all transcripts (Figures 2b and 3). Stages 4 to 8 see a sharp increase in this percentage, with over-representation of TFs in all tissues except the yolk and the pole cells. The yolk and the developing germ cells continue to express a low proportion of TFs in later embryonic development. This is in agreement with the yolk as a storage tissue, and the transcriptional quiescence of pole cells [53]. The other tissue that has a diminishing proportion of TFs is the amnio serosa; the degree of TF over-representation falls throughout embryogenesis to stages 13 to 16, when it only expresses one TF. This makes sense as it is an apoptotic tissue, which probably requires less regulation towards its end.
In stages 4 to 8, there is significant regulatory complexity in all the different germ layers and their derivatives. The ectoderm and the mesectoderm show the highest degree of TF involvement, reflecting the complex developmental competence of these germ layers. The mesoderm at stages 4 to 6 and endoderm at stages 4 to 6 exhibit a reduction of TF over-representation at later stages of development. In fact, at stages 9 to 10, only the trunk mesoderm and the posterior endoderm show significant TF enrichment. After stage 10, only the cardiac mesoderm has significant TF involvement.
This expression behavior is contrasted by a stable TF enrichment in most ectodermal (for example, procephalic ectoderm, dorsal ectoderm, and foregut anlage and primordium) and mesectodermal tissues (mesectoderm primordium, and ventral nerve cord anlage and primordium) into stages 11 to 12. This is in keeping with the findings of Reece-Hoyes et al. [35], who observed similar patterns in the worm C. elegans.
In the process of further differentiation, only the nervous system (for example, central brain, and peripheral nervous system) maintains high levels of TF enrichment, whereas TF over-representation in the dorsal and ventral epidermis and the tracheal system diminishes from stage 13 onwards. These results are in keeping with the pattern observable in Figure 2b.
The transcriptional programs necessary to initiate gastrulation and organogenesis cause the peak of TF over-expression. In the later embryonic stages of organ system differentiation, the high degree of TF involvement is limited to the nervous system. This is in agreement with the finding of Tomancak et al. [30] that many TFs are involved in nervous system development, and TFs are over-represented in these tissues. The exceptional position of the nervous system compared to all other tissues is especially interesting in the light of absolute TF numbers. Indeed, the number of TFs is highest in the embryonic brain (138) and the ventral nerve cord (139). Even the mechanoreceptors of the peripheral nervous system have significant over-representation of TFs (23 TFs). Perhaps the number of different cell types or spatial complexity influence the degree of TF over-representation, as the nervous system and the brain in particular have the highest diversity and structural complexity of all organs (see [54] for review).
TF genes are expressed more tissue-specifically than other genes
We were interested to see if these trends were alike for all of the major TF classes or if the apparent difference between the TFs and the non-TFs was primarily driven by a particular TF class. All of the large TF classes with at least about 20 members follow the previously described trends. Interestingly, the classes that we have discussed here with special importance in organogenesis (for example, Homeobox, HLH, Forkhead, bZIP1 and bZIP2) show the strongest tendency for specific expression.
'Specific' expression for most TF classes implies restriction to about one-quarter of terms used in the slim representation of embryonic anatomy. To further explore this finding, we were interested to see how narrow TF expression can be. When we excluded all TFs that are ubiquitously expressed at any one embryonic stage, we found that only about 10% of the remaining TF genes are expressed in less than three anatomical structures. Factors with expression in clearly different tissues with respect to developmental origin or function were manually excluded. The largest uniform group retained in this analysis is a set of 12 TFs expressed exclusively in the central brain and the ventral nerve cord (for example, Sox102F and a few previously uncharacterized factors), and the second largest comprises 5 TFs in the trunk musculature and related mesodermal structures (for example, Lame duck). Other tissues exhibit one or two exclusive TFs (for example: midgut, Estrogen-receptor related and Adult enhancer factor-1; head epidermis, PvuII-PstI homology 13; yolk, Cryptocephal). However, the latter gene also highlights the general difficulty of such analysis. While the BDGP annotators note a yolk-specific staining, a detailed study of the gene finds cryptocephal ubiquitously expressed [55].
Tissue-specific expression in the embryo versus the adult
As discussed above, most TFs expressed in the embryo are not specific to one but several tissues. Next, we ask whether this specificity in the embryo relates to expression of the TFs in the adult tissues of the FlyAtlas. In the adult, most TFs are broadly expressed, independent of what definition of expression is used to analyze the FlyAtlas data (Additional files 5 and 6). About two-thirds of the 687 TFs present in the adult show ubiquitous or very broad expression based on the FlyAtlas 'present' call (Additional file 6).
We then used the 'up' call to identify more tissue-specific adult TF expression as this should discern genes with significant expression in the respective body parts (Additional file 6). We identified seven clusters of TFs and classified them according to the adult tissues where they are expressed, as shown in Figure 4b. These seven clusters correspond to expression in ovary, testis, brain/ganglion or combinations of tissues (Additional file 6). The adult TF clusters with broad or ovarian expression do exhibit a broader embryonic expression than the groups with some adult specificity. However, although the adult tissue-specific clusters are also tissue-specific in the embryo, this does not necessarily pertain to the same or comparable tissue. This shows that adult TF expression is largely independent of embryonic expression. A good example is Trachealess, which is an inducer of the tracheal cell fate that is also expressed in the embryonic salivary gland, but also plays a role in the formation of the adult leg [56]. Tissue-specificity in terms of expression breadth is a property that seems inherent for some TFs; however, the tissues where they are expressed can be rather diverse and variable between embryo and adult.
Combinatorial use of transcription factor modules
Both anatomical complexity and absolute number of expressed TFs increase along the developmental time axis. At the same time, the tissue-specific expression of the TFs increases, ultimately leaving many differentiating organ systems except the nervous system with a significantly lower TF:non-TF ratio than, for example, their anlagen and primordial tissues. In addition, we show above that absolute tissue-specificity is virtually nonexistent. All this supports the concept that developmental and physiological gene expression programs are not regulated by individual factors, but by the unique combination of TFs present in a given biological context.
In line with this principle, we calculated how many of the possible pairs of TFs are expressed during embryonic development. From the BDGP data, the possible number of co-expressed TF pairs would be about 69,500 (3732/2). In Figure 5b, we show that the vast majority of these possible pairs are in fact co-expressed in at least one tissue and developmental stage. This means that they can potentially collaborate at genomic binding sites, an assumption that may be especially true if these TFs are persistently co-expressed in exactly the same tissues.
Next we ask how many of the co-expressed pairs and triplets of TFs are maintained as co-expressed modules across consecutive stages of embryonic development. In Figure 5c, we show that there is highly significant conservation of co-expressed pairs and triplets of TFs from one stage to another compared to a set of TF genes whose expression information has been randomly shuffled. However, similar levels of maintenance of co-expression are found in non-TFs. The only embryonic stages that show significantly higher conservation of co-expressed TF pairs and triplets compared to non-TFs is the transition from stages 11-12 to 13-16.
This analysis shows that overall co-expression of TFs is extremely plastic, with almost the entire space of possible TF pairs co-expressed at some point in embryonic development. Co-expression TF modules, however, represent potential functional entities that are much more abundant than to be expected at random. There is no evidence for these consistently co-expressed TFs to interact at cis-regulatory modules, even though it is known that TFs cooperate, as in the example of Twist and Mef2 above.
Functional genomics approaches provide unbiased insight into the spatial and temporal utilization of Drosophila genes on a genome-wide level. We integrated these data with information about the repertoire of site-specific TFs in the fly, and studied their expression dynamics in order to understand their role in development and physiology on a systems level. A potential shortcoming of this approach is that we are considering RNA levels as a proxy for TF activity. Because there are so few data available at the protein level, we are obliged to use the mRNA data. While mRNA levels will not always reflect protein levels, at least both the microarray and in situ datasets are relatively robust to transcriptional noise. This is achieved by both technical and empirical optimization of these methods.
A further limitation of both types of expression dataset is the granularity of both the sampling and the annotation. This implies that many of the co-expression clusters at the level of tissues may not actually be co-expressed at the level of individual cells. Therefore, our conclusions about re-shuffling of TFs between clusters may be even further enhanced once more fine-grained expression data become available.
Our data suggest that at least 50% to 60% of the TFs are maternally contributed (Figures 1 and 2a), which is in keeping with previous studies on the ascidian Ciona intestinalis, where a similarly high proportion was reported [58]. The early C. elegans embryo expresses only about 30% of its TFs [35], and it can be speculated the pre-defined developmental cell lineages do not require the same large number of maternal TFs. These studies are the only other works that address TF utilization in metazoan organisms on a genome-wide level, and therefore serve as an interesting comparison.
The number of early/maternal TFs is much larger than could be assumed based on a dozen or so anecdotal early patterning genes. This is a very important finding in the light of studies of early gene regulation. Early patterning in Drosophila is often viewed as a closed circuit of a handful of TFs regulating each other [1], and most of the recent network and perturbation analyses are based on this assumption. How does the plethora of maternally contributed TFs tie into the known and well-understood network? Recent studies suggest that the genome is plastered with binding sites occupied by the well-known early factors in blastoderm embryos [19]. Is this how they differ from the less prominent bulk of maternal TFs? Or is the early genome covered by hundreds of TFs, waiting in place to fulfill their function at a later stage?
The fraction of TFs utilized during embryonic development is about 95%, both in our study and in the analysis of Ciona TFs [58]. Given that these results are obtained by a variety of different assays (in situ hybridization, microarray) and in two different model organisms, this is unlikely to be an experimental artifact. The general time-course of TF expression suggests that the largest degree of combinatorial complexity occurs between 4 and 12 h AEL (Figure 2b), coinciding with the formation of germ layers and the onset of organogenesis. This is when the ratio of TFs to non-TFs is highest. Interestingly, the actual peak of TF expression is only achieved later in development, when both the number of body parts and TF specificity increase. Expressed TF numbers fall from 12 h AEL (that is, stage mid-15) onwards (Figure 2a), suggesting that the final differentiation of organ systems is a local event requiring less TFs per tissue. In the earlier phases of development large numbers of TFs seem to be required, either for inter-regulation or combinatorial regulation of target genes. The coding potential of fewer TFs appears to be sufficient once the embryo has been compartmentalized.
In this work we show that the Homeobox TFs make a large contribution to these local expression neighborhoods in late embryogenesis (Figure 1b; Additional file 4). Recent studies on the coding limits of TFs show that Homeobox TFs have by far the largest number of possible binding sites [59], which also supports the fact that a smaller number of TFs may achieve the same coding potential. Similar findings to our results regarding temporal utilization of TF families can also be found in Ciona, with Homeobox and HLH families generally showing a later onset of expression. This is in agreement with the developmental role these classes have been associated with: zinc finger TFs are a versatile class, while many other TF families (for example, HLH, Homeobox, T-box, Forkhead) are over-represented in organogenesis and differentiation (Additional file 7). This role is also reflected in their developmental expression, with zinc finger TFs showing ubiquitous expression more frequently than other classes (data not shown). This contrast is particularly strong in the light of TF overlap between tissues, where we show that zinc finger TFs are more likely to be shared between tissues than Homeobox TFs (Additional file 8).
We were surprised to see a general lack of true tissue-specificity, although TFs often have narrow expression domains. This is especially highlighted by the comparison of adult tissue-specificity and embryonic expression (Additional file 6; Figure 4b), where we found that those TFs that show some adult tissue-specificity are expressed in unrelated tissues during embryonic development. We did observe a biologically sensible correlation, however, between TFs expressed in the ovary and those present as maternal contribution. As many maternal TFs seem to play a role in embryonic nervous system development, there is also a group of TFs that show specific adult expression in the ovary and brain/central nervous system. Interestingly, many factors of the enigmatic group of zinc finger TFs with a zf-AD have this pattern, a group that shows strong lineage-specific expansion in holometabolous insects [39].
The combinatorial usage of site-specific TFs is an established concept. However, most of our current knowledge is based on a few examples like the 'stripe 2' enhancer [10]; we are only slowly accumulating sufficient data to address the combinatorial characteristics of regulatory modules on a broader scale [19]. Many previous studies concentrated on the inference of combinatorial TF usage from the co-occurrence of binding motifs [60, 61], but the expression of the TFs themselves was mostly neglected. Context-specific co-expression is one possibility for how combinatorial TF usage can be studied. While many TFs can be present in a particular tissue, the most consistent co-expression clusters are formed by TFs whose pattern precisely correlates in a variety of different tissues at a given stage. These clusters are rather dynamic and rarely stable between stages, and TFs are constantly exchanged between co-expression clusters along the developmental time axis (Figure 5c). However, they are more frequent than can be explained by chance and probably a good indicator of functional relevance. The characteristics of this reshuffling process have not been addressed on a systems level before, and it should be noted that their expression modularity is not much stronger than for non-TFs. This implies that a 'transcriptional logic' is probably not primarily conferred by the expression of TFs, as studies on early patterning might suggest.
Taken together, our study provides a first analysis of the system-wide behavior of Drosophila TFs. Our findings support the existence and predict a developmental role for almost the entire TF repertoire. The tissue-specific role of the HLH and Homeobox TF families, which is known anecdotally for a handful of genes, is supported by high-throughput gene expression data on a genome-wide level. The defined expression of TFs contributes to a constant reshuffling of TFs between clusters of co-expression, providing the outer bound for TF combinatorics. Expression modularity is not an inherent feature of TFs to a greater extent than non-TFs, which has implications for their regulatory logic.
Materials and methods
Gene expression datasets
Spatio-temporal gene expression was derived from the BDGP gene expression database [29, 30], pre-release version as of May 2006, using the Drosophila anatomical ontology or the slim representation of Tomancak et al. [30] to map individual body parts to developmentally related lineages. The BDGP contains information on 373 TFs, 51% of the TF repertoire in the FlyTF database [21].
Temporal gene expression information was derived from an embryonic gene expression time-course genome-wide microarray experiment [28], a microarray survey of early embryonic expression [32] and a time-course genome map of active transcription during embryonic development [27]. The transcript abundance per time point in the time-course gene expression experiment is relative to a pool of mRNA of all developmental stages, including larval, pupal and adult stages. Genes with no developmental change or those that are expressed at lower levels in the embryo than in other stages are therefore indistinguishable from genes that are not present in the embryo. Expression classes as observed by Hooper et al. [28] using a global convolution method were accessed through FlyMine [62] and overlap was determined using the FlyMine list manager. We chose an additional strategy to classify transcript abundance, and inferred three arbitrary classes ('low expression', value < -0.5; 'not classifiable/average', -0.5 < value < 0.5; and 'high expression', value > 0.5). These thresholds are based on the observation that the median expression value per time point is 0.72 for BDGP positive and 0.14 for BDGP negative genes, with a considerable spread of the data not allowing for more defined thresholds.
In the Affymetrix GeneChip experiment of early development by Pilot et al. [32] we counted a gene as expressed when it received at least two present calls in the three replicates. The 'present' call is based on the statistical analysis of all probes per gene and provides an indication of whether the observed signal stems from a transcript (if it is present or absent) or is part of a general background. We interpreted overlap of at least 10% of a gene's coding region with any transfrag from the active transcription map as a sign of its expression. This is less stringent than the criteria used by Manak et al. [27], who required at least 70% of a gene's coding sequence to be transcribed. However, lower transfrag coverage may simply be due to technical issues when the transcript is present at low abundance. Overall, our criteria produced similar results between the three temporal gene expression datasets.
Adult gene expression information was derived from FlyAtlas [31]. We rated a gene as expressed when it had at least three present calls in the four replicates per adult tissue, or if it was significantly up-regulated (received an 'up' call) in a tissue when compared to whole flies.
Additional file 9 provides a convenient lookup for the conversion between developmental time (hours AEL) and developmental stage.
Transcription factor over-representation in body parts
We used a slim representation of the anatomical ontology, using the tissue categories defined by Tomancak et al. [30], to allow comparability between the studies. We determined the non-redundant set of TFs (n) and non-TFs (m) expressed in body parts represented in each tissue category. Random sampling of n + m genes was used to calculate the number of TFs to be expected by chance, and a Z score was derived. We considered TFs to be over-represented in a tissue if Z ≥ 3. This approach is comparable with the anatogram summaries of Tomancak et al., where Z is the amplitude of the signal.
Significance of transcription factor overlap
The significance of TF repertoire overlap (Additional file 8) between two tissues with n and m TFs was determined by random simulation, comparing the actual overlap with overlaps obtained by randomly picking n and m genes from all TFs of the appropriate family with expression information.
Muscle transcription factor reshuffling
ChIP datasets [22] were obtained from the Furlong laboratory website. Filtered TileMap data were used to indicate reliable binding of Twi and Mef2.
There are no generally accepted criteria as to what defines the boundaries of a cis-regulatory module or TF binding site cluster. We therefore interpreted partial or complete overlap of ChIP peaks as an indication of common action of TFs within a genomic region.
We interpreted precise co-expression as a minimal requirement for modularity. If two or three TFs are consistently used in exactly the same developmental context (that is, precisely co-expressed at subsequent developmental stages), then they potentially form a module. We counted the potential pair-wise or triplet interactions between precisely co-expressed TFs in one stage and compared them to the potential interactions of the previous stage. The number of 'conserved' interactions between the two stages was counted. In a random experiment we used the same pairing matrix of the older stage, but each time reshuffled the order of the TFs for each of 1,000 iterations. In each of the iterations, we determined the number of 'conserved' interactions, which we used to calculate a Z score in comparison with the real data. These data were compared to the Z scores obtained by randomly sampling 373 genes and repeating this approach 100 times.
Gene Ontology analysis
Lists of candidate genes were uploaded to FlyMine and GO enrichment was analyzed using their GO widget with 'Benjamini and Hochberg' correction. For GO over-representation within the TFs, GO annotation was downloaded from FlyMine and over-representation for TF classes in comparison to a background of all TFs was calculated in GeneMerge v1.2 [63].
after egg lay
Berkeley Drosophila Genome Project
- ChIP:
chromatin immunoprecipitation
DNA-binding domain
Gene Ontology
gene regulatory network
transcription factor
- zf:
zinc finger
- zf-AD:
zinc finger-associated domain.
We thank Madan Babu, Steve Russell and Varodom Charoensawan for helpful discussions. BA was supported by an EMBO Long Term Fellowship.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Hi Bill,
Yes, you are getting the CDs. Your name was in the Autumn 2004 GATW under donations, but we discussed if you wanted to be switched over to the pre-purchase side, and that is my understanding of what you wanted.
The Golden Gate Geranium Society pre-purchase was listed in the Winter 2004 GATW.
I have a meeting with scan people on Monday afternoon and we should be finished with the master set. Then, they will be shipped off for the duplication. I have all the packaging here when they get back. Only have to do the cover insert. Can't believe it hasn't even been a year since the official kick-off at the annual meeting last June.
You will love the set. As I was typing the listing of photos in the 1953-2003 journals, found so many articles of interest and also lots of pictures of people whose names I had only read.
email@example.com wrote:
I just do not understand how mine can always arrive so late. Occasionally
I am getting my CDs right? I did not see my name on the pre purchase, or
the Golden Gate Group. Of course theirs was initially meant to be a
donation-but the CDs can be given a place in the Library-when the new bookcases are
made available.

subset: commoncrawl


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Build-A-Bear is on the hunt for a new e-commerce platform
CEO Sharon Price John says Build-A-Bear's old e-commerce system is a big reason for disappointing online sales in December.
Build-A-Bear is on the hunt for a new e-commerce platform
CEO Sharon Price John says Build-A-Bear's old e-commerce system is a big reason for disappointing online sales in December.
What is Wal-Mart's e-commerce strategy?
Wal-Mart's acquisition of Moosejaw is the latest in a series of recent moves that illustrate the retail giant's lack of a coherent e-commerce strategy, ...
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Since my early writing days, back around 2000, I know the power of people 'saying cool stuff about' you. As you can see from the quote I've made myself, it is always better to have someone else sing your praises that do it yourself.
To read the full article, click on the title or image.
Get your Free Business Plan Template here: http://bit.ly/1aKy7km
Via Neil Ferree, malek


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Merchandise from three prominent Virginia estates will be auctioned Feb. 4 by Tom's in Ivor, Va.
Hundreds of select, handpicked items from three prominent Virginia estates –in Franklin, Virginia Beach and Norfolk, plus other consignments -- will be sold at auction Saturday, Feb. 4, by Tom's Auctions & Appraisals in Ivor, Va., at 10 a.m..
At press time, over 600 lots of merchandise had been counted, but items were still being inventoried and the final tally could easily top the 700 mark. Offered will be items in a wide range of categories – period furniture, stoneware and pottery, duck decoys, stamps and coins, glassware, hand-made baskets, fountain pens, ephemera, decorative accessories and collectibles.
Headlining the period furniture category will be an early blue-painted dovetailed blanket chest, probably made in Pennsylvania in the early-to-mid 1800s; a burled mahogany 8-drawer highboy (circa 1780-1800) with ball-and-claw feet and arched shell carved into the center of the base; and a mid-1700s mahogany bachelor's chest with bracket feet and four graduated drawers.
Other furniture pieces will include a diminutive and unusual eastern North Carolina cupboard, not much larger that a child's piece and made circa mid-1800s; a period drop-leaf Pembroke dining table (possibly European); a huge Victorian-era wardrobe; and Empire pieces.
One lot certain to pique bidder interest is a 1927 diorama with squirrels, executed by the renowned American taxidermy artist Herman Grieb (Buffalo, N.Y., 1869-1928), about 48 inches wide and signed by the artist. Mr. Grieb's production was both prodigious and endearing. His natural creations are still actively sought out by collectors, more than 80 years after his death.
Another lot sure to get paddles wagging is a 19th century Carlsbad fish set, comprising a scalloped oval platter and twelve plates, all hand-painted and in perfect condition. All pieces carry the Carlsbad mark, ensuring their authenticity. Carlsbad is the Austrian-based china firm renowned for its hand-painted fish platter and plate sets. They are highly coveted by collectors.
Decorative accessories will feature a gorgeous green Northwood Carnival glass epergne (marked Northwood), around 20 pieces of stoneware (some of it beautifully decorated and highly sought after pieces from South Carolina, plus examples from New York, Pennsylvania and New Jersey), Roseville pottery, oil lamps, doorstops, sterling silver and a nice group of picture frames.
Also sold will be a circa 1880-1900 6-tune music box, probably Swiss, with damper and brass cylinder that plays six songs, a hand-illustrated cloth book of the classic Snow White and the Seven Dwarfs (circa 1880-1920), original paperwork from a washing machine made in 1900 (that cost just $10 new!), and a Virginia slave document transferring a slave from father to son.
The duck decoys are mostly unsigned, except for one signed "A. Wood," and the group of hand-made baskets includes a desirable miniature example. Stamps and coins will also cross the block (although none of the coins are gold). Other lots will include a Gem Roller organ that plays 16-inch cylinder rolls (cylinders included) and a rare sportsman's yearbook from 1940.
Previews will be held on Friday, Feb. 3, from 3-6 p.m., and Saturday, Feb. 4, the date of sale, from 8 a.m. until 10 a.m., when the first gavel comes down. Phone and absentee bids will be accepted. A 10 percent buyer's premium will be applied to all purchases. Terms will be cash and good checks only. No credit card purchases will be allowed. Refreshments will be served.
The old high school building in Ivor is located at 8430 Bell Avenue, off Route 460. To learn more about the auction, and for directions to the venue, log on to www.tomsauction.com. Many of the items to be sold have already been posted, and more images were being added at press time. A photo gallery of the items can also be viewed at Auctionzip.com (ID #1328).
Tom's Auctions & Appraisals is one of the premier auction firms in the Mid-Atlantic States. The firm is always accepting quality consignments for future sales. To consign an item, an estate or a collection, you may call them at (757) 539-2498, or (757) 617-9647. Or, you may send them an e-mail, at email@example.com. For more info, log on to tomsauction.com.
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Ken Hall writes pre-sale and post-sale press releases for auction houses, for a fee. He writes, submits and tracks stories for clients. Submissions are published in trade magazines, posted on industry websites and appear in local newspapers.
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Hotels in Busan, South Korea
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- Most bookedHotel Cieldemer Haeundae
854 people looked at this hotel in the last hour
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"Hard to find, but it's near to the beach and has a lot of cafes and restaurants nearby. Like the mood and feel of the room we were in."
A Traveller, Dec 2016, SG
642 people looked at this hotel in the last hour
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"Overall, it is a very good hotel but service is not consistent. For example at breakfast, i was not served coffee (yes coffee) despite my request to the waiter"
A Traveller, Nov 2016, AS
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"Main Building recently renovated and Annex Building is newer. Room was decent since the ocean view made up for other areas that were lacking. Bathtub not to standard to include the shower head.. especially for the price per night.. water kept switching from hot to cold and was no consistent. Othe..."
A Traveller, Nov 2016, US
480 people looked at this hotel in the last hour
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"I was amazed at how friendly every staff member I encountered was. They helped me exchange money, hail a cab, weigh my luggage, get directions to various sights, etc. Seriously, it was like having personal assistants at my service. The location is great (right on the beach). There are many re..."
A Traveller, Dec 2016, US
408 people looked at this hotel in the last hour
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"We were offered a room with cityview where we could see the beautiful Culteral Village at our right side. Thd room is specious and comfortable though the pillow is too soft."
A Traveller, Jan 2017, AS
385 people looked at this hotel in the last hour
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"I did give this hotel a good rating but there are some cons as well. PROS: - The lobby is beautiful and feels so luxurious. - The elevators going up to the rooms overlook Haeundae Beach and it's beautiful every time! - The front desk staff are really friendly and polite. They also speak E..."
K, Jan 2017, US
339 people looked at this hotel in the last hour
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"The hotel has the best shampoo & conditioner ever! Location is walking distance to tourist attractions like the BIFF sq night market and Lotte shopping mall. It's even near to a bustop that goes to taejongdae park! "
A Traveller, Jan 2017, SG
313 people looked at this hotel in the last hour
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"The room condition wasn't that great, it was worn out and old. The floor needs to be cleaned convenience store located right under the hotel and lots of restaurants around it. kitchen is included but utensils are not included in certain rooms(mine wasn't) so I couldn't cook anything. Room had ..."
A Traveller, Dec 2016, CA
311 people looked at this hotel in the last hour
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"My family and I stayed for two nights at this hotel. Very close to Haeundae beach and it is relatively close to the station. We were surprised by the size of the rooms, especially the corner rooms as they have a separate seating area from the beds and huge bathroom. The staff were very helpful an..."
A Traveller, Nov 2016, GB
143 people looked at this hotel in the last hour
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"Great hotel, nice place, full of culture. Staff was remarkably nice and generous! Will definitely be visiting again. Highly recommend!"
A Traveller, Mar 2016, US
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When you book your Busan stay with Hotels.com you may also earn free nights on participating hotels by joining the Hotels.com Welcome Rewards program. It's free to join and only takes 2 minutes to sign up and when you stay 10 nights you receive 1 night free*. So even a short weekend break in Busan can get you on your way to a free night.
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Thanks for making the 8th International Soul Society Festival a success!
Stay tuned on our social media to see footage of the festival events and participants from this year! Links to our social media can be found at the bottom of this page or at www.urbanartistry.org.
From June 12-19th, 2016, we held the 8th annual International Soul Society Festival in the Washington, DC and greater metropolitan area. The International Soul Society Festival is an all ages, week-long series of events celebrating the full spectrum of Hip-Hop, Urban Arts and its many influences.
Russell "Ironman" Campbell (Urban Artistry, CAB Cru) and Cody "Coflo" Ferreira (Circle of Fire, Soul Shifters, SourPatch Crew) getting down at our Juneteenth celebration at the Lincoln Memorial on the National Mall during our festival last year. Thanks to Michael Lipin for capturing this great footage!
About International Soul Society Festival
This action-packed festival creates the much needed opportunities for inspiration and collaboration amongst global artists right here in our Nation's capital. Through dance battles and workshops, DJ competitions, film viewings and graffiti exhibitions, there is something for everyone at the International Soul Society Festival, from age nine to ninety-nine.
2016 full events schedule is here!
Getting Around…
Getting around town is a breeze thanks to WMATA Trip Planner, just enter your destination and it will help you map the best route to travel. All of the International Soul Society venues are metro accessible.
About Urban Artistry
Urban Artistry, an International Culture and Education Project based in the DC Metro Area is a 501(c)(3) non-profit collective of dancers, artists, historians, musicians, folklorists and documentary filmmakers on a mission to preserve and continue the practice of urban dance, music and arts culture throughout the world.
A big thanks to our sponsor Joe's Record Paradise, who helped make our 2015 festival happen.
Donate to Urban Artistry now! It's a fast and easy way to support our mission. Without the generous support of our donors, we could not provide the holistic guidance and support to urban artists in our community. Please consider supporting Urban Artistry, Inc and the preservation of urban dance and arts culture today!
Contributions are tax-deductible to the fullest extent allowable by law. We also accept donations by mail at Urban Artistry, Inc, 932 Philadelphia Ave, 3rd Floor, Silver Spring, MD 20910. Please make checks out to Urban Artistry, Inc.
Stay tuned via Facebook, YouTube, Twitter, Tumblr, and Instagram!


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Juventus President Andrea Agnelli has given the green light for Gigi Buffon to be handed a new contract.
According to the Gazzetta dello Sport, the custodian will commit his future to the Bianconeri until the summer of 2016.
The 34-year-old, a doubt for this weekend's game against Napoli because of injury, has less than a year left on his present agreement.
As part of the new terms, the Italian international will accept a cut in his salary from €6m a term to around €4.5m.
Buffon is now the captain at club level, as well as international level, following the departure of Alessandro Del Piero in the summer.
Keep up to date with the latest news and action from Spain's Primera Division with Football Espana - from the team behind Football Italia.


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Neutral Bay, New South Wales, Australia 1892 – Sydney, New South Wales, Australia 1984
Looking astern
Collection Title: Sketchbook[26]. Scenes and figures en route and in England. Australia, Shipboard, Europe, 1948-50
Page: Leaf 3 recto.
Place made: Australia
Materials & Technique: drawings, sketchbooks, colour pencil; ink; paper drawing in pen and black ink and colour pencils Support: paper


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Pete Rose may be thinking about Derek Jeter, but Derek Jeter certainly isn't thinking about Pete Rose.
Rose said recently he didn't think Jeter would reach or surpass his all-time hits mark of 4,256 knocks.
Jeter was asked about Rose's comments before the Yankees' Game 3 matchup against Baltimore. In typical Jeter fashion, the Yankees captain deflected the question, saying it wasn't something he was focused on with the Yankees locked in a 1-1 series tie with the Orioles.
"Guys, I don't even know why we're talking about this," Jeter said. "I'm not talking about Pete Rose man, we're trying to win a game here."
When a reporter mentioned the possibility of a position switch to prolong his chance to reach Rose's mark, Jeter said, "Buddy, like I said, I'm trying to get through today."
Rose, who was banned from baseball for gambling on the game when he was manager of the Cincinnati Reds, retired in 1986 at age 45 with 4,256 hits. Rose broke Ty Cobb's record the year before he retired.
Rose told the website Sports On Earth recently he didn't think Jeter, who is 38 years old and 952 hits shy of his record, would touch his all-time mark.
"I don't think he will break the record," Rose said. "First of all, I don't think he wants to leave the Yankees. And the Yankees, they're about winning. Jeter had a great year this year, but he's what? Thirty-eight years old? And he's a shortstop? How many 40-year-old shortstops you see walking around? Not too many, right?"
For the full news story, click here.


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More Videos...
1972 Vega, 2007 Season recap.
11 Mad Engines You May Not Know About #2
Subscribe for plenty of car content coming: http://bit.ly/SubToVisioRacer SOCIAL: https://youtube.com/user/VisioRacer https://plus.google.com/+VisioRacer https://facebook.com/VisioRacer https://twitter.com/VisioRacer https://instagram.com/VisioRacer https://carthrottle.com/user/VisioRacer Subscribe to my friend's channel, IceOne01, dedicated to bikes and cars as well: https://www.youtube.com/channel/UC1wn3hDIISSUou_IX5WbvjQ?sub_confirmation=1 RELATED VIDEOS: https://youtu.be/dyUK4MHjP8g Mad Engines #1 DISCLAIMER: Under Section 107 of the Copyright Act 1976, allowance is made for "fair use" for purposes such as criticism, comment, news reporting, teaching, scholarship, and research. Fair use is a use permitted by copyright statute that might otherwise be infringing." The materials are used for illustrative and exemplification reasons, also quoting in order to recombine elements to make a new work. The original works were altered quantitatively or qualitatively and the video does not compete with the market for the original works. There were used small portions of the materials in a new context and expression for illustrative reasons only. I do NOT own the video materials and all credits belong righteous owners. In case of copyrights issues before taking action, please contact me immediately. CONTACT: firstname.lastname@example.com CREDITS: "Volvo BM 470 Bison med 38,8-liters V12" by Norrhyttan https://www.youtube.com/watch?v=Ha8V5cm0Yiw "Volkswagen Passat W8 Exhaust" by Lauren rpn https://www.youtube.com/watch?v=KFAp9-4CyXk "2003 Volkswagen Passat w8 straight piped" by agpetro https://www.youtube.com/watch?v=VqwDF9tp3ak "Fiat S76. 1911. 28.4 litres. 4 cylinders. Start-up 28th Nov 2014." by Graham Rankin https://www.youtube.com/watch?v=PlPbhGeMu4U "1911 28.4 litre Fiat S76 first start up - 28 November 2014" by Ant Lucas https://www.youtube.com/watch?v=BfYbH7926gk "CBR/Castrol Edge RX2 4 rotor 4 turbo start up" by CBRRX2 https://www.youtube.com/watch?v=xUXA1SIqJFY "Chainsaw Bike" by Jeff Rice https://www.youtube.com/watch?v=DDWqJe1dCgY "1953 F1 BRM V16: Fabulous drive in appalling conditions! Follyfilms" by follyfilms https://www.youtube.com/watch?v=umO3iqac_4c "V16 Hot Rod ( first drive)" by Sire Customs https://www.youtube.com/watch?v=34Rb_kw_NIs "V16 hot rod? rat rod? (twin V8)" by Sire Customs https://www.youtube.com/watch?v=vto1-J_CvI8 "V16 HOT ROD (Twin V8) doing a burnout, part 2" by SireCustoms https://www.youtube.com/watch?v=VfswVtaZJHU "12 Rotor Engine Running- Different Angles" by Tyson Garvin https://www.youtube.com/watch?v=Q2IFWveYBRs "Touareg V10 TDI muffler delete straight pipe" by Steve Berg https://www.youtube.com/watch?v=jvWrkTgvdkY "Rolls Royce V12 27litre Merlin engine PV12 FULL THROTTLE!" by cuda2266 https://www.youtube.com/watch?v=e1mh99ZLXcw "1972 Porsche 917/10 Can-Am Brumos" by alexander1485 https://www.youtube.com/watch?v=sdkICUG2Ar8 "Porsche 917 Shakedown for Rennsport Reunion V" by Porsche https://www.youtube.com/watch?v=mU_MZbgGpF4
1200 HP Alcohol Motor
Mike, John, and Mark
3,000 horsepower Z06 catches some air at 190 mph
Kelly Bise Racing and KP Racing are at it again! For the first time, Kelly Bise brought both corvettes (Double Trouble) to an event and they did not disappoint. Watch as Joe Huneycutt pilots this low flying aircraft past the finish line at 190mph, then gets sideways and ends up in the grass after a short flight that was NOT approved by the FAA.


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Album Vendo Amor en La Calle
Label Source Interlink
Released December 27, 2011
This site contains content from artists, fans, and writers from around the internet in it's natural form. Such content is not representative of Viacom Media Networks.
©2017 Viacom International Inc. All Rights Reserved. MTV and all related titles and logos are trademarks of Viacom International Inc.


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This week's excerpt from The Facts About Modern Manufacturing: More than any other sector, manufacturers bear the highest share of the cost of regulatory compliance. Many regulations have benefits for society, yet few people understand their costs and their impact on companies facing intense competition with overseas firms that often do not have similar costs. Manufacturers spend an estimated $192 billion annually to abide by economic, environmental and workplace safety regulations and ensure tax compliance -- equivalent to an 11% "regulatory compliance tax." Manufacturing Facts
Full Story:manufacturingfacts.org


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The Japanese dancer/choreographer and his company of dancers present the world premiere of a special version of their work for the festival, which blends contemporary dance with ballet.
Please use the event calendar to search for current events.
All events and artists subject to change without prior notice.
All ticket prices are subject to change based on demand. Purchase early to lock in prices and the best seats!


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About Shangana Cultural Village
At the core of Shangana Cultural Village is a busy African market where local craftspeople work and trade their crafts.
Trained guides take guests on informative and interactive trips to villages during the daytime as well as midday tours with lunch and the famous Evening Festival in the Chief's Kraal.
Shangana was created and built by the local Shangaan people and is a source of great pride. The village preserves their rich heritage and is an example of South Africa's great cultural diversity.
Added By: Kruger Park Lodge - Golf Safari SA
Directions to Shangana Cultural VillageShangana is an easy drive on excellent roads 5 km from Hazyview. We recommend driving slowly at night as there are often small animals such as honey badgers and mongoose crossing the road. From the centre of Hazyview, head north past Hippo Hollow over the Sabie River. Turn left onto the R535 towards Graskop – Shangana is 4 km from here on the right, clearly signposted.
Accommodation near Shangana Cultural Village
Kruger Park Lodge 514
Sleeps 6 in 3 rooms
Unit 514 is a free-standing, self-catering lodge sleeping up to six guests. The house is fully-equipped and serviced daily for guests'...
from R2000
per night for 2 guests
per night for 2 guests
Swallows House
Sleeps 4 in 2 rooms
Situated on the shores of the Da Gama Lake, this tranquil retreat is located in the most beautiful part of the Lowveld. It is close to...
from R1100
per night for 2 guests
per night for 2 guests
The Windmill Wine Shop & Cott...
Sleeps 22 in 6 rooms
Way out on the wine route is the Windmill Wine Shop & Cottages, including Sabie Valley Rider...
from R1090
per night for 2 guests
per night for 2 guests
Safari Kruger Cottages
Sleeps 30 in 4 rooms
Safari Kruger Cottages are 5 stylish self catering houses that sleep 23 guests in total and are situated in the heart of the lowveld....
from R650
per night for 2 guests
per night for 2 guests
Oppi Plaas
Sleeps 18 in 4 rooms
Oppi Plaas is situated just 8 km outside of Hazyview, making it easily accessible for our guests to go to various shops, supermarkets and...
from R580
per night for 2 guests
per night for 2 guests
Kwambali Riverside Lodge
Sleeps 54 in 11 rooms
Kwambali Riverside Lodge is located a mere four hours' drive from Johannesburg, Kwambali Riverside Lodge offers the ideal tranquil setting...
from R750
per night for 2 guests
per night for 2 guests
See all accommodation near Hazyview
Shangana Cultural Village Map
Shangana Cultural Village is 8.7km from the centre of Hazyview , the nearest airport 42.1km (Kruger Mpumalanga International Airport).
*Distances are shown as the crow flies and not necessarily the actual travelling
*Distances are shown as the crow flies and not necessarily the actual travelling
Other things to do near Hazyview
Visit an active silk farm and be amazed by the silk making process at this unique African...
Phabeni Gate is situated 10 km from Hazyview and serves as a convenient entry point for visitors...
Sample delicious orange wine from Rottcher Wineries at Casterbridge. Locally grown Valencia oranges...
See all things to do near Hazyview


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hotel info
Nearest Airport: YYJ
March 16, 2017 for 3 nights for 2 adults.
With the finest in both service and amenities, and intimate boutique-style accommodations, visit the award-winning Abigail's Hotel to...
March 16, 2017 for 3 nights for 2 adults.
A city filled with historic delights on British Columbia's Vancouver Island, Victoria is the ideal home for Abigail's Hotel. This member of Select Registry offers premier boutique-style accommodations, featuring heritage tudor architecture surrounded by colorful English gardens. Awash in soft and warm colors, the romantic guest rooms include a wealth of indulgences ranging from jetted tubs to wood-burning fireplaces. The gourmet breakfast menu changes each day, and evening wine and hors d'oeuvres are proof that genuine hospitality never goes out of style.
Abigail's Hotel is just three blocks from the colorful sailboats and elite yachts of picturesque Inner Harbour. It is superbly situated for exploring the city's many attractions, including the antique shops of Fort Street and the lively bistros and pubs of Government and Wharf Streets. You might also wander the 50 acres of floral finery at the wonderful Butchart Gardens, embark on a salmon fishing charter, or peruse the museum exhibits at Craigdarroch Castle. Be sure not to miss the pleasures of the hotel's very own Pearl Spa, where the finest holistic spa services are available at your whim. With the finest in both service and amenities, visit Abigail's Hotel to experience the best of downtown Victoria.
Luxury Link is a trademark of Luxury Link, LLC. ©Copyright 1997 - 2017


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Anatomy X5 Muscle
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Bodies come in all shapes and sizes. Most of us have a specific physique that we have in mind that we'd like to be. Some of us may fall on one end of the spectrum of bodies: skinny, light build, small joints, and stringy muscles. Others may fall on the other end of the spectrum: fat, flabby, spare tire around the waist, and thick arms & legs. Do you find yourself landing in one of these categories? Now you can undergo a complete body transformation thanks to Anatomy X5!!
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Anatomy X5 is a super dynamic, all-natural, muscle building supplement that will make you achieve a superior physique without any harsh side effects. You'll be able to achieve a muscular and strong body without heavy exercises at the gym or crash dieting.
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