Interactive Fly, Drosophila

grainy head


REGULATION

cis-Regulatory sequences

Use of a Drosophila genome-wide conserved sequence database to identify functionally related cis-regulatory enhancers

Phylogenetic footprinting has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). Currently, there is no systematic approach for enhancer discovery and analysis that takes full-advantage of the sequence information within enhancer CSCs. A Drosophila genome-wide database of conserved DNA has been developed consisting of >100,000 CSCs derived from EvoPrints spanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. The program first identifies conserved repeat elements within an input enhancer and then searches the database for CSCs that score highly against the input CSC. Scoring is based on shared repeats as well as uniquely shared matches, and includes measures of the balance of shared elements, a diagnostic that has proven to be useful in predicting cis-regulatory function. To demonstrate the utility of these tools, a temporally-restricted CNS neuroblast enhancer was used to identify other functionally related enhancers and analyze their structural organization. It is concluded that cis-Decoder reveals that co-regulating enhancers consist of combinations of overlapping shared sequence elements, providing insights into the mode of integration of multiple regulating transcription factors. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process (Brody, 2012).

DNA sequence conservation histograms of the Drosophila genome reveal that its non-coding DNA is made up of CSCs that are flanked by less-conserved ICR DNA. For example, a conservation histogram of the Drosophila melanogaster vvl gene transcribed region and 60 kb of 3′ flanking DNA (located on the 3L chromosome) identifies multiple peaks of conserved DNA that are flanked by less conserved DNA sequences. EvoPrint analysis reveals that the CSCs can be further resolved into multiple smaller conserved sequence blocks (CSBs). Most regions of chromosomes 2 and 3 gave a similar pattern of CSC density and distribution, while in general CSCs on the X and the 4th chromosomes exhibited less conservation among the twelve species. cis-Decoder alignment of CSBs constituting a CSC identifies both repeat and palindromic sequence (RPS) elements, of ≥ 6 bp in length, and reveals that these account for more than half of the CSC's conserved sequences. A 6.4-kb genomic region was selected because two of its CSCs (vvl-41 and vvl-43) were tested for their regulatory behavior in this study. A previous analysis of enhancer sequence conservation has shown that individual enhancers can be identified by the maintenance of their CSB cluster integrity across Drosophila species, while ICR regions show greater sequence length variability (Kuzin, 2009; Brody, 2012 and references therein).

As a first step in the identification of structurally related CSCs, a genome-wide database of Drosophila CSCs was created by EvoPrinting most of the euchromatic genome of Drosophila melanogaster and nearly all of the previously in vivo characterized enhancers that are included in the REDfly database. Database CSCs were extracted from more than 4,000 author-generated EvoPrints that generally spanned 15–30 kb of genomic DNA. EvoPrints of fewer bases were used depending on genomic context and availability of gap-free sequence data in the orthologous regions of the different species. Most EvoPrints included all of the available melanogaster group drosophilids (D. melanogaster, D. simulins, D. sechellia, D. yakuba, D. erecta, and D. ananassae), one of the obscura group (D. pseudoobscura or D. persimilis), and two to four orthologous regions selected from the more evolutionary distant species: D. willistoni, D. virilis, D. mojavensis, and/or D.grimshawi species. Most of the EvoPrints represented a combined evolutionary divergence of >150 My. Under these conditions, open reading frames that encode conserved protein domains do not show conservation in most of the codon wobble positions, indicating that the additive evolutionary divergence represented in each EvoPrint is sufficient to reveal with near base-pair resolution those sequences that are essential for gene function. EvoPrints of open reading frames, using different combinations of species, reveal that the lack of sequence conservation in the amino acid codon wobble position is not the result of different codon preferences between species (Brody, 2012).

To enhance the detection of conserved DNA and avoid alignment inaccuracies triggered by DNA sequencing errors, sequencing gaps, rearrangements, or genome assembly problems that were unique to any one of the species used in the analysis, relaxed EvoPrint readouts were employed to identify CSCs. A relaxed EvoPrint highlights sequences that are present in all or all but one of the orthologous DNAs used to generate the print. Species with sequencing gaps (identified as blocks of species-specific differences in the color-coded relaxed EvoPrint readouts or identified as gaps in the EvoPrinter scorecard) were avoided in generating EvoPrints, and second and third scoring pair-wise alignments were included in the analysis when rearrangements were detected (Brody, 2012).

To catalogue CSCs, EvoPrints were entered into the EvoPrint CSC cutter algorithm to isolate and annotate individual CSCs separated by at least 150 bp of less-conserved DNA. This program also assigns a file name and consecutive numbers to each CSC in an EvoPrint. In order to insure that enhancers that contain CSB separation gaps of 150 bases or more were not truncated, CSCs were also parsed independently two additional times using ICR cutoffs of 200 and 250 bp. Duplicates are given the same name but an additional notation to distinguish them. Therefore, clusters that were parsed multiple times (∼20% of the database CSCs), due to their having non-conserved intervals >150 or >200 but <250 bases, are present two or three times in the database. The database contains >100,000 non-redundant clusters. To expedite database searches, in addition to cataloging individual CSCs and their CSBs, RPS elements of 6 bp or longer were pre-identified by intra-CSC CSB alignments and stored in the database. Most CSCs that contain more than 150 bp of conserved DNA have RPS elements that account for >50 % of their sequences (Brody, 2012).

CSCs from all previously in vivo characterized enhancers were also included by EvoPrinting all entries in the REDfly database; these are identified in the CSC-database by their REDfly designations. Although most of these CSCs duplicate database entries, CSCs that represent the same region can be identified by their similar cis-Decoder scores and/or their similar identifying names. It should be noted that many REDfly entries were made from data that often did not delimit the exact boundaries of the enhancer. In addition many REDfly entries included multiple CSCs or truncated CSCs whose ends were restriction enzyme sites used for cloning purposes and were not within less-conserved ICRs. To reduce the number of truncated entries, EvoPrinted regions were expanded to include flanking ICRs. Also, since many REDfly entries are redundant, care was taken to eliminate this redundancy by eliminating repeated and overlapping entries (Brody, 2012).

The first step in a CSC database search is to enter into the cis-Decoder input window an EvoPrinted enhancer that spans a single CSC. cis-Decoder then parses and annotates constituent CSBs in forward and reverse/complement directions. By alignment of the CSBs to one another, the program next identifies multi-copy and palindromic elements that are ≥6 bp. A table is generated that shows the copy-number of each repeat, the element frequency in the database, and the number of database CSCs that contain two or more of each element. Based on an earlier analysis of known enhancers, matches of less than 6 bp in length were not considered, because searches with 5 bases or less yielded results that were not informative (Brody, 2012).

After identifying RPS elements, the cis-Decoder algorithm searches the CSC database to discover CSCs containing these repeats. The search algorithm also allows for user supplied mandatory sequences, to identify enhancers that are regulated by sequence-specific DNA-binding factors or families of transcription factors. Once database CSCs are identified, the program carries out individual CSB alignments between the input CSC and the database CSCs. Another set of algorithms then rates the individual database CSCs using the following similarity indices when compared to the input CSC: (1) A repeat balance profile, that assesses relative shared repeat copy numbers and weighs them according to the RPS length (shown as a pie chart and as a repeat balance map, which are accessible from the one-on-one alignment page; (2) A correlation coefficient, which reflects the relative frequency of shared sequence elements between the input and database CSCs; (3) The number of shared repeats (full-length RPS elements and shorter elements contained within longer input repeats); (4) Total number of shared elements including RPS and uniquely shared sequences; (5) Percent coverage of aligning input sequences, which reflects the number of conserved bases in the database CSC that align with the input enhancer CSBs, normalized to the total number of conserved sequences in the database cluster; (6) The number of user-specified required elements present in the database CSC; (7) The longest shared sequence between the input and database CSCs (viewed at the cis-Decoder scorecard by placing the cursor on the sequence length number); and (8) The total number of conserved bases within the database CSC. To allow the user to focus attention on any one of the rating criteria, the CSCs can be sorted by any of the similarity indices in addition to sorting by CSC file name. Sorting by file name allows for the rapid identification of closely associated, neighboring CSCs that are structurally related to the input enhancer (Brody, 2012).

To demonstrate the utility of cis-Decoder database search algorithms to identify tissue- and temporal-specific enhancers, one of the late-temporal network NB enhancers (database CSC cas-6) was used that controls the embryonic expression of the gene encoding Cas, a zinc-finger transcription factor expressed during late embryonic CNS NB lineage development. Like endogenous cas mRNA expression, the cas-6 enhancer activates reporter transgene expression in CNS NBs and ventral cord midline cells during embryonic stage 10 and in additional ventral cord and cephalic lobe NBs during stages 11–13. EvoPrint analysis reveals that the cas-6 CSC is made up of 46 CSBs of 6 bp or more and contains 720 conserved base pairs in 1,613 bp of genomic sequence. Mutational analysis of the cas-6 CSC via 5' and 3' deletions revealed that the entire cluster was required for full reporter activity (A. Kuzin, unpublished results cited in Brody, 2012). The cas-6 CSC is located 392 bp 5' to the cas gene predicted transcriptional start site. As described above, one of the first steps in the cis-Decoder analysis is parsing CSBs from the input EvoPrinted enhancer in both forward and reverse directions, and then aligning the CSBs with one another (self-alignment) to discover RPS elements. More than 65% of the conserved bases in the cas-6 CSBs were represented in RPS elements; an alignment revealed that these are either separate, adjacent, or overlapping each other. Core DNA-binding motifs for known transcription factors within CSBs are indicated in the figure (Brody, 2012).

Prominent among the cas-6 RPS elements are three 10mer repeat motifs [TTATGCAAAT], which contain a POU-homeodomain-octamer-binding site [ATGCAAAT]. The highest copy number element [ATGCAAA], containing 7 of the 8 octamer motif sequences, was found 5 times. It is considered a sub-repeat element, since there is only one instance of the heptamer in the CSBs that is independent of longer elements. Also present are multiple elements containing the core ATTA sequence for Antennapedia class homeodomain containing transcription factors. Also present in the RPS elements are two palindromic E-box sequences, CAATTG and CAGCTG, while three additional E-boxes are present in conserved non-repeated sequences. The cas-6 enhancer CSBs also contains Hunchback and Cas core DNA-binding sequences. Given that many of the cas-6 RPS elements are novel sequences, they most likely contain additional binding sites for as yet uncharacterized transcription factors that modulate enhancer regulatory behavior (Brody, 2012).

To identify database CSCs that share repeat and unique elements with the cas-6 CSC, a search was initiated by first identifying CSCs that contained at least three copies of the ATGCAAA element. Although asking for a mandatory sequence is not required, the cas-6 RPS table revealed that the highest copy number element, ATGCAAA, was present 7,208 times in the CSC database and 371 CSCs contained two or more of these elements. The cis-Decoder scorecard for this search revealed that the database contained 104 CSCs with 3 or more of this element. Thus, the search was focused on this limited set of CSCs. Once these CSCs were identified, one-on-one alignments between the input and database CSBs were automatically performed to discover additional shared sequence elements. As expected, the highest scoring database CSC for most of the indices was cas-6 itself. Other high-scoring enhancers were considered as candidate late temporal network NB enhancers and were tested in enhancer-reporter transgenes. For example, while cg7229-5 scored highest for the correlation coefficient, other CSCs scored higher for each of the other metrics (Brody, 2012).

Although the search required the hepamer sequence ATGCAAA to be present at least three times in the database CSC, most of the highest-scoring CSCs (both for correlation coefficients and shared RPS elements) contained at least three RPS elements with the full octamer motif [ATGCAAAT], including cg7229-5, grh-15, vvl-41, and tkr-15. In addition, many of the CSCs that contained octamer motifs also shared, with cas-6, single or different combinations of bHLH E-box DNA-binding sites and repeated HOX-binding sites, including shared sequences flanking the core ATTA motif. One-on-one alignments between cas-6 and related database enhancers reveal different multi-copy repeats are nested within larger unique matches. For example, RPS elements corresponding to a HOX site are seen overlaping a POU-octamer site. This view of overlapping shared motifs represents a map of the substructure of an enhancer in terms of the transcription factor–binding sites that integrate multiple regulatory inputs (Brody, 2012).

cis-Decoder also generates lists sequence elements that are shared between the input and database CSC. Fifty-seven percent of the cg7229-5 conserved sequences aligned with cas-6 conserved sequences. In addition, cis-Decoder also identifies RPS elements within the input and database CSC that are not shared between the two CSCs, and these elements are also listed on the one-on-one alignment page (Brody, 2012).

The relative frequency of appearance of sequences in cg7229-5 that correspond to cas-6 RPS elements is shown by color-coded highlights. This comparison is termed a “repeat balance map,” a visual representation that illustrates the relative frequency of appearance of each of the shared motifs in the comparison between the input and database enhancers. Forty-six percent of the aligning bases within the cg7229-5 CSC are present in the same ratio in the cas-6 CSC. The predominance indicates that many of the shared elements in the two enhancers are present at equal frequency. Another example of a CSC identified in this search that shares balanced RPS elements with the input cas-6 is the grh-15 CSC, also a temporal network NB enhancer (see below) (Brody, 2012).

To test the in vivo cis-regulatory activity of CSCs, CSCs were selected that contained both repeat and unique sequence elements found in the cas-6 enhancer. The CSCs were selected based on rating criteria described above. Enhancer-reporter transgene transformants for the individual CSCs were generated using the targeted φC31 integration system to ensure that the regulatory behavior for each was assessed in the same genomic environment. Although not an exact match, the expression pattern of the cg7229-5 enhancer transgene shares many of the expression dynamics of the cas-6 enhancer-transgene. As with cas-6, onset of cg7229-5 expression is in a subset of midline cells and a single lateral NB at stage 10, and expression in subsequent stages closely matches, but is not identical to, expression of the cas-6 reporter. The insert shows that cg7229-5 reporter GFP expression overlaps but is not identical to that of cas-6 red fluorescent protein reporter (Brody, 2012).

Many of the tested CSCs yielded detectable CNS expression and function as late temporal network CNS neuroblast enhancers. Eleven were expressed in late temporal network ventral cord NBs and three were expressed in other CNS precursors or neurons. Comparing these expression patterns to the cas-6 reporter expression, it is apparent that each functions as a late temporal network enhancer. An indication of the specificity of the search for cas-6-like enhancers is that the search did not identify early temporal NB enhancers, nor did it identify broadly expressed NB enhancers such as that of deadpan (Brody, 2012).

Although the cas-6-related enhancers are active in overlapping neural precursor cells, each has its own unique cis-regulatory identity. Each has a different pattern of expression in subsets of NBs, GMCs, and/or nascent neurons. For example, three identified enhancers (nab-1, CG6559-28, and tkr-15) exhibit early expression in a subset of ventral cord midline cells, while sqz-11 and vvl-41 (identified using cas-8 as the input CSC) exhibit onset in a larger number of midline cells while other enhancers do not activate reporter expression in the midline precursor cells. The cas-8 CSC activated reporter expression in many more precursors at stage 11 than any of the other reporter constructs. tkr-15 is expressed in many cells at stage 11. Since these cells are too small to be considered NBs, they are most likely GMCs or nascent neurons. Comparing different transgene reporter expression patterns in lateral ventral cord cells at stage 11 reveals that for certain CSCs, in particular sqz-11, ct-3, [identified using the pdm-2 NB enhancer as input, fewer lateral cells express, or they exhibit uniquely different spatial expression patterns. This is also true for ct-14 (identified using combined cas-6 and CG6559-28 as input) and vvl-41 (identified cas-8 as input). cas-6 and cas-8 enhancers both drive reporter expression in overlapping subsets of cells that represent sub-patterns of endogenous cas expression (Brody, 2012).

These studies also revealed that there is no apparent consistency in the ordering, overlap, or orientation of shared elements between functionally related enhancers. For example, RPS elements shared between cas-6, cg7229-5, and grh-15 appear in unique contexts within each enhancer. This lack of consistency in positioning of shared elements has also been noted in early sub-lineage NB enhancers (Brody, 2012).

During the functional analysis of database CSCs that share RPS elements with cas-6, one of the CSCs, vvl-43, was found to share 92 RPS and unique sequence elements with cas-6. It did not, however, drive transgene reporter expression in NBs but activated expression instead in the embryonic ectoderm. cis-Decoder analysis of the shared RPS elements revealed that the balance of PRS elements was markedly different between cas-6 and vvl-43. Notable is the large number of conserved HOX motifs within vvl-43 in comparison to cas-6. Expression of vvl-43 in the embryonic ectoderm is segmental, and although temporally late, there is no embryonic CNS expression. Previous studies demonstrate that the vvl-encoded protein, a POU homeodomain factor, is expressed in the CNS and in the ectoderm of embryos, suggesting that vvl-43 functions as an ectodermal enhancer for vvl expression. The disparity of shared element frequencies between cas-6 and vvl-43 is in marked contrast to the similarity of frequencies when comparing cas-6 and cg7229-5. That lack of balance in shared element copy numbers between enhancers suggests that they may have different regulatory behaviors (Brody, 2012).

Another example of how unbalanced RPS elements indicate functionally different enhancers can be seen in the comparative analysis of vvl-41 with vvl-43 CSCs. Like the previous comparisons to cas-6, the vvl-41 and vvl-43 CSCs share similar elements; vvl-41 shares 96 RPS and unique elements with vvl-43 CSCs, and 68% of the vvl-43 conserved sequences are covered by these shared elements. Although these two CSCs have extensive overlap of shared elements, the repeat balance index and correlation coefficient reveal that their shared elements are not balanced in copy number. Consistent with the imbalance in their shared elements, these enhancers displayed markedly different regulatory behaviors in the embryo. Nevertheless, these two enhancers drive reporter expression in different sets of larval neurons. Whereas most of the cells expressing the vvl-41 reporter transgene are sub-esophageal ganglion interneurons, vvl-43 enhancer drives reporter expression in a subset of ventral cord motor neurons. Thus the presence of identical elements in different clusters does not necessarily lead to similar regulatory behaviors, and comparing shared element copy-numbers has a better predictive value for determining enhancer behavior (Brody, 2012).

To further test the ability of cis-Decoder database searches to identify different families of functionally related enhancers and to compare cis-Decoder search protocols to other enhancer search algorithms, database searches were initiated with different well-characterized enhancer types. Using the Krüppel gap enhancer Kr_CD1, the giant gt_(−10) enhancer was identified. Besides sharing HOX sites with different flanking bases, the two enhancer CSCs also share a 14-bp sequence, TGAACTAAATCCGG. Remarkably, this 14-bp element within the Krüppel enhancer was identified as a site of competitive binding by the activator Bicoid and the repressor Knirps transcription factors. The conservation of interlocking or overlapping docking sites for Bicoid and Knirps within both of these gap enhancers supports the contention that large CSBs (containing 7 to 10 bp or more) most likely function as the point of integration of multiple transcription factors in the regulation of enhancer behavior (Brody, 2012).

The search using the Kr_CD1 also identified the kni_(+1) intronic gap enhancer. Shared sequence motifs between Kr_CD1 and kni_(+1) include multiple polyA/polyT motifs, presumably targets of Hunchback, that are found in even balance (five copies) between the two enhancers. Other shared sequences include several HOX-binding sequence elements (Brody, 2012).

Previous work has shown that many segmentation genes utilize multiple enhancers that regulate gene expression in nearly identical patterns. These enhancer pairs have been termed (1) primary enhancers, found closely associated with the transcriptional start site, and (2) “shadow” enhancers, found at a distance from the structural gene. Starting with the primary vnd ventral neuroectoderm enhancer CSC, a cis-Decoder search identified its shadow enhancer based on the balanced copy number appearance of its RPS elements and uniquely shared sequences. In addition to other shared elements, both of these enhancers contain 2 copies of the CACATGA bHLH motif, which matches the optimal DNA-binding site for the transcriptional regulator Twist (Brody, 2012).

The cis-Decoder search algorithms were tested to see if it would be possible to detect enhancers regulated by Notch signaling. Previously identified Notch-targeted enhancers include those associated with the E(spl) complex genes. Multiple alternative binding sites within these enhancers have been identified for Suppressor of Hairless [Su(H)], the transcription factor utilized by the Notch pathway. A cis-Decoder search was initiated with one of the CSCs (Espl-1) to discover other similarly structured CSCs, using as required sequences a single Su(H)-binding site (TGGGAA) and a single bHLH-binding site (CAGCTG). This search resulted in 101 database hits, including CSCs from known Su(H) targets m2, m6, and mγ as well as putative enhancers for the neural determinants Dichaete, deadpan, nervy, tailless, castor, Fps85D, Notum, and extra macrochaetae. In addition, searching with the Notch-targeted deadpan NB enhancer (cis-Decoder CSC dpn-3), that contains two alternative Su(H)-binding sites (GTGAGAA), other putative Notch pathway targeted enhancers were identified: CG7229-5, cas-8, a HLHmβ-associate CSC (HLHmbeta-2), and the m4 PNS enhancer. Thus, cis-Decoder searches can identify functionally related enhancers that regulate gene expression during different phases of development and in different tissues (Brody, 2012).

Each of the embryonic NB enhancers identified above were also tested for regulatory activity during later stages of development, and many were observed to activate transgene reporter expression in the third instar larva and/or adult CNS. Three of the tested enhancer transgene reporters, cg6559-28, grh-15, and tkr-15 exhibited expression in a similar pattern within brain neural precursor cells, thoracic neuromeres and posterior neural precursors of the third instar larva CNS, while the cas-6 and cas-8 enhancers were not active in larvae. The ct-3 and ct-14 CSCs drove expression in small subsets of neurons in the sub-esophageal ganglion and in the ventral cord abdominal neuromeres. Additionally, nab-1 expression was similar to that of the dnabe310 enhancer-trap expression in third-instar larvae CNS. In the adult, many of the enhancers were expressed in a subset of central brain neurons, and in the optic lobe. Specifically, cg6559-28, vvl-14, and nab-1 reporters were expressed in the mushroom body. While cas-6 was not expressed in the adult brain, cas-8 reporter expression was detected in the ellipsoid body in a pattern similar to cas adult expression. The embryonic and adult reporter expression was tested of another 60 CSCs, chosen by a variety of criteria. Many of these activate transgene reporter expression in both the embryonic and adult CNS. Given the fact that CSC sub-regions of these multiuse enhancers have not been tested for reporter activity, it cannot be ruled out that different regions within the cluster have autonomous functions and represent discrete enhancers. However, functional analysis of the nerfin-1 NB enhancer and the cas-6 enhancer CSCs has revealed that full enhancer function requires the complete cluster. The EvoPrinter algorithm provides a methodology for testing for the close apposition of independent enhancers (Brody, 2012).

Although each of the cis-Decoder scorecard indices provides useful information in judging the relationship of the input enhancer to database CSCs, the repeat balance index and the correlation coefficient are more accurate indices when searching for functionally related enhancers, since they take into account not only the number of shared elements but also the RPS copy number balance between the input enhancer and database CSC. The percent alignment coverage is likewise an important indicator of the relationship between the input and database CSCs. Thus, sorting the scorecard by the repeat balance index or by the correlation coefficient increases the likelihood that functionally related enhancers rank at the top of the list. For example, all of the late temporal NB enhancers identified in this study had repeat balance index scores of greater than 1.0, correlation coefficient rankings of above 0.4, and percent coverage of ≥40% (Brody, 2012).

To estimate the number of false-positive predictions and functionally related enhancers that were missed in cis-Decoder searches, the cas-6 was used as the input enhancer. The search returned 111 database hits, of which 27 that shared many repeat elements with cas-6 were tested for enhancer activity in flies. Of these, 12 proved to be late temporal network enhancers, with each being expressed in a different subset of midline, brain, and/or ventral cord neuroblasts. Eleven were expressed exclusively either in adult brain, larval precursors, or in embryonic neurons, and four were considered negative, since their reporter expression was undetectable or found in other tissues other than the nervous system. As for enhancers that were missed in the search, late temporal network enhancers were identified that do not contain three or more complete or partial octamer sequences, or do not score highly using cas-6 as input. The low-scoring enhancers included sqz-11 and vvl-41, which were discovered using cas-8 as the input CSC (mentioned above). Likewise, ct-3 and ct-14 did not contain three octamer sequences, and they also proved to be late temporal network NB enhancers. Finally, five other late temporal network enhancers were identified that do not contain octamer motifs but do contain other repeated elements found in late temporal network enhancers. It is clear from these results that a search for enhancers using a mandatory sequence, such as the octamer motif, is insufficient to detect the full genomic repertoire of late temporal network enhancers. To identify as many functionally related enhancers as possible, multiple database searches using different search criteria, are recommended. Current understanding of the role of octamer motifs in conferring temporal gene expression is incomplete, in that it was not possible to fully distinguish between embryonic late temporal network enhancers, and octamer-site rich larval or adult brain enhancers. Nevertheless, the fact that only four of the 27 clusters tested were not expressed in the CNS, speaks to the efficacy of cis-Decoder search algorithms in detecting neural enhancers (Brody, 2012).

Ideally, it would be useful to make direct comparisons of the cis-Decoder algorithm with other web-based tools for discovery and analysis of cis-regulatory elements. However, not all search programs use evolutionary comparisons, and those that do use different levels of evolutionary divergence to identify conserved sequences in enhancers. The comparative analysis of enhancer discovery programs nevertheless points to factors present in various computational formats that appear to be important for successful cis-regulatory element prediction. These include sequence conservation between related species, motif clustering, and availability of prior information on the presence of known transcription factor–binding sites. In this context, combined use of cis-Decoder methodology with Chip-Seq data, that shows occupancy of cis-regulatory modules by specific transcription factors, will improve identification of functional motifs within enhancers that are bound by specific transcription factors, and resolves additional functionally important flanking sequences. The libraries of repeat and uniquely shared sequences generated by cis-Decoder are useful for sub-structural analysis of enhancers; for example, discovery of the unique element shared by Krüppel and giant gap enhancers demonstrates the ability of cis-Decoder to reveal combinatorial interactions by analysis of blocks of conserved sequences. Other aspects of cis-regulatory biology will also be relevant; for example, the configuration of the chromatin as detected by DNase1 hypersensitivity indicates accessibility of enhancer sequences to transcriptional regulators. The knowledge of chromatin state is invaluable for prediction of enhancer activity, and information concerning specific CSCs can be accessed via the UCSC browser (Brody, 2012).

Efficacy of cis-Decoder in predicting enhancers can be compared to a study that used known cis-regulatory modules to develop a training set of computationally predicted transcription factor–binding sites to predict genomic cis-regulatory modules (Rouault, 2010). That study predicted neural expression of the same cg7229 enhancer that was identified using cis-Decoder. Likewise an algorithm known as Ahab, which uses transcription-factor-binding-site information for known regulators of cellular blastoderm enhancers, successfully predicted the gt_(−10) and kni(+1) gap enhancers (Schroeder, 2004) that also scored highly in a search using the Kr_CD1 gap enhancer as the input CSC. It is important to point out that cis-Decoder search protocols make direct use of CSC information for enhancer prediction, while other resources, such as Genome Surveyor, use site conservation as a criterion, but do not provide information to infer enhancer boundaries. Given that multiple enhancer prediction programs that employ different search criteria are available, it would be advisable to employ several discovery programs before settling on a final list of candidate genomic regions for analysis in enhancer-reporter transgenic studies (Brody, 2012).

The comparative analysis of enhancers described in this report and an additional 60 enhancers, have yielded the following observations considering enhancer structure and behavior: (1) Functionally related enhancers can be identified based on their balanced copy numbers of shared conserved repeat elements. (2) Enhancers that have extensive shared conserved sequence elements (often >60%), but do not have balanced shared repeat copy numbers, may display significantly different regulatory behaviors. (3) Shared repeat and unique elements between functionally related enhancers are not found in any fixed order or orientation. (4) Similarly regulating families of enhancers need not share specific sets of conserved sequence elements, since different enhancers can accomplish the same regulatory behavior with different but overlapping sets of conserved elements. (5) Enhancers that share conserved repeat elements and perform related cis-regulatory functions also contain unique sets of repeat elements that are only partially shared with other related enhancers (Brody, 2012).

These observations have revealed that Drosophila CNS developmental enhancers are highly complex, based on their conserved sequence composition, and many have proven to be multifunctional. The observed complexity of enhancers, specifically with regard to multi-copy repeat motifs, also suggests that enhancer function is realized through a complex process involving combinatorial interactions among many factors and cannot be easily explained by single activator/repressor transcription factor switches. In addition, the fact that functionally diverse enhancers can display such extensive overlap in their conserved sequences underscores the combinatorial complexity of cis-regulation. Because of the lack of fixed order and orientation of shared elements between related enhancers, only the alignment flexibility of the cis-Decoder CSB aligner can rapidly detect the extent and makeup of shared conserved sequences between different enhancers. Until now, enhancer boundaries have, for the most part, been resolved by reporter transgene deletion analysis. The addition of evolutionary clustering of conserved sequences to this identification process will aid in enhancer identification and allow for an assessment of their structure and spatial constraints. cis-Decoder algorithms also allow one to generate libraries of conserved sequence elements that are shared among enhancers; this dataset will be useful for understanding the combinatorial complexity of tissue-specific gene regulation (Brody, 2012).

Structure and cis-regulatory analysis of a Drosophila grainyhead neuroblast enhancer

Evolutionary analysis of cis-regulatory DNA reveals that enhancers consist of clusters of conserved sequence blocks (CSBs) that are made up of both unique and repeated sequence elements. This study set out to address aspects of Drosophila neuroblast (NB) enhancer structure, including the basis for enhancer spatial and temporal regulation and the functional significance of their individual CSBs. A search for temporally restricted CNS NB enhancers identified an enhancer within the transcription factor grainyhead (grh) gene locus. The intronic enhancer, grh-15, contains two separable semi-autonomous activities, one that drives expression predominantly within the developing brain NBs and another in ventral cord NBs. To gain insight into the function of the CSBs constituting the brain-specific enhancer, each CSB was systematically deleted, and the activity of the altered enhancer was compared to that of the full brain-specific enhancer. While the results indicate that information regulating enhancer activity is highly redundant, individual CSBs were found convey expression in subsets of larval lineages that are generated from either Type I or Type II NBs. These studies also highlight how evolutionary sequence conservation can be used as a guide the functional analysis of cis-regulatory DNA (Kuzin, 2018).

The goal of this study was to functionally test the role of CSBs within the grh-15a brain NB enhancer. To assure accurate comparison of the full grh-15a enhancer activity to enhancers with CSB deletions, third instar transgenic larvae carrying two transgenes were examined: the full enhancer and the deletion constructs. The readouts were dual-labelled larval brain preparations in which the pattern of full grh-15a enhancer activity was compared to grh-15a enhancers with deleted CSBs (Kuzin, 2018).

It was found that grh-15a brain enhancer CSBs exhibit overlapping but distinct roles in controlling gene expression in the larval CNS during development. The full grh-15 enhancer consists of 24 clustered CSBs that potentially serve as docking sites for one or more transcription factors and identified two semi-independent clusters within grh-15 that drove expression in embryonic brain and ventral cord NBs (Kuzin, 2018).

In order to examine the evolutionary integrity of the grh-15 NB enhancer, the structure of its conserved sequence cluster (CSC) was examined by EvoPrint analysis using twelve Drosophila species. The grh-15 enhancer contains 24 CSBs and is located in the grh 10th intron (genomic position chr2R: 17829129-17829979, BDGP release 6), downstream of the 3rd transcriptional start site. This analysis also revealed a non-conserved region in the middle of the D. virilis, D. mojavensis and D. grimshawii enhancer orthologues. The no-conserved region is not observed in D. willistoni or other species used in EvoPrint analysis, suggesting that there may be two independent enhancer activities. To test for the presence of independent enhancer activities within the D. melanogaster grh-15 CSC, the cis-regulatory activity of the separate CSB clusters was examined in embryos and larvae. The full grh-15 enhancer drove reporter expression in the brain and ventral cord of the embryo in late temporal network NBs and in ~30 lineages of the 3rd instar larval brain and ventral cord. The distal part of the enhancer (grh-15a) drove reporter expression in the embryo in late temporal network brain NBs and in a single NB in each hemisegment of the ventral cord, while the proximal half (grh-15b) activates expression in a ventral cord NBs, with only weak expression in the brain. Similarly, in the third instar larval CNS the complete grh-15 CSC activated expression in brain and ventral cord lineages; grh-15a drove reporter expression in ~25 brain lineages, only partially overlapping the lineages marked by the full CSC, and in 14 ventral cord lineages; grh-15b drove expression in a small number of ventral cord neurons. grh-15a was expressed in fewer brain lineages than the full grh-15 CSC, and grh-15b did not activate the full expression in a pattern corresponding to the ventral cord pattern of grh-15. These results suggest that the grh-15 CSC consists of two semi-autonomous enhancers, one able to drive reporter expression mainly in brain lineages, while the second half activates expression mainly in ventral cord NB linages (Kuzin, 2018).

To compare grh-15a to endogenous grh protein expression, third instar larvae expressing a grh-15a-CD8-GFP transgene was costained with antibodies to grh protein and to GFP. While all lineages marked with CD8-GFP exhibited nuclear grh protein, additional Grh+ cells were apparent that were not associated with CD8-GFP, suggesting that grh-15a drives expression in only a subset of Grh+ lineages. This result suggests that more than one grh NB enhancer is required to control the full NB expression of the grh gene (Kuzin, 2018).

The previous identification of another grh NB enhancer, led to an examination of the presence of CSB clusters within the fragment identified in the Prokop study (1998) and its enhancer activity was compared to grh-15a. The CSC, termed grh-9, contains 21 CSBs and is located in the third intron (genomic position chr2R: 17814850-17816520, BDGP release 6). A reporter transgene was generated using the CSC contained within this fragment. The embryonic expression pattern indicated that it was sufficient for the embryonic expression of the previously identified larger fragment. Transgenic enhancer-reporter construct of the grh-9 cluster also drove expression in numerous 3rd instar larval brain and ventral cord NB lineages When cis-regulatory activity of grh-15a was compared to the grh-9 enhancer it was found that grh-9 activated expression in more larval lineages than grh-15a and with only partial overlap between grh-15a and grh-9 lineages. (Kuzin, 2018).

There are two types of NBs in the larval brain: Type I NBs undergo continued generation of GMCs as they self-renew, and Type II produce intermediate neural progenitors that undergo several self-renewing divisions to amplify the number of progeny produced by each NB. Type II NBs are marked by expression of deadpan (dpn), while Type I NBs are marked by co-expression of asense (ase) and dpn. To determine whether the NBs marked by the expression of grh-15a are Type I or Type II, third instar larval brains were triple-stained for grh-15a-GFP activity and with Ase and Dpn antibodies. Examination of serial sections revealed four Dpn+ Ase- NBs (Kuzin, 2018).

Expression of grh-15a in Type II NBs was examined. The Dll reporter expressed in dorso-medial NBs. Each of these were identical to cells marked by grh-15a-CD8-GFP. In the current study the Dll reporter expressed in five lineages. Co-expression of Dll-Gal4 and grh-15a identified a subset of Grh+ NBs as dorsomedial (DM) Type II NBs (Kuzin, 2018).

The grh-15a enhancer drives expression in discrete sets of neural lineages. In the following description of grh15a-CD8-GFP expression, six different lineages are referred to: 1) the anterior-medial group (AM) of 5 lineages that includes one anterior-medial cell that projects across the midline; 2) the Dorsal group (D) of 10 lineages, which included four dorsal Type II NB lineages that, for the most part, project towards the midline; 3) the anterior group (A) of 5 lineages that project to the center of the brain lobe; 4) a posterior group (P) of about 5 lineages that projects anteriorly; 5) four lineages posterior and ventral to the central brain that are presumably in the sub-esophageal zone; and 6) paired medial ventral cord lineages. Full-sized enhancers activating either GFP or RFP were analyzed in heterozygote flies, confirming that each of the lineages expressed the full-length GFP and RFP reporters to the same extent in third instar larval brain of transgene flies (Kuzin, 2018).

Each CSB was individually deleted from grh-15a to examine its role in enhancer function. Except for a few distinctive lineages that contain unique axonal projections, it was not possible to uniquely identify all of the NB lineages. Nevertheless, each deletion/deficiency produced a distinctly different reproducible pattern, allowing generalizations to be made about the effects of CSB deletions (Kuzin, 2018).

Deletion analysis revealed that some of the CSBs resulted in distinct lineage-specific effects. Df-5, which lacks the longest of the ten CSBs examined in this study, is the only deletion/deficiency that resulted in lack of expression in the distinct anterior-medial lineage that projects posteriorly, then towards the midline and then crosses the midline. Df-5 enhancer/reporter construct also lacked expression in the clearly identifiable dorsal lineage whose projection joins AM-1 near the point it turns towards the midline. Df-5 also resulted in loss of expression in several additional dorsal and posterior lineages, but not in any of the anterior lineages. Finally, Df-5 resulted in heightened expression in several lineages, suggesting that the fragment contains repressive sequences. Whereas Df-1 lacked of expression in some anterior-medial lineages, most dorsal lineages, and all posterior lineages, GFP reporter was expressed in the two distinctive lineages and in the anterior lineages that project downwards to the center of the larval brain. Df-1 was the only deficiency that did not drive expression in most ventral cord lineages (Kuzin, 2018).

Only Df-2 resulted in lack of expression in a group of four clearly identifiable lineages that are located outside the larval brain lobe in a position that appears to be the sub-esophageal zone. Df-2 also appears to be irregularly upregulated (increased RFP expression) in anterior lineagesand in some dorsal lineages. Except for occasional lack in a few dorsal lineages, Df-3 did not show consistent lack of expression in other lineages (Kuzin, 2018).

Removal of CSBs containing POU domain TF binding sites, ATGCAAAT octamer motifs, Df-4, Df-6 and Df-10, resulted in the absence of reporter expression in many anterior medial, dorsal and posterior lineages. The smallest effect occurred for Df-4, which reduced expression in two AM lineagesand less than half the dorsal lineages. Expression was unaffected in the AM-1 lineage and in anterior lineages but was absent in two lineages that flank the AM-1 lineage. In most Df-6 brains, expression in D-1 and several other dorsal lineages appeared to be diminished. There was a marked apparent increase in expression in anterior lineages of Df-6, suggesting that the CSB removed by Df-6 might act to restrain expression in this lineage. Df-10 exhibited full expression in AM-1, but lacked expression in all AM lineages flanking AM-1. Expression of DF-10 was lacking in all dorsal lineages except for D-1 and in all posterior lineages (Kuzin, 2018).

Deficiency of CSB 7 resulted in lack of expression in the D-1 lineage, but this was not true for all replicates. Other anterior, dorsal and posterior lineages lacked reporter expression, and occasional lineages exhibited ectopic expression (Kuzin, 2018).

CSBs 8 and 9 both contain bHLH TF binding sites, CAGTTG and CAATTG respectively. Reporter expression of Df-8 revealed ectopic activation in dorsal lineages that project towards the midline; these expressed only light GFP in the wild-type construct. Similarly, relatively higher expression was often observed in other dorsal or posterior lineages, but this observation varied in different replicates. No alteration was observed in any of the clearly identifiable lineages. Df-9 showed lack of or reduced reporter expression in about half of the AM and dorsal lineages and occasional ectopic activation in a few dorsal lineages (Kuzin, 2018).

This study documents the different effects engendered by CSB deletion including lost reporter expression, a decrease or increase in expression level, and/or ectopic expression. In terms of number of CSBs within the enhancer, the grh-15 enhancer is comparable in complexity to other late temporal network NB enhancers this laboratory has analyzed. For example the cas5 NB enhancer, consisting of >60 CSBs of seven bases or greater, was separable into three sub-fragments that activated reporter expression in different subsets of NBs but not in all NBs that express the full cas-5 reporter transgene. Similarly the vvl-49 NB enhancer consisted of 20 CSBs, comparable in sequence complexity to the full grh-15 enhancer with 24 CSBs (Kuzin, 2018).

Currently the TFs that regulate grh-9 and grh-15a enhancer activities are unknown. Their overlapping expression domains could be due in part to their shared conserved TF binding sites. For example, both grh-15a and grh-9 contain three conserved POU-domain TF binding sites. The three octamer motifs in grh-15a are in CSBs 4, 6 and 10; deletion of any of these modifies enhancer activity in multiple lineages. grh-9 also contains an octamer motif with an overlapping HOX binding site (ATTATGCAAAT). grh-15a has two consensus E-box bHLH binding sites (CAGTTG in CSB 8, with the 6th base not conserved in D. grimshawi and CAATTG in CSB 9, conserved in all species). Deletion of CSBs containing these motifs also modified expression in multiple lineages. The grh-9 enhancer also has two conserved bHLH consensus E-boxes, one overlapping an octamer motif (CATTTGCAT) and a second in a longer conserved sequence (TTCAGGTG, bHLH motif). Potential candidate trans-acting factors binding to the octamer sites include Ventral Veins Lacking, POU domain transcription factors Nubbin and Pdm-2 and Pdm-3. In addition to the transcription factors, the Achaete-Scute bHLH TFs should also be considered candidate regulators of the grh enhancers. It is possible that different members of these families bind these motifs in different tissues. Further studies are needed to identify the TFs binding these motifs (Kuzin, 2018).

The full grh-15 CSC has a more complex structure of conserved sequences compared to other characterized Drosophila melanogaster enhancers. For example, the sparkling enhancer, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye, has 11 CSBs that contain three types of binding sites. Other examples include the ASE5 Suppressor of Hairless that has 10 CSBs; m-alpha, that has eight CSBs, and nerfin-1 NB enhancer, with 12 CSBs. The grh-15a fragment analyzed in this paper consisted of 10 CSBs comparable to these other smaller enhancers described in the literature (Kuzin, 2018).

Enhancer structural complexity can be described in terms of the multiple functions, e.g., the control of gene expression in different cell types and tissues or at different stages of development. This study found evidence of grh expression in a diversity of cells, including NBs, GMCs and neurons and also stage-specific expression during embryonic and larval development. Previous studies has similarly documented complex enhancer activity for other enhancers: nerfin-1 NB enhancer drives expression both in the embryo and larva, the cas-1 enhancer drives expression in the embryo, larva and adult and the vvl-41 CNS enhancer activates expression in the embryo, larva and adult. It is proposed that CSBs serve as enhanceosomes, in that they consist of overlapping binding sites for multiple TFs, whose functions integrate to produce the full expression pattern of the enhancer (Kuzin, 2018).

The term 'spatial regulation' can be used to describe gene expression in specific NB lineages that are spatially arrayed in the larval brain. Spatial regulation is the sum of activation and repression by transcription factors binding their cognate sites in tissue specific enhancer. This study has shown that removal of individual CSBs effects the grh-15a reporter expression in specific sub-populations of NBs and their lineages. Each CSB contributes to the full expression pattern. This study has documented the different effects engendered by CSB deletions including lost reporter expression, a decrease or increase in expression levels, and/or ectopic expression. It was observed the grh-15a CSBs act in a combinatorial fashion to give rise to the complete repertoire of NB gene expression. One of the goals of the study was to identify regulatory elements that determine the temporal expression of grh, a late temporal network determinate. No single CSB within grainyhead's brain enhancer was found to be key for temporal regulation: that is, deletion of single CSBs did not change the temporal window of enhancer activity, suggesting that the temporal regulation is coded for in a redundant fashion, e.g., more than one CSB contains the temporal information. Given these findings, it is speculated that CSBs of length greater than 10 to 20 bases are likely to integrate the activity of multiple transcription factors. Determination of the temporal factor(s) regulating this enhancer is a subject for further investigation (Kuzin, 2018).

Transcriptional Regulation

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but in the cells that surround it and will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).

The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).

To follow the movements of the spiracular chamber cells as they invaginate, constructs were examined that were made with particular enhancers of the cut, ems, and grh genes, each of which drive expression of beta-gal in a subset of cells that express the cut gene at stage 11. These enhancers do not drive the whole spiracular expression of their genes, but are good tools for studying cell specification and the morphogenetic movements of the posterior spiracle cells. The expression of cut in the posterior spiracle is controlled by at least three different enhancers, two of which have been used in this study. From stage 13, the ct-A4.2 enhancer marks the precursors of the four spiracular hairs. The grh-D4 enhancer of the grh gene is expressed in a single group of cells in this area. The expression of ems in the spiracle is driven by at least by one enhancer: ems-1.2. From stage 11 this enhancer marks a group of cells abutting the tracheal pit. Double stainings of the cut-D2.3, ems-1.2, and grh-D4 lacZ constructs show that they are expressed in non-overlapping subsets of cells. The correlation of the expression of these three constructs allows the fate mapping of the spiracular chamber primordium when it is a two dimensional sheet of cells. The different spatial expression of these enhancers at stage 11 shows that the two-dimensional sheet of cells is already patterned and that the cells invaginate to precise positions during development (Hu, 1999 and references therein).

The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).

Hedgehog signaling acts with the temporal cascade to promote neuroblast cell cycle exit

In postembryonic neuroblasts, transition in gene expression programs of a cascade of transcription factors (also known as the temporal series) acts together with the asymmetric division machinery to generate diverse neurons with distinct identities and regulate the end of neuroblast proliferation. However, the underlying mechanism of how this 'temporal series' acts during development remains unclear. This study shows that Hh signaling in the postembryonic brain is temporally regulated; excess (earlier onset of) Hh signaling causes premature neuroblast cell cycle exit and under-proliferation, whereas loss of Hh signaling causes delayed cell cycle exit and excess proliferation. Moreover, the Hh pathway functions downstream of Castor but upstream of Grainyhead, two components of the temporal series, to schedule neuroblast cell cycle exit. Interestingly, Hh is likely a target of Castor. Hence, Hh signaling provides a link between the temporal series and the asymmetric division machinery in scheduling the end of neurogenesis (Chai, 2013).

This study shows that Hh signaling functions during later postembryonic development and acts together with the NB temporal transcription factor cascade to regulate NB cell cycle exit. It was further demonstrated that hh is a downstream target of Cas, a member of temporal series that determines the time at which NBs terminate proliferation via down-regulation of Grh. While increased Hh signaling results in increased cell cycle length and premature NB cell cycle exit, loss of Hh signaling decreases NB cell cycle length and also prolongs the duration of NB proliferation (Chai, 2013).

Hh family proteins can act as short- or long-range morphogens covering distances as few as ten cell diameters (20 µm), or as far as a field containing many more cell diameters (200 µm). In the postembryonic brain, hh is expressed predominantly in the NBs and the newborn GMCs, whereas the expression of its target gene reporter, ptc-lacZ is observed in a narrow area covering the adjacent NB and the sibling GMCs, indicating a limited response to and suggesting a limited spread of Hh ligand. In addition, Hh protein is always found to be enriched and clustering around the NBs in a punctuated form rather than forming a gradient. These data, together with the lineage autonomous phenotype of hh mutant NB clones, strongly suggest that Hh acts locally at short range in the larval brain. This is consistent with the structural arrangement of the larval brain, where each NB lineage comprising of the NB itself, GMCs, and neurons, is encapsulated by a meshwork of glial processes that form a three-dimensional scaffold that potentially acts as a stem cell niche. Such a spatial arrangement may serve as a barrier to restrict spread of the ligand and confine signaling events within a particular lineage so that an individual NB lineage can development with considerable independence from its neighbouring lineages. Indeed, a NB clone derived from a hh null allele exhibits the GMC pool expansion phenotype even though GMCs from its neighbouring lineages are competent in producing Hh ligand (Chai, 2013).

While it is tempting to assume that Hh can also act on the GMCs in an autocrine mode of action judging from the presence of ptc-lacZ expression, no noticeable GMC fate transformation or change in their proliferative capability was seen in ptcS2 and smoIA3 clones. The higher mitotic rate in hh loss-of-function NBs could largely explain the amplification of the GMC pool and enlarged clone-size; however, a possible delay in GMC differentiation cannot be ruled out. The proposition that Hh ligand, which is produced by the NB and daughter GMCs, feeds back on the NB to control its own proliferative capacity and the timing of cell cycle exit is interesting but not totally unfamiliar. Similar feedback signalling mechanism has been demonstrated in the mouse brain in which post-mitotic neurons signal back to the progenitor to control cell fate decisions, as well as the number of neurons and glia produced during corticogenesis (Chai, 2013).

Hh signal reception is detectable in NBs as early as in L2 and persists throughout larval life and in early pupae when NBs undergo Pros-dependent cell cycle exit. This delay of approximately 96 h between the start of Hh reception and the ultimate outcome of cell cycle exit may be due to a requirement for cumulative exposure of NBs to increasing local concentrations of Hh. Such a graded response will enable the wt postembryonic NBs to progress from high to low proliferative stages before ceasing division, in line with the development of the larva. Evidence supporting this notion includes gradual accumulation of Hh on the NBs, lengthening of NB cell cycle time, as well as the necessity of high levels of Hh signaling to trigger cell cycle exit. It is worthwhile to note that even at pre-pupal stage during which most NBs are starting to undergo cell cycle exit, fewer than 20% of them are associated with Hh puncta at any point of time. One likely explanation is that not all the NB lineages within the larval central brain respond synchronously to Hh-mediated temporal transition. However, unlike the embryonic central nervous system in which hh expression is localized to rows 6-7 of the neuroectoderm, this study found it difficult to pinpoint a specific expression pattern in the postembryonic central brain due to the disorganized array of NB lineages. It is equally possible that different NBs exit cell cycle progression at different time points. This is also consistent with the structural organization of individual NB into different 'trophospongium' or stem cell niches (Hoyle, 1986). Nevertheless, the possiblility cannot be ruled out that Hh signal activation primes another yet-to-be-identified developmentally regulated signal/event to schedule NB cell cycle exit (Chai, 2013).

Interestingly, a recently proposed 'cell cycle length hypothesis' postulates that cell cycle length, particularly the length of G1 phase in neural stem cells acts as a switch to trigger the transition from proliferative to neurogenesis mode (Salomoni, 2010). In fact, experiments have shown that manipulation of cdk4/cyclinD1 expression and cdk2/cyclinE activity that result in the lengthening of G1 is sufficient to induce precocious neurogenesis; while inhibition of physiological lengthening of G1 delays neurogenesis and promotes expansion of intermediate progenitors. The curren results show that Drosophila postembryonic NBs in the central brain exhibit a comparable trend of cell cycle lengthening from young to old larval stages. Interestingly, NBs with excess Hh signaling have an extended cell cycle time, consistent with the idea that there is a forward shift of the 'perceived' age, leading to premature cell cycle exit. In contrast, Hh loss-of-function NBs have a shorter cell cycle time compared to their wt counterparts of the same actual age; hence, they have a younger 'perceived' age and are able to maintain their proliferative phase over a longer period of time. Consistent with this, it was shown that persistent NB proliferation in smoIA3 clones as well as the early termination of ptcS2 NBs proliferation, are always associated with the presence and absence of CycE expression, respectively. However, loss of Hh signaling in NBs merely extends their proliferative phase but is not sufficient to ensure perpetual proliferation as no mitotic NB is observed in the adult brain. It is also noted that a previous report suggested that the cell cycle time of the larval NBs reduced during their growth and reached a peak at late third instar with a minimum cell cycle time of 55 min. However, this study was conducted on thoracic NBs from the neuromeres T1 to T3, which have a very distinctive proliferative profile to the central brain NBs assayed in the current study. Indeed, has been shown in that abdominal NBs exhibit significantly different cell cycle times compared to their thoracic counterparts (Chai, 2013).

In Drosophila, the precise timing of NB cell cycle exit is governed by a highly regulated process that involves sequential expression of a series of transcription factors: Hb->Kr->Pdm1->Cas, known as the temporal series. It is known that the temporal series probably utilizes Grh in the postembryonic NBs to regulate Pros localization or apoptotic gene activity, thus determining the time at which proliferation ends. In addition, the temporal series also regulates postembryonic Chinmo->Br-C neuronal switch, which specifies the size and the identity of the neurons. The current data show that Hh signaling does not regulate early to late neuronal transition as Chinmo and Br-C expression timings appear unaffected in both ptc and smo mutant clones. In contrast, excess Hh signaling leads to a variety of features associated with NB cell cycle exit: (1) premature down-regulation of Grh, (2) nuclear localization of Pros (in NBs), and (3) reduction of NB size. Taken together with the extended proliferative duration of Hh loss-of-function NBs, it is apparent that Hh signaling is a potent effector of the temporal series and functions late to promote NB cell cycle exit (Chai, 2013).

The results from the current genetic interaction assays with Hh pathway components and grh reaffirmed the conclusions from previous studies that Grh is necessary to maintain the mitotic activity of the postembryonic NBs. The loss of Hh signaling keeps the central brain type I NBs in their proliferative state and this is largely contributed by persistent grh expression past their normal developmental timing at around 24 h APF. Even though Grh is necessary to extend the proliferative phase of these NBs, it is not sufficient to rescue all aspects of the premature cell cycle exit phenotype seen in ptc mutant NBs. Hence, down-regulation of grh by over-activating Hh signaling is not solely responsible for NB proliferative defects, and this implies that Hh signaling may terminate NB cell cycle via other mechanisms in addition to Grh (Chai, 2013).

The expression of hh appears to be dependent on the pulse of Cas expression at the transition between L1 and L2, as induction of cas mutant clones after that stage does not significantly affect hh expression. Moreover, ChIP assays suggest that Cas binds the hh genomic region, thereby placing Hh as a direct downstream target of the temporal series. However, it is intriguing to speculate on how the early pulse of Cas can mediate hh expression, which only comes on later during larval development. One possible explanation involves a relay mechanism in which that pulse of Cas activates an (or a cascade of) unknown components, which persist and eventually turns on the later hh expression. Yet, in such a model, Cas need not interact directly with the hh locus as the ChIP assay clearly suggests. Moreover, there are at least two pulses of hh expression during larval brain development, and the earlier, shorter pulse that is required for the activation of quiescent NBs appear to be independent from Cas regulation as Cas is only switched on in the larval NBs upon reactivation. Most importantly, the data show that mis-expression of cas abolishes, rather than triggers ectopic hh expression. Thus, the findings do not favour the continuous expression of a hh activator downstream of Cas. Alternatively, Cas may be involve in the epigenetic modifications of the hh locus such that it is primed for expression at a much later stage. This may also explain why saturating the system with Cas for prolonged period of time via mis-expression can negatively affect subsequent hh expression because of to its potential aberrant association with the chromatin. Although such a function has not been reported for Cas, previous studies have postulated that components of the temporal series, such as Hb (or mammalian homolog Ikaros) and Svp (or mammalian homolog COUP-TFI/II), play a role in modulating chromatin structure, hence modifying the competency of downstream gene expression subsequently (Chai, 2013).

The relationship between svp and Hh signaling within the postembryonic temporal series cascade is interesting yet unexpected. svp was thought to be a downstream component of cas on the basis of studies in postembryonic NBs in the thoracic segment of the ventral nerve. This is supported by the observations that the pulse Svp occurs at 40-60 h ALH following the pulse of Cas at 30-50 h ALH. Moreover, both svp and cas mutant clones affect Chinmo/Br-C neuronal target transition, apart from causing NBs' failure to exit the cell cycle at early pupal stage. However, examinations of Svp and Cas expression patterns in the central brain region in this study reveal that the Cas expression window overlaps with the peak of the Svp expression window, even though the latter has a much wider expression window in which low expression levels can still be detected in the NBs at 96 h ALH. Moreover, the data show that abolishment of cas function starting from the embryonic stage does not reduce Svp expression in the NBs at 24 h ALH. Hence, previous interpretation that svp functions downstream of cas in the thoracic postembryonic NBs may not be easily extrapolated to NBs in other brain regions. On the basis of the current results, it is tempting to postulate that Cas and Svp constitute two parallel pathways within the temporal series and Hh signaling is regulated by Cas but not Svp. Nevertheless, such a hypothesis warrants more in depth studies (Chai, 2013).

The precise generation of diverse cell types with distinct function from a single progenitor is important for the formation of a functional nervous system during animal development. It has been shown that, in Drosophila, the developmental timing mechanism (the temporal series) is tightly coupled with the asymmetric machinery. However, the underlying mechanism of this coordination remains elusive. The current data suggest that on the one hand, Hh signaling is under the control of the temporal series (hh expression is directly regulated by Cas), while on the other hand, Hh signaling participates in asymmetric segregation of Mira/Pros during NB division. Introduction of ectopic/premature Hh signaling (in ptc mutant clones) during developmental stages in which NBs are proliferating results in cytoplasmic localization of Mira/Pros during mitosis, reduction of NB size, and slow-down of NB cell cycle progression, reminiscent of the final division of NBs in early pupa just before cessation of proliferation. Consequently, these NBs exit the cell cycle prematurely. It is speculated that Pros may be a direct or indirect target of Hh signaling as elevated pathway activity invariantly leads to increased pros expression in the NBs. Furthermore, reducing the level of Pros protein by removing one copy of function pros is able to rescue the Mira delocalization phenotype seen in ptc mutant NBs. Thus, it is plausible that Hh signaling impinges on the asymmetric division apparatus, likely through Pros, to diminish NB fate gradually (as seen with the absence of Dpn and Mira delocalization) prior to the final cell cycle exit. Despite the results indicating a tight correlation between nuclear entry of Pros into the NBs and the eventual cell cycle exit of these NBs during pupal stage, it should be considered that Pros may not be the direct causative agent in controlling NB cell cycle exit. Therefore the actual role of Pros in this process is purely speculative as far as this study is concerned (Chai, 2013).

In contrast, loss of Hh signaling (e.g., in Smo mutant clones) maintains NBs in their 'younger' proliferating stage far beyond the time when they normally exit the cell cycle. Thus, Hh signaling couples the developmental timing mechanism (the temporal series) with the NB intrinsic asymmetric machinery for the generation of a functional nervous system (Chai, 2013).

In vertebrates, constitutive activation of the Sonic hedgehog (SHH, a homologue of Drosophila Hh), signaling pathway through inactivation mutations in PTCH1, activating mutations in SMO, as well as other mutations involving SHH, IHH, GLI1, GLI2, GLI3, and SUFU, has been implicated in a vast array of malignancies. The proven association of Hh signaling pathway with tumourigenesis and tumour cell growth fuel the view that Hh constitutes a mitogenic signal that promotes pro-proliferative responses of the target cells. Moreover, Hh acts as a stem cell factor in somatic stem cells in the Drosophila ovary, human hematopoietic stem cells, and mouse embryonic stem cells, possibly by exerting its effects on the cell cycle machinery (Chai, 2013).

This report provides an opposing facet of Hh signaling where it is required for timely NB cell cycle exit in the postembryonic pupal brain. This may sound astonishing, but the essential roles of Hh signaling as a negative regulator of the cell cycle has been eclipsed by the common bias that it stimulates proliferation, given the many examples of malignancies with the Hh pathway dysregulation. Indeed, studies have indicated that cell cycle exit and differentiation of a number of cell types, such as absorptive colonocytes of the mammalian gut, zebrafish, and Drosophila retina, require Hh activities. SHH signaling pathway is highly activated in human embryonic stem cell (hESC) and such activity is crucial for hESC differentiation as embryoid bodies. The opposing functions of Hh signaling pathway in different cell types reveal that the ultimate effect of this pathway is likely to be tissue specific, depending on its interaction with other regulatory pathways. The current data indicate that in Drosophila postembryonic NBs of the brain this does indeed appear to be the case, because in this system, Hh signaling pathway interacts with NB-specific temporal series and likely the asymmetric cell division machinery to promote pros nuclear localization to trigger cell cycle exit (Chai, 2013).

Targets of Activity

Activation of the Torso RTK (receptor tyrosine kinase) at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless gene. The Torso response element (TOR-RE) in the tll promoter indicates that the key activity modulated by the TOR RTK pathway is a repressor present throughout the embryo. The TOR-RE has been mapped to an 11-bp sequence. The proteins GAGA and NTF-1 (also known as Elf-1, product of the grainy head gene) bind to the TOR-RE. NTF-1 can be phosphorylated by Rolled, also known as MAPK (mitogen-activated protein kinase). tll expression is expanded in embryos lacking maternal NTF-1 activity. These results make NTF-1 a likely target for modulation by the TOR RTK pathway in vivo. Thus activation of TOR RTK at the poles of the embryo leads to inactivation of the repressor (GRH) and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo (Liaw, 1995).

The Dorsal morphogen is a transcription factor that activates some genes and represses others to establish multiple domains of gene expression along the dorsal/ventral axis of the early Drosophila embryo. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). A corepression element has been identified in decapentaplegic (dpp), a zygotically active gene that is repressed by the Dorsal morphogen. This dpp repression element (DRE) is located within a previously identified VRR and close to essential Dorsal-binding sites. A factor from Drosophila embryo extracts has been identified that binds to the DRE but not to mutant forms of the DRE that fail to support efficient repression. This protein also binds to an apparently essential region in a VRR associated with the zerknullt (zen) gene. One of the DREs in the dpp VRR overlaps the binding site for a potential activator protein suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. The DRE-binding protein is identical to NTF-1 (equivalent to Elf-1, the product of the grainyhead gene), a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters. NTF-1 mRNA is synthesized during oogenesis and deposited in the developing oocyte where it is available to contribute to ventral repression during early embryogenesis. Previous studies have shown that overexpression of NTF-1 in the postblastoderm embryo results in a phenotype that is consistent with a role for this factor in the repression of dpp later in embryogenesis (Huang, 1995).

GRH was isolated on the basis of its binding to element I, a proximal promoter site that has a major role in nervous system expression of Dopa decarboxylase (Bray, 1989). Nevertheless, GRH does not mediate the nervous system expression of Dopa decarboxylase, even though GRH has been shown to bind to the promoter site (Bray, 1991). GRH binds to and regulates the proximal promoter of Ubx (Dynlacht, 1989), and a neurogenic enhancer region of ftz, 1.7 kb upstream of the structural gene (Dynlacht, 1989). GRH also regulates engrailed (Soeller, 1988).

GRH is a cofactor in the repression of decapentaplegic and zerknüllt. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). A corepression element in dpp is located within a previously identified VRR and close to essential Dorsal-binding sites. One of the Dorsal response elements in the dpp VRR overlaps the binding site for a potential activator, suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. The Dorsal response element binding protein is identical to GRH. (Huang, 1995). GRH protein binds to and regulates an essential ventral repression region associated with the zerknüllt gene (Huang, 1995).

The Drosophila Proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including the upstream regulatory element (URE), a DNA replication-related element, and E2F recognition sites. In addition to DRE and E2F sites, the PCNA promoter contains three CFDD (common regulatory factor for DNA replication and DREF genes recognition) sites. Among these three, at least site 1 could be demonstrated to play an important role in promoter activity in both cultured cells and living flies. In addition to the PCNA gene, multiple CFDD sites have been found in promoters of the DNA polymerase and DREF genes. In nuclear extracts of Drosophila embryos, a protein factor, the URE-binding factor (UREF), has been detected that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies reveal that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allows isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (Grh/NTF-1). The nucleotide sequence required for binding to Grh is indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to Grh reacts with UREF in embryo nuclear extracts. From these results it is concluded that GRH is identical to UREF. Although Grh has been thought to be involved in regulation of differentiation-related genes, this study demonstrates for the first time the involvement of a Grh-binding site in the regulation of the DNA replication-related Proliferating cell nuclear antigen gene (Hayashi, 1999).

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).

These results demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).

An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head

Wounded Drosophila embryos were used to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier (Mace, 2005).

Animals have evolved biological armor, an epidermally derived integument, to protect their bodies from physical injury and dehydration and have evolved control pathways to regenerate this barrier after wounding. A key component of this barrier in mammals is the stratum corneum of the skin, and a key component of the barrier in insects is the cuticle. In invertebrates, the immediate barrier response to wounding involves the formation of a temporary plug at wound sites, along with the activation of melanization and cross-linking enzymes that encapsulate invading microbes and help seal wound openings. In vertebrates, the immediate humoral response to vascular wounding results in the activation of proteases leading to the formation of a fibrin clot to help seal wound openings (Mace, 2005).

In both invertebrates and vertebrates, introducing infectious microbes through wounds results in the induction of the innate immune pathways. In two branches of these pathways, Toll-family transmembrane receptors or Imd-dependent signals trigger a signaling cascade that allows transcription factors from the NF-kappaB family to enter the nucleus, where they directly activate the transcription of genes that provide a first line of defense against pathogens (Mace, 2005).

Another response to epithelial wounds is mediated by wound healing pathways that re-epithelialize the breach. Genes that are required for regenerating the epithelial sheet after laser-induced or mechanically induced wounds in the Drosophila epidermis include those encoding Rho, Cdc42, and Jun N-terminal kinase (JNK). Additional Drosophila genes have been implicated in the process of epithelial repair by means of their requirement for epidermal dorsal closure during late embryogenesis. These include genes encoding the Drosophila Jun and Fos transcription factors as well as Dpp, Ras, and Puckered. Homologs of most of these proteins, in addition to many others, are associated with the process of epithelial regeneration in vertebrates (Mace, 2005).

By comparison, the genetic pathways that respond to aseptic breaks in the barrier integument and provide for its regeneration are poorly understood. Although the integuments of both mammals and insects depend on a dense, highly cross-linked matrix of proteins and other macromolecules, heretofore there has been no reason to suspect common genetic control pathways in the repair of mammal skin and insect cuticle (Mace, 2005).

Drosophila embryos that lack all Hox gene function in a body region, or that are mutant for the Hox-interacting gene spen, develop ectopic sclerites (hard, melanized cuticular structures) in the trunk of first instar larvae. These sclerites were proposed to be ectopic head skeleton, but the sclerites in these mutant larvae often look like the cuticular scar tissue that often surrounds the healed hole generated by a sterile needle in late-stage embryos (Mace, 2005).

Therefore, whether the sclerites observed in spen or Scr Antp double-mutant larvae were associated with breaks in the epidermal integument was tested. To assay this, rhodamine-labeled dextran was injected into the perivitelline space of stage 17 wild-type, spen mutant, and Scr Antp mutant embryos. As a positive control, rhodamine-dextran directly was injected into the body cavity of a wild-type embryo. It was observed that the fluorescent dye penetrated the body cavity of spen mutant and Scr Antp double-mutant embryos but not control wild-type embryos. It is concluded that there are localized failures of epidermal integrity in late-stage embryos that lack the function of spen or both Scr and Antp (Mace, 2005).

Next, whether two genes required for normal cuticular sclerotization were activated in the wound regions that developed scars was tested. The genes were Ddc, which encodes dopa decarboxylase, and pale (ple), which encodes tyrosine hydroxylase. These proteins contribute to the formation of the larval and adult cuticular skeleton in epithelial cells through the production of catecholamines that are converted to quinones by phenol oxidases. The reactive quinones then cross-link protein polymers and chitin polymers to generate the largely impermeable integument of insects. First instar larvae that are doubly mutant for Ddc and ple have almost no melanization and sclerotization of the head skeleton. A key regulatory step in the localized deposition of hard, dark cuticle is exerted at the transcriptional level of these genes, given that the hard skeleton-producing cells of the late embryo and early larva accumulate abundant Ddc and ple transcripts (Mace, 2005).

In aseptically wounded late embryos, transcripts from both Ddc and ple accumulate to high levels in the epidermal cells near the wound site. Transcripts from these genes can be detected within 30 min after injury, suggesting that these genes are direct targets of a wound-induced signal transduction pathway. Transcription of Ddc and ple is also abundant in the defective epidermal regions that develop sclerotic scar tissue in spen mutants and Scr Antp double mutants. Control in situ hybridizations were done to eliminate the possibility that the increased Ddc and ple signals at wound sites were an accessibility artifact in late embryos (Mace, 2005).

To dissect transcriptional regulatory inputs involved in the activation of epidermal wound response genes, the regulatory regions of Ddc were analyzed. The expression pattern provided by a 7.5-kb segment of DNA that included a hemagglutinin-tagged Ddc protein coding sequence was examined. This 7.5-kb region provides the normal Ddc expression pattern during embryogenesis and is also activated near wound sites in late-embryonic epidermal cells (Mace, 2005).

Deletion analyses of lacZ and/or green fluorescent protein (GFP) reporter constructs fused to the hsp70 basal promoter show that sequences between -1.4 kb to the Ddc transcription start are sufficient for a wound response, but sequences from -381 base pairs (bp) to the transcription start are not. The -1.4-kb Ddc-GFP reporter is activated over many cell diameters near wound sites, and the extent of activation increases with larger wounds and longer incubations after injury. The graded nature of the response suggests that a signal is produced at the injury boundaries that activates the wound response enhancer in a dose-dependent fashion in nearby epidermal cells. The -1.4-kb Ddc-GFP reporter was also tested in Scr Antp double-mutant embryos and it was found found that the wound response reporter is activated in regions where cuticular scars develop. Similar results were obtained in spen mutants (Mace, 2005).

To determine whether the aseptic wound response pathway (as defined by the Ddc wound response enhancer) and the infectious wound response pathways overlap, tests were performed for activation of the -1.4-kb Ddc-GFP wound response reporter after aseptic wounding of zygotic mutants of the innate immunity signaling pathway genes Toll, tube, imd, and 18-wheeler as well as in zygotic mutants of the innate immunity transcription factor genes rel, Dif, and Dl (Dl and Dif were tested as a double-mutant combination). In all of these mutant backgrounds, the -1.4-kb Ddc-GFP reporter was activated near wounds, as it was in wild-type embryos. Toll maternal/zygotic mutants and tube maternal/zygotic mutants were also tested, and activation of the wound response reporter was observed at the breaks in the epidermal integument that occur in these mutants. (Mace, 2005).

Previous studies have indicated that the JNK pathway is required for the process of wound healing in embryos and adults. It was found that the -1.4-kb Ddc-GFP wound response reporter is still activated at aseptic wound sites of zygotic mutants in either Drosophila JNK, Jun, or Fos and is also activated in cells at and near the dorsal epidermal leading edge 'wound boundaries' that form when dorsal closure fails in these mutant backgrounds. The wound response reporter is not activated in dorsal epidermal leading edge cells in wild-type embryos. It is concluded that the zygotic functions of JNK, Jun, and Fos are dispensable for the activation of the -1.4-kb Ddc wound response reporter, even in the cells where the zygotic functions of these genes are required for the migration and sealing of epithelial cell sheets during dorsal closure (Mace, 2005).

The Drosophila grainy head (grh) gene encodes two major transcription factor isoforms (Grh-N and Grh-O). Grh-N is expressed in regions of the central nervous system, whereas Grh-O is expressed in barrier epithelia such as the embryonic epidermis, the foregut, the hindgut, and the tracheal system. Zygotic mutants in grh die at the embryonic/larval transition with weak epidermal cuticle, malformed head skeletons that are composed of discontinuous grainy sclerites, and abnormal tracheal trunks. Clones of grh mutant cells in the adult epidermis have defects in pigmentation, cell polarity, and epidermal hair differentiation (Mace, 2005).

The similarity of the grh mutant phenotype to Ddc and ple mutants prompted a determination of whether grh function is required for activation of -1.4-kb Ddc wound response element, which has two evolutionarily conserved Grh binding sites. In aseptically wounded grh mutant embryos, the -1.4-kb Ddc wound response reporter is at most weakly activated in a few cells immediately adjacent to the wound border. This is consistent with the abnormal wound healing in grh mutant larval cuticle. When compared with wild-type wounds, the grh mutant wound sites are deficient in normal cuticle regeneration, as well as in displacement of the melanized plug that forms immediately after wounding. A similar phenotype is seen in wounded Ddc mutants, although the remaining plug is less melanized (Mace, 2005).

The -1.4-kb Ddc wound response enhancer has consensus transcription factor binding sites for Grh, NF-kappaB/Rel proteins, adenosine 3',5'-monophosphate response element-binding protein (CREB)-A proteins, and AP-1-like/basic-leucine zipper proteins, which are candidate sites required for the function of the wound response enhancer. Transgenic embryos for the -1.4-kb Ddc-GFP reporter with point mutations in the six consensus sites for NF-kappaB family proteins show normal wound-induced activation, whereas a similar reporter with point mutations in the single CREB-A and the three AP-1-like consensus sites show a marked reduction in wound-induced activation compared with wild-type reporter controls (Mace, 2005).

Further deletion analyses define a minimal Ddc epidermal wound response element from -472 bp to the start of transcription, in which sequences from -472 to -381 bp are required for wound response function. The -0.47-kb Ddc wound response element from D. melanogaster has only five blocks of marked sequence conservation (a perfect match of 6 bp or greater) with a D. virilis Ddc promoter proximal fragment from base pairs -392 to +13, which provides a wound response when attached to reporter genes in D. melanogaster embryos. The blocks of sequence conservation are each 12 to 13 bp and they include one Grh site, one AP-1 consensus site, one ETS consensus site, a GGGGGATT motif (which overlaps with one of the NF-kappaB consensus sites), and the TATA box region. In D. melanogaster, the conserved GGGGGATT motif, AP-1-like site, and ETS site are all within the interval of -472 to -381 bp that is required for wound response function. The conserved Grh site, which is closer to the promoter, is required for the -472-bp element function, given that its mutation abolished wound response reporter activation (Mace, 2005).

To identify potential wound response enhancers at the D. melanogaster ple gene, the sequences from 10 kb upstream to 10 kb downstream of the ple transcription start were scanned for clusters of evolutionarily conserved Grh, AP-1, ETS, and GGGGGATT sites, and two regions were selected. One is a 3.0-kb DNA fragment beginning 2.9 kb upstream of the ple transcription start, which contains two Grh sites, two AP-1 sites, four ETS sites, and three GGGGGATT sites. The second is a 995-bp fragment just upstream of the ple transcription start, which conserves one Grh, one AP-1, and three ETS sites but no GGGGGATT motif. Both fragments were tested in red fluorescent protein (DsRed) reporter construct and the 3.0-kb ple fragment was found to robustly activate reporter expression around aseptic wounds, whereas the 995-bp fragment shows a very weak and slow response (Mace, 2005).

The involvement of mitogen-activated protein (MAP) kinases in epithelial injury response prompted a test for receptor tyrosine kinase or MAP kinase activation in cells that induce wound response enhancers. Using antibodies directed against phosphotyrosine (p-Tyr), an increase was found in p-Tyr staining in the cells near aseptic wounds, as well as in the wounded thorax of spen mutants, when compared with controls. This increase in p-Tyr correlates well with cells that activate the -1.4-kb Ddc-GFP wound response construct, although at the times both could be tested (2 hours postwounding), some cells showed activation of the wound response enhancer without a detectable increase in p-Tyr (Mace, 2005).

Regulation of post-embryonic neuroblasts by Drosophila Grainyhead

The Drosophila post-embryonic neuroblasts (pNBs) are neural stem cells that persist in the larval nervous system where they proliferate to produce neurons for the adult CNS. These pNBs provide a good model to investigate mechanisms regulating the maintenance and proliferation of stem cells. The transcription factor Grainyhead (Grh), which is required for morphogenesis of epidermal and tracheal cells, is also expressed in all pNBs. This study shows that grh is essential for pNBs to adopt the stem cell program appropriate to their position within the CNS. In grh mutants the abdominal pNBs produced more progeny while the thoracic pNBs, in contrast, divided less and produced fewer progeny than wild type. Three candidates were investigated to determine whether they could mediate these effects; the neuroblast identify gene Castor, the signalling molecule Notch and the adhesion protein E-Cadherin. Neither Castor nor Notch fulfills the criteria for intermediaries, and in particular Notch activity is dispensable for the normal proliferation and survival of the pNBs. In contrast E-Cadherin, which has been shown to regulate pNB proliferation, is present at greatly reduced levels in the grh mutant pNBs. Furthermore, ectopic expression of Grh is sufficient to promote ectopic E-Cadherin and two conserved Grh-binding sites were identified in the E-Cadherin/shotgun flanking sequences, arguing that this gene is a downstream target. Thus one way Grh could regulate pNBs is through expression of E-cadherin, a protein that is thought to mediate interactions with the glial niche (Almeida, 2005).

The transcription factor Grh is present in all the postembryonic neuroblasts (pNBs). The characteristics of its expression pattern in the ventral ganglion, which reflect the segmental differences in the number of neuroblasts that persist in this part of the larval CNS, are summarized here. (1) Many more Grh-expressing cells can be detected in the thoracic region (these are reported to have 23 pNBs per hemisegment) than in the abdominal neuromeres (where only 3 pNBs, vm, vl and dl, persist). (2) Grh expression in the abdominal region disappears by late third instar (96 h after-hatching) corresponding to the time when the abdominal pNBs cease dividing and die. (3) Grh is also detected in smaller cells associated with the pNBs, which appeared to be the ganglion mother cells (GMCs) based on their position and co-labelling with a cell division marker phospho-Histone H3 (pH3). (4) Grh expression does not persist in the post-mitotic progeny of pNB lineages which can be labelled with Gooseberry-proximal (Gsb-p, transiently expressed in the progeny of some lineages and overlapped with Grh in the GMC but not in other cells). The more mature progeny also express high levels of Prospero, which is present at much lower levels in the Gsb-p expressing cells and the GMCs. The expression of Grh in the pNB and GMC suggests that it could have a role similar to that of neuroblast identity genes. Therefore, whether it confers specific properties on the thoracic and abdominal pNBs of the ventral ganglion was investigated (Almeida, 2005).

Null alleles of grh are embryonic lethal. Therefore, to investigate the role of Grh in the pNBs advantage was taken of the grh370 allele, which has a frame-shift in the CNS specific transcript resulting in termination upstream of the DNA binding and dimerisation domain of Grh. The grh370 animals survive to post-embryonic stages because Grh is still present in other tissues, although it is absent from the pNBs. This allele was used in trans to a deletion that removes the grh gene [Df(2R)Pcl7B], which produces a slightly more severe phenotype than grh370 homozygotes, indicating that the grh370 allele is not completely null for CNS function (Almeida, 2005).

The proliferation of pNBs in wild-type and grh370 was examined by labelling cells in S-phase with bromodeoxuridine (BrdU). When wild-type and grh370 larvae were fed BrdU, several differences in the pNB lineages were detected: (1) there were 1-2 extra ventral clusters per hemisegment in the abdominal segments in grh370; (2) there were more progeny in the abdominal clusters; (3) there appeared to be fewer progeny in the thorax. Thus removal of grh appears to result in complex phenotypes in the pNB stem cells, with opposite effects on abdominal and thoracic pNBs (Almeida, 2005).

To confirm the defects observed with BrdU labelling, the expression of Gsb-p and Prospero was characterized in the CNS of wild-type and grh mutant larvae. In the embryo, the paired-homeodomain transcription factor Gsb-p is present in eight lineages per hemi-segment, where it confers positional identity. In the larval CNS, it is also present in eight lineages per thoracic hemisegment (6 ventral and 2 dorsal) and in 2 of the 3 abdominal lineages (vm and vl). Gsb-p expression was detected in a similar number of larval lineages in wild-type and mutant CNS, and where it was expressed in the subset of progeny located closest to the pNB (Almeida, 2005).

In wild-type CNS, Gsb-p expression disappeared from the abdominal regions at about the time when the abdominal pNBs normally die, suggesting Gsb-p marks a transient stage in the development of the pNB progeny. In grh370 expression continues until much later times (>96 h after hatching) in the abdominal segments and more labelled cells are detected in each abdominal cluster. At their peak (approximately 72 h after hatching) there are on average 7-9 Gsb-p cells per cluster in the mutant compared with 3-4 in wild-type. These data confirm the BrdU labelling and indicate that the abdominal pNBs proliferate more extensively and for longer. In contrast, the thoracic clusters of Gsb-p expressing-cells become progressively smaller over the course of larval development. By late third instar there are many fewer Gsb-p expressing cells in each thoracic cluster than in wild-type, suggesting that mutations in grh result in reduced proliferation or premature differentiation in these lineages (Almeida, 2005).

To further investigate the change in the pNBs behaviour in grh mutant, Prospero expression was monitored at late L3 CNS. Prospero protein is present in the progeny of both wild-type and grh370 pNBs. However, in grh370 the density of Prospero expressing cells in thoracic segments was clearly decreased, consistent with the decreased Gsb-p expression and BrdU incorporation in these lineages. Within the anterior of each segment a subset of pNB lineages showed a more marked reduction in the number of Prospero expressing cells. This was similar to the effect on Gsb-p, where two of the six clusters in each hemi-segment showed a more profound reduction in size and suggested that the precise effects of grh mutation differ according to the lineage (Almeida, 2005).

The change in proliferation patterns was born out when mitotic activity was analyzed using anti- phospho-histone H3 (pH3) antibody to give a snap-shot of the number of cells in mitosis. There was increased pH3 labelling in the abdominal region of grh mutant CNS with on average 13 mitotic cells in A2-A6 of grh370 CNS compared to <1 mitotic cell in wild-type. In contrast, there were fewer mitotic cells present in the thoracic region confirming that grh370 leads to reduced proliferation in thoracic pNB lineages, in contrast to the effects in the abdomen. The changes in proliferation in the thoracic lineages could reflect delays in the re-activation of the pNBs or an alteration in the subsequent maintenance/proliferation. To investigate this an examination was performed to see at what stage Gsb-p-expressing progeny first appear in wild-type and grh370 larvae. In both cases, Gsb-p expressing progeny were first detected in the thoracic neuromeres of late L2 CNS (30-45 h after hatching) and in the abdominal neuromeres of early L3 CNS (50-60 h after hatching) (Almeida, 2005).

In summary, therefore, reduced proliferation was observed of the thoracic pNBs in grh370 larvae. This contrasts with the effects in the abdominal segments, where the pNBs continue proliferating for longer. These complex defects suggest that grh is likely to regulate pNBs through a number of different mechanisms. Three possible candidates for downstream effectors Castor, Notch and Cadherins were examined. The former is a transcription factor expressed in the embryonic neuroblasts prior to Grainyhead. The latter are cell surface proteins implicated in stem cell regulation in several other systems (Almeida, 2005).

Previous studies have shown that E-Cadherin is necessary for normal pNB proliferation. These studies show that expression of a dominant negative E-Cadherin in the neural and glial cells reduces the number of progeny produced by pNBs to <25% of wild type. Expression in the ensheathing glia alone led to more minor reduction, arguing that the protein is needed in both glia and pNBs. As reported previously, strong expression of E-Cadherin was detected in the pNBs and their adjacent progeny. In grh370 however, the levels of E-Cadherin in the thoracic region of the CNS were dramatically reduced. Several pNBs lack significant E-Cadherin expression all together, others retained some expression but at much lower levels compared to wild type. Similar effects were seen in clones mutant for another loss of function grh allele, grhB32. In lineages homozygous for grhB32 there was a variable reduction in E-Cadherin compared to neighbouring wild-type lineages (Almeida, 2005).

The effects on Cadherin contrast with those on Notch, where expression in the thoracic pNBs remains robust in grh370, arguing against an indirect effect resulting from changes in size. To further test this, it was asked whether Grh is sufficient to promote E-Cadherin expression when expressed elsewhere in the CNS. A pros::Gal4 driver line was used that directs high levels of expression in neurons and lower expression in the pNB lineages. When this was used to drive expression of the CNS isoform of grh, high levels of ectopic E-Cadherin were detected, particularly in many of the embryo-derived neurons that are normally devoid of E-Cadherin expression at these stages. Neither Castor nor Notch expression was altered under these conditions. Therefore Grh appears to be an activator of E-Cadherin expression. However, ectopic Grh was not sufficient to direct additional proliferation under the conditions tested (Almeida, 2005).

The genomic sequence flanking the E-Cadherin gene (shotgun, shg) was examined for consensus Grh binding sites using two different strategies. Grh binds as a dimer. In recent studies of Grh family proteins a consensus target-site was derived (WCHGGTT). Eight matches to this consensus are present in the genomic region spanning from 1 kb upstream of the shg transcript (another gene, CG10540, starts 944 bp upstream of shg) to 5 kb downstream. A second search using a weighted matrix revealed 13 matches within 5 kb of shg. A comparison of the two sets of putative sites identified four common matches: AAACAGGTTA (−300); AAACAGGTAA (+275); ATACTGGTTT (2650 bp downstream, Shg2); CAACAGGTAG (3131 bp downstream, Shg1). The latter two are 100% conserved between D. melanogaster and the five other Drosophila species for which sequence is available. To confirm that these two sites are recognised by Grh, a stringent assay was used where their ability to compete with a well-characterised, high affinity site, Gbe2 from the Dopa decarboxylase gene, was tested. Both sites were able to compete, reducing the amount of probe bound by 51% (Shg1) and 75% (Shg2) when present at 40× molar excess. The presence of these conserved sites indicates therefore that shg/E-Cadherin is likely to be a direct target of Grh. However, E-Cadherin cannot be the only target, since it was not possible to rescue the grh370 mutant phenotype by supplying E-Cadherin via an exogenous driver (GrhNB::Gal4/UAS::E-Cadherin) (Almeida, 2005).

Although the data show that Cadherin is regulated by Grh, they do not resolve unequivocally whether it is a direct target. Examination of genomic sequence revealed two binding-sites close to the shotgun/E-Cadherin gene that are conserved in other Drosophila species and that are bound by Grh in vitro. Future studies will show whether these sites are essential for shg expression. However, these results are exciting because they provide a link between grh function in the pNBs and in other tissues. Changes were observed in E-Cadherin levels in response to grh in other parts of the animal. There is also an interaction between shg and several genes that act together with grh in epidermal morphogenesis (although no direct genetic interaction was seen between shg and grh itself). Given that the precise levels of E-Cadherin proteins can be critical in shaping the sorting and interactions between cells it will be important to determine whether Grh is required for this regulation. It will also be important to establish whether Cadherins are targets of Grh in other animals, for example in mice where mutations in Grhl3 result in defects in neural tube closure and epidermal integrity (Almeida, 2005).

Despite the fact that Castor appeared a likely target for Grh no evidence was found that it is deregulated in the larval CNS of grh mutants. There was no ectopic Castor in thoracic pNBs at the stage when they first reactivate, as might be predicted if Grh was essential for castor repression. No repression of Castor was found when Grh was ectopically expressed with pros::Gal4. Therefore, Castor does not appear to be a primary target for the effects of Grh in the pNBs. However, in wild type CNS Castor was present transiently in many if not all of the pNBs and it remains possible that in grh mutants Castor is activated prematurely or for more prolonged periods in some of the pNBs. It was not possible to dissect in sufficient detail the timing of expression in individual thoracic lineages to resolve this. Nevertheless, it is clear from these studies that Castor is re-activated in post-embryonic lineages. This indicates that the temporal cascade of transcription factor expression does not extend simply into the post-embryonic stages. It is possible that the quiescent period during early larval stages could reset the temporal clock so that embryonic factors can be reused. However, the presence of Castor in some pNBs at the time when they first reactivate would argue against the post-embryonic series recapitulating the embryonic one, since Castor is expressed at late stage in embryonic pNBs (Almeida, 2005).

Whether or not Notch is regulated by Grh, it is certainly a prime candidate to maintain the pNB stem-cells. Previous studies had shown that Notch is present on the pNBs and an E(spl)mγ-GFP reporter showed definitively that Notch is activated in these cells. However, surprisingly, mutations in Notch failed to perturb any of the aspects of pNB behaviour that could be easily assayed. For example, one simple model suggested that signals from the progeny to the pNB mediated by Notch would prevent the pNB from differentiating prematurely. However, in Notch mutants the pNBs were found to persist as normal throughout larval stages. The abdominal pNBs also disappeared at the normal stage, indicating that the timing of their apoptosis is independent of Notch, even though Notch does regulate cell death elsewhere. Likewise, no evidence was found that Notch controls proliferation of the pNBs because the number of progeny produced and their maturation was unaffected by Notch mutations. It has not been possible however to evaluate whether the ultimate fates of the progeny are altered in Notch mutants so it remains possible that it regulates the neuronal or glial cell types produced. Now that lineage maps are being generated for the pNBs it should be possible to start investigating this possibility. Nevertheless, it is clear that the pNBs retain their stem-cell characteristics in the absence of Notch activity (Almeida, 2005).

The defects in the pNB lineages of grh mutants are position dependant. Thus, the thoracic pNBs produce fewer progeny whereas abdominal pNBs proliferate for a more prolonged period. In general, such A/P position dependent patterning is co-ordinated by the homeotic genes and indeed abdA has been shown to regulate the timing of cell death and hence the period of proliferation in the abdominal pNBs, as well as regulating the number of pNBs that persist in abdominal segments. However the phenotype of abdA mutants is significantly different from that of grh; for example the abdominal clusters are much larger and there are no defects in thoracic clusters, so it is unlikely that grh is upstream of abdA. Furthermore, in parallel studies Cenci (2005) has shown that the initiation of AbdA expression still occurs in grh mutants. Therefore it is more likely that grh acts in parallel to the homeotic genes to co-ordinate the pNB program (Almeida, 2005).

Grainy head promotes expression of septate junction proteins and influences epithelial morphogenesis

Transcription factors of the Grainy head (Grh) family are required in epithelia to generate the impermeable apical layer that protects against the external environment. This function is conserved in vertebrates and invertebrates, despite the differing molecular composition of the protective barrier. Epithelial cells also have junctions that create a paracellular diffusion barrier (tight or septate junctions). To examine whether Grh has a role in regulating such characteristics, an epidermal layer in the Drosophila embryo was used that has no endogenous Grh and lacks septate junctions, the amnioserosa. Expression of Grh in the amnioserosa caused severe defects in dorsal closure, a process similar to wound closure, and induced robust expression of the septate junction proteins Coracle, Fasciclin 3 and Sinuous. Grh-binding sites are present within the genes encoding these proteins, consistent with them being direct targets. Removal of Grh from imaginal disc cells caused a reduction in Fasciclin 3 and Coracle levels, suggesting that Grh normally fine tunes their epithelial expression and hence contributes to barrier properties. The fact that ectopic Grh arrests dorsal closure also suggests that this dynamic process relies on epithelia having distinct adhesive properties conferred by differential deployment of Grh (Narasimha, 2008).

To test the role of Grh in regulating epithelial characteristics, the epidermal splice forms (N/K) were specifically expressed in the amnioserosa, an epithelial tissue normally devoid of Grh (using c381::Gal4 and G332::Gal4). This was sufficient to block dorsal closure, an effect previously seen with ubiquitous Grh overexpression. The effects were most penetrant with c381::Gal4 (hereafter referred to as ASc381>grh) which resulted in 100% of embryos having dorsal holes at stage 17/hatching, when all wild-type embryos had completed dorsal closure. Defects were already evident earlier (stage 14-16). In ASc381>grh embryos, the amnioserosa was less contracted than wild type and contained cells with abnormal morphology. In addition, the epidermal edges failed to meet at the poles. Thus, expression of Grh disrupted the ability of amnioserosa cells to function in dorsal closure, suggesting that it altered their fundamental properties and/or perturbed their interactions with the epidermis (Narasimha, 2008).

One explanation for the defects caused by Grh expression in amnioserosa cells is that these cells acquire epidermis-like characteristics, such as septate junctions (SJs) characteristic of conventional barrier epithelia. Proteins that localise to SJs include the FERM-domain protein Coracle, the immunoglobulin-family adhesion protein Fasciclin 3 (Fas3), the transmembrane protein Neurexin (Nrx), and the claudin-related proteins Sinuous (Sinu) and Megatrachea. In addition, the Discs large (Dlg)-Scribble-l(2)gal (Lgl) complex initially localises basolaterally and becomes incorporated into SJs. To investigate whether Grh regulates such components their expression was examined in ASc381>grh embryos. Expression of Fas3, Coracle and Sinu was strikingly upregulated in the amnioserosa of ASc381>grh embryos in comparison to control embryos. Nrx and Atpα (Na/K-ATPase subunit) were more weakly upregulated, and Dlg was upregulated in a patchy manner, although this effect was less penetrant. Thus, levels of several different SJ proteins are increased by Grh expression in the amnioserosa. Of these, Fas3 was the earliest that could be detected (Narasimha, 2008).

In conventional epithelia, junctional proteins are localised to discrete domains in the lateral membrane. In ASc381>grh embryos, the sub-cellular localisation of SJ proteins was abnormal. Fas3, Coracle and Dlg were more diffuse than in wild type and frequently expanded along the apical and/or basal surface. For example, Fas3 proteins were present in a more apical plane than the adherens junction component E-cadherin, and Dlg was expanded throughout basal and apical regions. Because E-cadherin itself was still localised at apical junctions in AS>grh embryos, the underlying apical/basal polarity appears unaffected. Thus, the altered distribution of Fas3, Coracle and Dlg suggests that Grh is sufficient to promote expression of SJ proteins, but not to ensure the correct organisation of these proteins within the apical-basal axis (Narasimha, 2008).

The upregulation of SJ proteins caused by ectopic Grh expression is complementary to the apical membrane expansion detected in grh loss-of-function mutants. However, SJ proteins (e.g., Coracle) are still present in the mutant tracheal and epidermal cells. Thus, Grh is apparently not essential for expression of SJ proteins, although it can clearly promote their expression ectopically. One way to reconcile these differences is if Grh fine-tunes the expression of such proteins to increase or strengthen lateral junctions in mature epithelia. This was tested by generating clones of grh-mutant cells in the wing imaginal disc, in which the juxtaposition of wild-type and mutant cells aids detection of subtle changes in expression levels. SJ proteins were still present in mutant wing disc cells, as they were in mutant tracheal cells. However, using this approach it was possible to detect a reduction in the levels of Fas3 and Coracle in cells lacking grh. This was most consistent for Fas3: the majority (11/16) of clones scored had a detectable reduction in Fas3. With Coracle, the effects were more variable, but 5/16 grh-mutant clones had subtle decreases in its levels. The fact that the effects were subtle and variable could be a consequence of timing, because the consequences of removing Grh was assayed at a relatively early stage in the maturation of these epithelia. Nevertheless, removal of Grh was not sufficient to compromise the barrier properties of the tracheal epithelia in the embryo, as measured by dextran exclusion experiments. Fluorescent dextrans injected into wild-type and grh-mutant embryos failed to enter the lumen of the trachea, indicating that they are unable to pass through the junctions. By contrast, when injected into mutant embryos in which SJs were compromised, dextran rapidly spread throughout the tracheal lumen. Thus, Grh is not essential for the establishment of SJs, although it can influence the levels of SJ proteins (at least in the wing disc) and is sufficient to promote their expression ectopically. These data suggest a model in which Grh in Drosophila elevates the expression of SJ proteins in a similar manner to the effects of GRHL3 in mice on claudins and occludins, proteins found in the analogous tight junctions (Yu, 2006). In neither animal is there complete loss of these proteins in the grh/Grhl3 mutants, but their levels and distribution are altered in a manner that could alter the robustness of an epithelial barrier (Narasimha, 2008).

To investigate whether genes encoding SJ proteins could be direct targets of Grh, the coracle and fas3 genes were analysed for sequences that had good matches to a weighted matrix derived from known Grh-binding sites and that were conserved in the cognate genes from highly diverged drosophilids (D. pseudoobscura, D. virilis, D. mojavensis). There were two conserved matches to the Grh-binding-site consensus in the first intron of fas3 (fas3A, 5'-ACCGGTTT-3'; fas3B, 5'-ACCAGTTT-3') and in the first intron of coracle [coraA, 5'-ACCAGTTT-3' (–strand); coraB, 5'-ACCGGTTT-3' (–strand)]. These four sites were recognised by Grh in vitro in a competition assay in which their binding affinities were compared with a high-affinity Grh target site, Gbe2, from the dopa decarboxylase gene. Putative sites from fas3 and coracle significantly reduced binding to the labelled Gbe2 probe, and were even more effective than a similar excess of the cognate Gbe2 site, demonstrating that they are high-affinity binding sites for Grh. Thus, both fas3 and coracle have the potential to be direct targets of Grh (Narasimha, 2008).

To further test their potential for regulation by Grh, fragments encompassing the Grh-binding sites were inserted upstream of a minimal promoter fused to luciferase and expression was assayed in the presence and absence of Grh in transient transfection assays. In total, 3/4 fragments conferred Grh responsiveness (>2.5x) on the reporter. In addition, two fragments from sinu that encompassed putative Grh-binding sites [sinu1, 5'-ACCTGTTC-3' (–strand); sinu2, 5'-TCCGGTTT-3'] were tested in the same assay, and sinu2 also showed a response to Grh. Together, these data suggest that the effect of Grh on SJs involves direct regulation of component-encoding genes (Narasimha, 2008).

Because ectopic expression of Grh has a profound effect on dorsal closure, the morphology of ASc381>grh cells and the distribution of other adhesion complexes were examined more closely. In ASc381>grh embryos there were large variations in shape and size of amnioserosa cells and the contacts with the adjacent dorsal epidermis were dramatically different. A subset of epidermal cells had expanded contact with an amnioserosa cell at the expense of their neighbours, which became bunched together. It appeared, therefore, that many Grh-expressing amnioserosa cells had maximised the contact with a single epidermal cell, rather than making contact with five to six cells, as in wild type. Ultimately, some amnioserosa cells appeared to lose contact with the epidermal cells (Narasimha, 2008).

The change in morphology in ASc381>grh embryos was accompanied by altered distribution of β-position-specific (βPS) integrins and E-cadherin. In wild-type embryos, these co-localise to prominent dots at the interface between amnioserosa and epidermal cells and are present in overlapping domains associated with amnioserosa cell-cell contacts. In ASc381>grh cells, βPS integrins appeared diffuse and frequently spread across the apical surface. Furthermore, less βPS integrin accumulated at the interface between epidermal and AS cells; instead, it was concentrated in regions with bunched epidermal cell contacts and no longer localised with E-cadherin. Thus, Grh perturbs other adhesive characteristics of amnioserosa cells. This could be an indirect consequence of the increase in SJ proteins, causing altered distribution of apical and basal adhesion receptors, or Grh could additionally regulate the expression levels of cadherin and integrins. Whichever the mechanism, amnioserosa cells acquire altered adhesion properties with neighbouring epidermal cells, which could explain why dorsal closure is perturbed (Narasimha, 2008).

The results obtained from ectopic Grh expression have helped uncover functions that are not easily evident from loss-of-function experiments and suggest that Grh is normally involved in fine-tuning the expression levels of proteins, such as Fas3, Coracle, Sinu, Nrx and Dlg, which are involved in conferring robust barrier function on the epidermis. Given the observation that GRHL3 also fine-tunes the levels of junction proteins in mice (Yu, 2006), it appears that this represents a highly conserved aspect of Grh function. In addition, Grh might be intrinsic to the observed cross-talk between the extracellular matrix and junctional complexes, because it plays a role in regulating both elements (Narasimha, 2008).

Grh transcription factors are also components in a conserved mechanism for wound healing, in part via their effect on extracellular matrix deposition/synthesis. The results suggest that regulation of cell junctions might also be important for epidermal 'sealing'. They further suggest that differences in Grh levels or activity could regulate morphogenesis within an epithelium, as well as the ability of epithelia to adhere to one another, by influencing the levels and distribution of septate/tight junction proteins and other adhesion molecules. This could also explain the role of GRHL3 during neural tube closure in mice, an epithelial fusion event that shares features with dorsal closure (Narasimha, 2008).

Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues

The generation of distinct neuronal subtypes at different axial levels relies upon both anteroposterior and temporal cues. However, the integration between these cues is poorly understood. In the Drosophila central nervous system, the segmentally repeated neuroblast 5-6 generates a unique group of neurons, the Apterous (Ap) cluster, only in thoracic segments. Recent studies have identified elaborate genetic pathways acting to control the generation of these neurons. These insights, combined with novel markers, provide a unique opportunity for addressing how anteroposterior and temporal cues are integrated to generate segment-specific neuronal subtypes. Pbx/Meis, Hox, and temporal genes were found to act in three different ways. Posteriorly, Pbx/Meis and posterior Hox genes block lineage progression within an early temporal window, by triggering cell cycle exit. Because Ap neurons are generated late in the thoracic 5-6 lineage, this prevents generation of Ap cluster cells in the abdomen. Thoracically, Pbx/Meis and anterior Hox genes integrate with late temporal genes to specify Ap clusters, via activation of a specific feed-forward loop. In brain segments, 'Ap cluster cells' are present but lack both proper Hox and temporal coding. Only by simultaneously altering Hox and temporal gene activity in all segments can Ap clusters be generated throughout the neuroaxis. This study provides the first detailed analysis of an identified neuroblast lineage along the entire neuroaxis, and confirms the concept that lineal homologs of truncal neuroblasts exist throughout the developing brain. Also this study provides the first insight into how Hox/Pbx/Meis anteroposterior and temporal cues are integrated within a defined lineage, to specify unique neuronal identities only in thoracic segments. This study reveals a surprisingly restricted, yet multifaceted, function of both anteroposterior and temporal cues with respect to lineage control and cell fate specification (Karlsson, 2010).

To understand segment-specific neuronal subtype specification, this study focused on the Drosophila neuroblast 5-6 lineage and the thoracic-specific Ap cluster neurons born at the end of the NB 5-6T lineage. The thoracic appearance of Ap clusters was shown to result from a complex interplay of Hox, Pbx/Meis, and temporal genes that act to modify the NB 5-6 lineage in three distinct ways (see Summary of Hox/Pbx/Meis and temporal control of NB 5-6 development). In line with other studies of anterior-most brain development, it was found that the first brain segment (B1) appears to develop by a different logic. These findings will be discussed in relation to other studies on spatial and temporal control of neuroblast lineages (Karlsson, 2010).

In the developing Drosophila CNS, each abdominal and thoracic hemisegment contains an identifiable set of 30 neuroblasts, which divide asymmetrically in a stem-cell fashion to generate distinct lineages. However, they generate differently sized lineages -- from two to 40 cells, indicating the existence of elaborate and precise mechanisms for controlling lineage progression. Moreover, about one third of these lineages show reproducible anteroposterior differences in size, typically being smaller in abdominal segments when compared to thoracic segments. Thus, neuroblast-specific lineage size control mechanisms are often modified along the anteroposterior axis (Karlsson, 2010).

Previous studies have shown that Hox input plays a key role in modulating segment-specific behaviors of neuroblast lineages. Recent studies have resulted in mechanistic insight into these events. For instance, in the embryonic CNS, Bx-C acts to modify the NB 6-4 lineage, preventing formation of thoracic-specific neurons in the abdominal segments. This is controlled, at least in part, by Bx-C genes suppressing the expression of the Cyclin E cell cycle gene in NB 6-4a. Detailed studies of another neuroblast, NB 7-3, revealed that cell death played an important role in controlling lineage size in this lineage: when cell death is genetically blocked, lineage size increased from four up to 10 cells. Similarly, in postembryonic neuroblasts, both of these mechanisms have been identified. In one class of neuroblasts, denoted type I, an important final step involves nuclear accumulation of the Prospero regulator, a key regulator both of cell cycle and differentiation genes. In 'type II' neuroblasts, grh acts with the Bx-C gene Abd-A to activate cell death genes of the Reaper, Head involution defective, and Grim (RHG) family, and thereby terminates lineage progression by apoptosis of the neuroblast. This set of studies demonstrates that lineage progression, in both embryonic and postembryonic neuroblasts, can be terminated either by neuroblast cell cycle exit or by neuroblast apoptosis. In the abdominal segments, it was found that the absence of Ap clusters results from a truncation of the NB 5-6 lineage, terminating it within the Pdm early temporal window, and therefore Ap cluster cells are never generated. These studies reveal that this truncation results from neuroblast cell cycle exit, controlled by Bx-C, hth, and exd, thereafter followed by apoptosis. In Bx-C/hth/exd mutants, the neuroblast cell cycle exit point is bypassed, and a thoracic sized lineage is generated, indicating that these genes may control both cell cycle exit and apoptosis. However, it is also possible that cell cycle exit is necessary for apoptosis to commence, and that Bx-C/hth/exd in fact only control cell cycle exit. Insight into the precise mechanisms of the cell cycle exit and apoptosis in NB 5-6A may help shed light on this issue (Karlsson, 2010).

Whichever mechanism is used to terminate any given neuroblast lineage -- cell cycle exit or cell death -- the existence in the Drosophila CNS of stereotyped lineages progressing through defined temporal competence windows allows for the generation of segment-specific cell types simply by regulation of cell cycle and/or cell death genes by developmental patterning genes. Specifically, neuronal subtypes born at the end of a specific neuroblast lineage can be generated in a segment-specific fashion 'simply' by segmentally controlling lineage size. This mechanism is different in its logic when compared to a more traditional view, where developmental patterning genes act upon cell fate determinants. But as increasing evidence points to stereotypic temporal changes also in vertebrate neural progenitor cells (Okano, 2009), this mechanism may well turn out to be frequently used to generate segment-specific cell types also in the vertebrate CNS (Karlsson, 2010).

These findings of Hox, Pbx/Meis, and temporal gene input during Ap cluster formation are not surprising -- generation and specification of most neurons and glia will, of course, depend upon some aspect or another of these fundamental cues. Importantly however, the detailed analysis of the NB 5-6T lineage, and of the complex genetic pathways acting to specify Ap cluster neurons, has allowed this study to pin-point critical integration points between anteroposterior and temporal input. Specifically, cas, Antp, hth, and exd mutants show striking effects upon Ap cluster specification, with effects upon expression of many determinants, including the critical determinant col. Whereas Antp plays additional feed-forward roles, and exd was not tested due to its maternal load, it was found that both cas and hth mutants can be rescued by simply re-expressing col. This demonstrates that among a number of possible regulatory roles for cas, hth, Antp, and exd, one critical integration point for these anteroposterior and temporal cues is the activation of the COE/Ebf gene col, and the col-mediated feed-forward loop. Both col and ap play important roles during Drosophila muscle development, acting to control development of different muscle subsets. Their restricted expression in developing muscles has been shown to be under control of both Antp and Bx-C genes. Molecular analysis has revealed that this regulation is direct, as Hox proteins bind to key regulatory elements within the col and ap muscle enhancers. The regulatory elements controlling the CNS expression of col and ap are distinct from the muscle enhancers, and it will be interesting to learn whether Hox, as well as Pbx/Meis and temporal regulatory input, acts directly also upon the col and ap CNS enhancers (Karlsson, 2010).

One particularly surprising finding pertains to the instructive role of Hth levels in NB 5-6T. At low levels, Hth acts in NB 5-6A to block lineage progression, whereas at higher levels, it acts in NB 5-6T to trigger expression of col within the large cas window. It is interesting to note that the hth mRNA and Hth protein expression levels increase rapidly in the entire anterior CNS (T3 and onward). In addition, studies reveal that thoracic and anterior neuroblast lineages in general tend to generate larger lineages and thus remain mitotically active for a longer period than abdominal lineages. On this note, it is tempting to speculate that high levels of Hth may play instructive roles in many anterior neuroblast lineages. In zebrafish, Meis3 acts to modulate Hox gene function, and intriguingly, different Hox genes require different levels of Meis3 expression. In the Drosophila peripheral nervous system, expression levels of the Cut homeodomain protein play instructive roles, acting at different levels to dictate different dendritic branching patterns in different sensory neuron subclasses. Although the underlying mechanisms behind the levels-specific roles of Cut, Meis3 or Hth are unknown, it is tempting to speculate that they may involve alterations in transcription factor binding sites, leading to levels-sensitive binding and gene activation of different target genes (Karlsson, 2010).

The vertebrate members of the Meis family (Meis1/2/3, Prep1/2) are expressed within the CNS, and play key roles in modulating Hox gene function. Intriguingly, studies in both zebrafish and Xenopus reveal that subsequent to their early broad expression, several members are expressed more strongly or exclusively in anterior parts of the CNS, in particular, in the anterior spinal cord and hindbrain. Here, functional studies reveal complex roles of the Meis family with respect to Hox gene function and CNS development. However, in several cases, studies reveal that they are indeed important for specification, or perhaps generation, of cell types found in the anterior spinal cord and/or hindbrain, i.e., anteroposterior intermediate neural cell fates. As more is learned about vertebrate neural lineages, it will be interesting to learn which Meis functions may pertain to postmitotic neuronal subtype specification, and which may pertain to progenitor cell cycle control (Karlsson, 2010).

In anterior segments -- subesophageal (S1-S3) and brain (B1-B3) -- a more complex picture emerges where both the overall lineage size and temporal coding is altered, when compared to the thoracic segments. Specially, whereas all anterior NB 5-6 lineages do contain Cas expressing cells, expression of Grh is weak or absent from many Cas cells. The importance of this weaker Grh expression is apparent from the effects of co-misexpressing grh with Antp -- misexpression of Antp alone is unable to trigger FMRFa expression, whereas co-misexpression with grh potently does so. It is unclear why anterior 5-6 lineages would express lower levels of Grh, since Grh expression is robust in some other anterior lineages (Karlsson, 2010).

In the B1 segment two NB 5-6 equivalents have been identified. However, the finding of two NB 5-6 equivalents is perhaps not surprising, since the B1 segment contains more than twice as many neuroblasts as posterior segments. Due to weaker lbe(K)-lacZ and -Gal4 reporter gene expression, and cell migration, these lineages could not be mapped. However, irrespective of the features of the B1 NB 5-6 lineages, bona fide Ap cluster formation could not be triggered by Antp/grh co-misexpression in B1. Together, these findings suggest that the B1 segment develops using a different modus operandi, a notion that is similar to development of the anterior-most part of the vertebrate neuroaxis, where patterning and segmentation is still debated. On that note, it is noteworthy that although Hox genes play key roles in specifying unique neuronal cell fates in more posterior parts of the vertebrate CNS, and can indeed alter cell fates when misexpressed, the sufficiency of Hox genes to alter neuronal cell fates in the anterior-most CNS has not been reported -- for instance, Hox misexpression has not been reported to trigger motoneuron specification in the vertebrate forebrain. Thus, in line with the current findings that Antp is not sufficient to trigger Ap cluster neuronal fate in the B1 anterior parts, the anterior-most part of both the insect and vertebrate neuroaxis appears to be 'off limits' for Hox genes (Karlsson, 2010).

The Hox, Pbx/Meis, and temporal genes are necessary, and in part sufficient, to dictate Ap cluster neuronal cell fate. However, they only do so within the limited context of NB 5-6 identity. Within each abdominal and thoracic hemisegment, each of the 30 neuroblasts acquires a unique identity, determined by the interplay of segment-polarity and columnar genes. In the periphery, recent studies demonstrate that anteroposterior cues, mediated by Hox and Pbx/Meis genes, are integrated with segment-polarity cues by means of physical interaction and binding to regulatory regions of specific target genes. It is tempting to speculate that similar mechanisms may act inside the CNS as well, and may not only involve anteroposterior and segment-polarity integration, but also extend into columnar and temporal integration (Karlsson, 2010).

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. This study identified stitcher (stit: FlyBase name Cad96Ca), a new Grh target in Drosophila. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses (Wang, 2009).

Epidermal cells and their apical extracellular matrix provide a vital shield against physical damage and pathogens. Despite remarkable structural differences between mammalian skin and insect cuticles, Grh emerges as a conserved regulator of epidermal wound healing across phylogeny. In wounded fly embryos it rapidly activates Ddc and ple (the genes encoding DOPA decarboxylase and tyrosine hydroxylase, respectively) to facilitate cuticle repair. Similarly, Grhl-3 (mouse Grainy head-like 3) induces transglutaminase-1 expression in neonatal skin wounds to crosslink the stratum corneum. Grhl-3 has also been implicated in epithelial remodelling after wounding, but the mechanism by which Grhl-3 controls re-epithelialization is unclear (Wang, 2009).

Two conserved Grh-binding elements (Gbes) were used to search the Drosophila genome for target regions containing clustered binding sites by using the CIS-ANALYST algorithm. A cluster of four putative Gbes was found within the second intron of CG10244. CG10244 was selected for further analysis because it was strongly expressed in all ectodermal tissues, like grh, and it encoded an uncharacterized protein with homology to receptor tyrosine kinases (RTKs). CG10244 was renamed stitcher (stit) to reflect its function. stit expression initiated at stage 12 in epithelial tissues and was severely decreased in the epidermis of grh mutants in comparison with the wild type. The relative amount of stit mRNA in grh mutants only reached about 50% of wild-type levels by stage 16. Conversely, Grh overexpression in epidermal stripes of en>grh embryos activated striped stit expression, indicating that Grh controls stit during development. Electrophoretic mobility-shift assays with the Grh DNA-binding region fused to glutathione S-transferase revealed direct Grh binding to Gbe3 of stit. To determine whether Grh binding activates stit transcription, the ability of stit Gbes to drive epidermal expression of a green fluorescent protein (GFP) reporter was tested. A 5.4-kilobase (kb) DNA fragment encompassing five Gbes directed GFP to the epidermis of transgenic embryos. In grh mutants, this expression was severely reduced and limited to only a few cells. The 5.4-kb DNA segment was split into a 3.4-kb fragment and a 2-kb fragment containing the Gbe cluster. Both fragments directed epidermal GFP expression in transgenic embryos. However, whereas stitβ/-GFP expression (see Schematic illustration of the stit locus) remained robust in grh mutants, stitγ/>-GFP levels were greatly decreased. The stit regulatory region therefore contains a Grh-dependent enhancer. To test the contribution of Gbes to stitγ-GFP expression, stitδ-GFP embryos were created with all four putative Gbes mutated. Epidermal GFP expression was markedly decreased in these embryos in comparison with stitγ-GFP. Therefore Grh directly activates stit during development. Does it also control stit expression during wound healing? Aseptic wounding of wild-type embryos induced a rapid accumulation of stit transcripts at wound sites. Wild-type and grh mutants carrying either the stitα-GFP or the stitγ-GFP reporter were punctured. The GFP levels from both reporters were robustly increased around the wounds of wild-type embryos. This induction was severely decreased in grh mutants. Finally, wild-type embryos carrying the stitδ-GFP reporter showed hardly any GFP induction on wounding. Grh therefore activates epidermal stit expression during development and wound closure (Wang, 2009).

To elucidate stit function, two null alleles were characterized. Homozygous animals for either allele and also the transheterozygotes die at late pupal stages. The lethality of stit mutants could be partly rescued by stit transgenic expression at low levels in ectodermal tissues by using the 69B-GAL4 driver. High-level Stit expression caused embryonic lethality. Unlike grh mutants, stit embryos showed no developmental defects in ectodermal tissues. Does Stit therefore have a role in wound healing? Wild-type and stit embryos were punctured and survival was examined after 20 h. Most wounded wild-type animals survived, whereas 30% of the mutants succumbed to wounding. stit therefore facilitates survival on wounding. To address stit function in epithelial closure after puncture, an actin-binding GFP–moesin marker (GFP–moe) was expressed in the epidermis. 400-800-microm2 puncture wounds on 69B>GFP-moe and stit 69B>GFP-moe embryos and they were imaged in parallel. Whereas 69B>GFP-moe embryos sealed their wounds within 60 min, 26% of stit 69B>GFP-moe embryos failed to close their wounds within 3 h. The remaining mutants took on average 110 min to seal their wounds. To ensure that it was stit deficiency that caused the delayed wound closure, Stit was re-expressed in stit mutants at low levels by using 69B-GAL4. Such embryos closed their wounds twice as fast as stit mutants treated in parallel. Thus, Stit is required for rapid re-epithelialization; 25% of the mutants showed at least a threefold delay in epithelial repair, and the other 75% of stit mutants required double the time needed for wound closure in wild-type embryos (Wang, 2009).

To define stit function further, changes in cell shape in wounded embryos were examined by confocal microscopy. A Neuroglian-GFP (Nrg-GFP) protein-trap marker expressed at endogenous levels in epithelial cell membranes. Wild-type embryos expressing Nrg-GFP repaired their wounds within 73 min. Epidermal cells at the margins first extended and then constricted to seal the puncture. In 27% of the stit mutants, margin cells also elongated towards the centre of the wound but failed to close it for at least 5 h. No obvious defects were detected in cell elongation in the remaining mutants, which also showed a delayed wound closure phenotype in comparison with the wild type. Wide-field epifluorescence microscopy was used to assess the cell elongation process in living wild-type and stit mutant embryos expressing Nrg-GFP. Both wild-type and stit mutants showed characteristic elongated cell shapes during the second half of re-epithelialization. This suggested that Stit functions during the second half of re-epithelialization (Wang, 2009).

Epidermal healing relies on dynamic filopodia and lamellipodia and on the formation of a continuous actin cable at the wound perimeter. Confocal microscopy was used to analyse cytoskeletal dynamics in wounded wild-type and stit mutants with the GFP-moe marker. No gross defects were detected in the cellular protrusions at wound margins of stit mutants. Halfway through wound healing, GFP-moe revealed a pronounced actin cable around the wound edge of wild-type embryos. In contrast, in stit mutants the GFP-moe ring was either discontinuous or showed severely reduced intensity. To quantify cytoskeletelal changes during wound closure, 18 wild-type and 27 wounded stit mutant embryos were recorded. The relative fluorescence intensity (RFI) of GFP-moe at the wound edge was calculated for each embryo at defined time points of the wound closure period in each phenotypic class. The rate of increase in cytoskeletal RFI during wound closure was much decreased in stit mutants in comparison with the wild type. In addition, the RFI at the wounds of stit mutants was significantly lower during the second half of the wound healing process than in the wild type. Thus, Stit coordinates the formation of the actin cable at the wound perimeter during re-epithelialization (Wang, 2009).

The predicted Stit protein contains an amino-terminal signal peptide, an extracellular cadherin domain, a transmembrane motif and an intracellular tyrosine kinase domain. This domain combination is distinctive for members of the Ret RTK family. RTK activation normally depends on ligand-induced dimerization and the autophosphorylation of tyrosine residues in the intracellular domain. A series of V5-tagged constructs were generated to test whether Stit could function as a RTK in S2 cells. To force ligand-independent dimerization, the Stit extracellular region was replaced by the Lambda cl repressor dimerization domain. To eliminate the predicted kinase activity StitK504A, StitD628A and StitγIntra were generated. These constructs and Stit-V5 were expressed in S2 cells and tyrosine phosphorylation was analyzed by immunoprecipitation and western blotting. LambdaStit and overexpressed Stit underwent ligand-independent autophosphorylation, as expected of constitutively active RTKs. Ni phosphotyrosine on the StitγIntra, StitK504A and StitD628A proteins was detected, indicating that Stit itself is a tyrosine kinase. Is the Stit kinase activity needed for Stit function in vivo? The ability of a kinase-defective stitK504A transgenic construct and a wild-type stit transgene to rescue the lethality of stit mutants was compared. Only the wild-type construct provided rescuing activity of the lethality phenotype, indicating that the tyrosine kinase activity is required for Stit function (Wang, 2009).

RTK activation typically leads to phosphorylation of members of the mitogen-activated protein kinase family. Transgenic strains were used, expressing either en>stit or the kinase-defective en>stitK504A in embryonic epidermis. en>stit, but not en>stitK504A, produced ectopic phosphorylation of tyrosine and ERK in stripes. Thus, Stit can activate ERK, a common RTK effector. To define its endogenous localization, anti-Stit antisera was generated. Double stainings for the membrane markers CAAX-GFP, Crumbs (Crb) and Fasciclin III (FasIII) indicated that Stit was predominantly localized apically in epidermal cells. Stit staining was severely decreased in stit mutants, indicating the specificity of the antiserum. The intensity of Stit staining was markedly increased at the wound edges of wild-type embryos, indicating that Stit levels are elevated at wound sites. In summary, Stit is a wound-responsive, apical membrane tyrosine kinase that can induce ERK activation in vivo. ERK activation results in the Grh-dependent induction of Ddc and ple on wounding. Is Stit also required for ERK activation at wound sites? Wild-type and stit mutants were pierced and double staining was performed for dpERK and FasIII to reveal surrounding epidermal cells. dpERK staining is not detectable in epidermal cells of stage 16 wild-type embryos. Wounding induced a robust dpERK signal extending two to three cell diameters around the puncture site of wild-type embryos. By contrast, in stit mutant embryos, this dpERK signal was weaker and restricted to cells immediately adjacent to the edge. Similarly to dpERK, the inducible phosphotyrosine staining was decreased at the wounds of stit mutants compared with that in the wild-type. Stit is therefore required for the expansion of ERK phosphorylation at wound sites. In wild-type embryos, Ddc transcripts readily accumulated in cells located several cell diameters away from the puncture site. This response was limited to far fewer cells in stit mutants. The induction of the Ddc-GFP reporter in wild-type and stit mutants was quantified. Among Ddc-GFP embryos, 85% showed a broad or moderate GFP induction, and only 15% presented a weak signal. By contrast, 41% of 234 wounded stit mutants showed a restricted and weak induction and 59% showed broad or moderate GFP activation. Stit was also required for the extended activation of the ple-DsRed wound response reporter. Conversely, Ddc mRNA was induced ectopically in en>stit embryos but remained unaffected in en>stitK504A embryos. This induction was abolished in en>stit; grh mutants. Collectively, Stit regulates ERK phosphorylation and Grh-dependent activation of cuticle repair genes. The genetic analysis places stit both downstream and upstream of grh, suggesting a positive feedback loop mechanism in wound repair. Because stit mRNA is induced at wound sites, whether the Grh-dependent stitα-GFP induction requires endogenous Stit was tested. In contrast with the robust GFP induction in wild-type embryos, stitα-GFP expression in wounded stit mutants remained unchanged. Accordingly, stitα-GFP was activated in stripes in en>stit embryos, and this increase was abolished in en>stit; grh mutants. Thus, Stit function is required for the transcriptional upregulation of stit at the wound (Wang, 2009).

Stit presents the first candidate receptor in Drosophila epidermal wound healing. The functional analysis of stit proposes a two-tier mechanism in wound healing. Epidermal wounds generate signals, which presumably activate Stit and other receptors to initiate the expression of cuticle repair genes and cytoskeletal rearrangements. Stit also further induces its own transcription and a second tier of responses by a Grh-dependent positive feedback loop. The proposed amplification mechanism ensures efficient epidermal wound repair and relies on transcriptional regulation without the need for further extracellular stimulation. Several RTK have been implicated in mammalian epidermal wound healing, suggesting that similar mechanisms may coordinate and amplify wound responses in insects and vertebrates (Wang, 2009).

Grainyhead and Zelda compete for binding to the promoters of the earliest-expressed Drosophila genes

Maternally contributed mRNAs and proteins control the initial stages of development following fertilization. During this time, most of the zygotic genome remains transcriptionally silent. The initiation of widespread zygotic transcription is coordinated with the degradation of maternally provided mRNAs at the maternal-to-zygotic transition (MZT). While most of the genome is silenced prior to the MZT, a small subset of zygotic genes essential for the future development of the organism is transcribed. Previous work has identified the TAGteam element, a set of related heptameric DNA-sequences in the promoters of many early-expressed Drosophila genes required to drive their unusually early transcription. To understand how this unique subset of genes is regulated, a TAGteam-binding factor, Grainyhead (Grh), was identified. Grh and the previously characterized transcriptional activator Zelda (Zld) bind to different TAGteam sequences with varying affinities, and Grh competes with Zld for TAGteam occupancy. Moreover, overexpression of Grh in the early embryo causes defects in cell division, phenocopying Zld depletion. These findings indicate that during early embryonic development the precise timing of gene expression is regulated by both the sequence of the TAGteam elements in the promoter and the relative levels of the transcription factors Grh and Zld (Harrison, 2010).

To understand how a subset of genes are uniquely transcribed in the pre-cellular blastoderm (pre-CB) Drosophila embryo when the remainder of the genome is not, attempts were made to identify proteins that bind to TAGteam elements in the regulatory regions of pre-CB-expressed genes. Nuclear extract prepared from wild-type Drosophila embryos were fractionated and assayed for activity using DNase I protection of a portion of the early-expressed Sxl establishment promoter, SxlPe, containing two overlapping CAGGCAG sites. Partially purified protein(s) protected multiple regions of SxlPe from DNase I digestion, including the two TAGteam elements. As a final purification step, fractions were applied to a DNA-affinity column composed of oligonucleotides corresponding to four repeats of a portion of the zen ventral repression element (VRE), a TAGteam-containing sequence shown to regulate the pre-CB expression of zen. Using the zen VRE for the DNA-affinity column rather than SxlPe ensured that the purified protein(s) would bind to at least two sequences driving pre-CB gene expression. Two polypeptides of ~130 kD and 120 kD specifically eluted from the column. Mass spectrometry identified these two polypetides as the products of two splice isoforms generated from the single gene, grainyhead (grh). This identification was confirmed by immunoblotting (Harrison, 2010).

Grh is a transcription factor conserved from worms to humans that acts in mediating both transcriptional repression and activation. Previous work has demonstrated that Drosophila Grh can bind to the promoters of three additional pre-CB expressed genes, fushi tarazu (ftz), tailless (tll), and decapentaplegic (dpp), and the evidence suggests that Grh binding to these promoters results in transcriptional repression. However, this study is the first to demonstrate that Grh binds to TAGteam sites, greatly increasing the number of pre-CB genes Grh may regulate. In the early embryo, Grh may act as a repressor, in part, through its interactions with Polycomb-group proteins. Later in both fly and mammalian embryonic development, Grh is expressed in the epidermis and functions as an important transcriptional activator during the wound-healing response. Thus, whether Grh binding results in transcriptional activation or repression depends on developmental context (Harrison, 2010).

While the immunoblots confirmed that Grh bound to the zen VRE DNA-affinity column, it was important to determine if Grh provided the DNA-binding activity present in embryonic nuclear extract. Anti-Grh antibodies raised against the DNA-binding domain disrupted the SxlPe DNA-binding activity present in nuclear extract, whereas non-specific IgG did not, confirming that Grh was responsible for the activity. In addition, purified full-length recombinant Grh (rGrh) provided DNase I protection of the zen VRE and SxlPe indistinguishable from that of the activity in nuclear extract (Harrison, 2010).

Because Grh bound to TAGteam elements in both SxlPe and the zen VRE, it was determined whether Grh specifically required TAGteam sequences for binding. Heparin-fractionated nuclear extract and rGrh were used for DNase I protection assays with a fragment of SxlPe identical to that used in the purification described above except that the overlapping CAGGCAG elements were mutated. Grh binding to the mutated CAGGCAG elements was severely inhibited demonstrating that these TAGteam sequences are essential for Grh binding (Harrison, 2010).

Grh bound to additional sequences outside the TAGteam elements in SxlPe and the zen VRE as determined by DNase I protection assays. These additional binding sites do not contain sequences highly similar to the TAGteam sequences. While a consensus Grh binding site has been defined as ACYGGTT(T), there is considerable variability among previously defined Grh binding sites. MEME searches on the Grh binding sites defined by DNase I protection experiments failed to identify a strong consensus site, despite some similarity between the previously defined consensus site and TAGteam elements (Harrison, 2010).

The focus of these studies was placed on the TAGteam-binding activity of Grh as these sequences have been shown to have important functions in the pre-CB embryo. The TAGteam elements have been defined as a group of related sequences including CAGGTAG, CAGGCAG and TAGGTAG, with CAGGTAG being the most enriched in the promoters of pre-CB genes (De Renzis, 2007; Li, 2008; ten Bosch, 2006). To determine if Grh could bind to the prevalent CAGGTAG sequence, rGrh was used in protection assays on a region of the sc promoter containing three CAGGTAG elements and one CAGGCAG element. These experiments demonstrated protection of the CAGGCAG element as well as at least two of the three CAGGTAG elements. Electromobility shift assays (EMSAs) were used to test the affinity of Grh for different members of the TAGteam family. EMSAs with probes that only differed by the sequence of the TAGteam element showed that Grh binds strongly to CAGGTAG and CAGGCAG elements, but only weakly to TAGGTAG sequences, demonstrating the importance of the initial cytosine in Grh recognition. Previous work analyzing the ability of Grh to bind to the closely related sequence GCAGGTAA also showed the importance of the cytosine in Grh recognition. Furthermore, this cytosine was critical for the pre-CB ventral repression of a transgene reporter driven by the dpp ventral repression region (VRR). Together these data show that Grh specifically binds to TAGteam elements within the promoters of three genes expressed in the early embryo, although preferentially to specific TAGteam sequences (Harrison, 2010).

Given that Grh can bind to TAGteam elements, it was asked if Grh was present in the early embryo. Using RT-PCR it was shown that grh transcripts are present in early embryos as well as in egg chambers (ovaries). In agreement with these data, in situ hybridizations had previously identified grh mRNA in these tissues. There are two well-characterized examples of alternative splicing of the grh pre-mRNA, and it was determined that alternative splicing results in multiple mRNAs present in the early embryo. Immunoblots showed that these mRNAs are translated producing Grh proteins. Notably, Grh protein appears absent or at very low levels in late-stage egg chambers, suggesting that maternal grh mRNA, but not protein, is deposited into the embryo (Harrison, 2010).

While this study was characterizing the TAGteam-binding factor Grh, another TAGteam-binding protein, Zelda (Zld), was identified. Zld is a zinc-finger protein that binds to TAGteam elements in the zen VRE and is required for the proper activation of more than 100 genes in the pre-CB embryo. To test whether Grh and Zld have similar binding profiles for the zen VRE, full-length recombinant Zld was purified and used in DNase I protection assays. Interestingly, whereas Grh showed strong protection of the CAGGCAG element and little to no protection of the TAGGTAG element, Zld showed protection of the TAGGTAG and not the CAGGCAG element. To further determine if Zld and Grh had different binding affinities for TAGteam family members, the affinity of Zld for distinct TAGteam elements was tested using EMSAs. Similar to the binding profile for Grh, Zld bound most strongly to oligonucleotides containing the canonical CAGGTAG element. However, the affinity of Zld for the two additional TAGteam elements was reversed from that of Grh: Zld bound the TAGGTAG element more strongly than the CAGGCAG element. These data show that at least two TAGteam-binding factors are present in the early embryo, and that the affinities of these factors for various TAGteam elements differ. While it was previously unknown if all of the related TAGteam elements are equally effective in driving gene expression, the data demonstrating that the transcriptional activator Zld as well as the transcription factor Grh have different affinities for the related TAGteam sequences suggest that it is unlikely they are. Although all three TAGteam sequences are enriched in promoters of pre-CB expressed genes, their differential recognition by these two transcription factors may result in distinct effects on the levels or timing of gene expression (Harrison, 2010).

Because both Grh and Zld bind to TAGteam elements, tests were performed to see whether both proteins could bind these sequences simultaneously or whether instead they compete for binding. For these assays it was imperative probe bound by Zld could be distinguised from that bound by Grh. While Grh is smaller than Zld, it binds DNA as a dimer, and binding of the Grh dimer in EMSAs resulted in shifted species that were difficult to distinguish from those shifted by Zld binding. Therefore a C-terminal portion of Grh containing the DNA-binding and dimerization domains (amino acids 603-1032) was expressed and purified. This truncated form of Grh binds to TAGteam-containing sequences, but its binding is easily distinguishable by EMSA from that of Zld. EMSAs performed with a probe containing two overlapping CAGGCAG elements from SxlPe, and low amounts of Grh(603-1032) resulted in a single shifted species. Increasing amounts of protein produced a slower migrating species, likely due to the binding of a second Grh dimer, suggesting that Grh binds to each of the CAGGCAG elements in the probe. Probes corresponding to portions of the zen VRE or sc promoter containing TAGteam elements yielded similar results. Higher levels of Grh(603-1032) were required to bind both TAGteam elements in the zen VRE probe than for the SxlPe or sc probes, as expected from previous findings that Grh binds weakly to the TAGGTAG variant. The full-length rGrh protein showed similar binding behavior, suggesting that the DNA-binding and dimerization domains alone control binding-site specificity. Additionally, it is noted that attempts to co-immunoprecipitate full-length Grh and Zld from embryonic extracts or a mixture of purified epitope-tagged proteins were negative, indicating that interactions between the two full-length proteins through direct protein/protein interactions is not likely. Having shown that rGrh and Grh(603-1032) have similar binding profiles and binding of the truncated protein to oligonucleotide probes is easily distinguished from Zld binding, Grh(603-1032) was used in EMSAs to test for cooperativity or competition (Harrison, 2010).

The minimal amount of rZld required to saturate binding and eliminate free probe was determined experimentally. Reactions supplemented with increasing quantities of Grh(603-1032) showed reduced amounts of probe complexed with Zld and a concomitant increase in the amounts of probe bound by two Grh dimers, demonstrating that Grh competes with Zld for TAGteam binding. As predicted from previous results, Grh competed most weakly with Zld for binding to the zen VRE probe, which contains a TAGGTAG site to which Grh binds more weakly than Zld. Therefore Grh is capable of competing with the transcriptional activator Zld for binding to TAGteam sites, and these data suggest that Grh acts to repress transcription from TAGteam-containing promoters in the pre-CB embryo. Similarly, Grh has been shown to compete with an unidentified activator for binding to a TAGteam-related sequence in the dpp VRR. Thus, one possible mechanism for the previously reported Grh repression in the pre-CB embryo is competition with an activator for DNA binding (Harrison, 2010).

Given that at approximately equal molar amounts Grh and Zld compete for TAGteam binding, the relative levels of each protein was determined at different times during early embryonic development to learn whether they would have an opportunity to compete in the early embryo. Embryos were harvested at one-hour time intervals after egg laying (AEL), and levels of Grh and Zld were compared using quantitative immunoblots. Grh levels were constant in the early embryo. By contrast, levels of Zld were low in the 0-1 hour embryos and increased in the 1-2 hour embryos, when early gene expression initiates. To allow for a comparison between the relative amounts of each protein in the early embryo, approximate protein concentrations for Zld and Grh were determined by comparison of the immunoblot signals with the signal obtained from known amounts of recombinant protein. Each embryo contains approximately 1.8 × 109 molecules of Grh regardless of age, equating to ~9 × 108 molecules of Grh dimers with DNA-binding activity. This estimate is based on the fact that as determined by gel filtration chromatography little or no Grh protein exists as a monomer. Zld levels were ~7 × 108 molecules per embryo in the 0-1 hour embryo and increased to ~1.5 × 109 molecules per embryo in the 1-2 and 2-3 hour embryos. Thus, in the early embryo when there is no zygotic transcription, Grh levels are higher than Zld levels. These data, in combination with the fact that Grh may have a slightly higher affinity for CAGGTAG sites than Zld, suggest a model wherein Grh is likely bound to TAGteam elements and helps to maintain a transcriptionally silent state in the pre-CB embryo. At the time that early zygotic transcription initiates Zld levels have increased, raising the possibility that Zld now outcompetes Grh for TAGteam binding and thus helps drive gene expression (Harrison, 2010).

Validating the suggestion that Grh is not an essential activator of early gene expression, maternal depletion of grh does not result in obvious defects in cellular blastoderm formation or viability. Furthermore in situ hybridizations have not shown any obvious effects of maternal depletion or overexpression of Grh on the expression patterns of zen or tll in the stage 5 embryo; it is unclear whether this is because grh expression is only being perturbed in the maternal germline. It is possible that premature expression of pre-CB genes resulting from the maternal depletion of grh will not result in a significant phenotypic consequence unless the embryo is subject to stress or Zld levels are perturbed, resulting in a failure to detect abnormal expression patterns for zen and tll. In addition, the extra maternally deposited Grh in the overexpression experiments may be overcome by the increase in Zld levels that occurs one hour after fertilization. Alternatively, the additional Grh binding sites in the pre-CB promoters might have other functions in regulating gene expression that confound these experiments where the expression of the native genes was observed. Importantly, no expansion of tll expression was observed in embryos maternally depleted for grh despite previously published reports to the contrary. The only difference between these experiments and the published experiments were that the FLP-FRT system was used to generate embryos lacking maternal grh, while the previous work relied on X-ray induced mitotic recombination. Thus, it is suggested that the expansion of tll observed ib previous may have been due to an unrelated defect caused by the irradiation (Harrison, 2010).

It is noted that overexpression of grh in the maternal germline leads to defects in nuclear division in the blastoderm embryo reminiscent of the defects observed in zld mutant embryos or when Zld levels are decreased by RNAi, supporting the model that Grh acts as a transcriptional repressor by competing with Zld for DNA binding. Anaphase bridges between dividing nuclei, aberrant cell divisions perpendicular to the normal plane of division, and a lack of synchronicity in cell divisions were detected in about 50% of the blastoderm embryos generated from mothers of two different lines overexpressing grh in the maternal germline. None of these defects were noted in wild-type siblings. Interestingly, Grh overexpression in the pre-CB embryo resulted in ~50% reduction in hatching, indicating that these cell-division defects may ultimately decrease embryo viability. These observations are consistent with the competition model suggested by the in vitro experiments. When Grh is overexpressed in the maternal germline the resulting abnormally high levels of Grh in pre-CB embryos may disrupt the ability of Zld to function properly in the very early embryo by competing for TAGteam-binding sites (Harrison, 2010).

In summary, these data suggest that the concentrations of at least two TAGteam-binding factors (Grh and Zld), as well as the sequence variants of the TAGteam elements in the promoters, regulate gene expression in the pre-CB embryo, ensuring that transcription does not initiate prematurely. In its simplest form the model from existing data is that Grh acts as to inhibit premature transcription in the pre-CB embryo during the first hour following fertilization by blocking the ability of Zld to bind to TAGteam sites and activate gene expression. As Zld levels increase during the second hour, Zld now successfully competes against the constant level of Grh for TAGteam binding and activates gene expression. This competition between Grh and Zld can ensure that despite minor fluctuations in Zld levels or other stochastic activating events, expression of pre-CB genes will not initiate prematurely. This model is supported by previous work showing that Grh binds to repressive elements in the tll and dpp promoters, mutation of the Grh binding site can cause an expansion of dpp expression, and Grh competes with an unidentified activator for binding to sites in the dpp promoter. Furthermore, as Zld and Grh bind differentially to discrete TAGteam variants, activation at different promoters can be fine-tuned by the combination of TAGteam sequences present. This differential binding preference may explain, in part, how different pre-CB genes initiatetranscription at precise nuclear cycles. Thus Grh, Zld and the TAGteam elements could combinatorially regulate transcription in the pre-CB embryo, establishing the foundation for proper future embryonic development (Harrison, 2010).

Lateral gene expression in Drosophila early embryos is supported by Grainyhead-mediated activation and tiers of dorsally-localized repression

The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. This study shows that a short 12 base pair sequence ('the A-box') present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, affinity chromatography using the A-box element was conducted and a number of DNA-binding proteins and chromatin-associated factors were found using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. The results suggest that maternally expressed Grh acts as an activator to support expression of ind, which was surprising since this factor was identified using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through the A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a 'switch-like' response that helps to sharpen the ind dorsal boundary. Furthermore, the results also demonstrate that TGF-beta signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo (Garcia, 2011).

Other studies have shown combinatorial interactions are necessary to support patterns of gene expression along the DV axis. For instance, one study showed Dorsal and Zelda function together to produce the broad lateral domain of sog. Mutation of either the Dorsal sites or the Zelda sites in the sog CRM produced a pattern that was narrower than the wild-type expression pattern. It was concluded that both Dorsal and Zelda must be present to produce a proper Sog pattern. It is also well appreciated that Dorsal can act cooperatively with the bHLH transcription factor Twist to support expression in ventral and ventrolateral regions of the embryo. It is proposed that Grh and Dorsal act together to support the ind expression pattern. While the ind CRM containing a mutant Dorsal site did support some expression, the expression pattern contained a gap and was weaker in posterior regions; in contrast, in Dorsal mutants, ind expression is completely absent. This result may be explained if both indirect as well as direct functions for Dorsal are required to support ind expression. For instance, Dorsal has other target genes including rho, which is required to support Egfr signaling. Furthermore, mutation of the A-box/Grh binding site within the ind CRM caused expression of the reporter that was expanded dorsally and weak, suggesting this site mediates repression and also activation. Similar to Dorsal mutants, the phenotype observed when the A-box sites were mutated is different than the phenotype in the Grh mutants, thus it cannot be ruled out that Grh may act through other sites as well as the A-box and/or that Grh may act indirectly to influence ind expression by regulating the expression of other transcription factors. A model is proposed that is most consistent with the current data which is that ind is activated in regions where Dorsal is present as well as optimal levels of Grh; it is then refined by Snail and Vnd in ventral regions and Cic and Schnurri/Mad/Medea (SMM) in dorsal regions (Garcia, 2011).

grh and cic genes are both maternal and ubiquitously expressed, thus, another input is necessary to explain how localized expression of ind is supported. This positional information could be provided in part by competition between Grh and Cic proteins for the A-box binding site and in part by ventrolaterally-localized Egfr signaling. A model in which Egfr signaling supports activation of ind via inhibition of a ubiquitous repressor (e.g. Cic) is supported by the results which demonstrate that A-box mediated repression is expanded in Egfr mutants. A recent study also showed expanded expression of an ind CRM fragment reporter in ras cic double mutants in which neither Egfr signaling or Cic repressor is present, suggesting that Egfr may function by inhibition of an 'inhibitor' to promote activation. This data suggests that the putative A-box repressor, Cic, may not be dorsally localized but that its activity is regulated by Egfr signaling which provides the positional information necessary for a sharp boundary. However, the domain of dpERK activation (as detected by anti-dpERK, an antibody to the dual-phosphorylated from of ERK) does not exactly overlap with the ind expression domain at cellularization, as would be expected in the simplest model (Garcia, 2011).

Ajuria (2011) suggested that Egfr signaling supports ind expression through inhibition of Cic, and it is added that it is also plausible Egfr signaling impacts activation of ind through Grh. In fact, a recent study showed that Grh activity during wound response is modulated by ERK signaling. Specifically, both unphosphorylated and phosphorylated Grh were shown to be able to bind DNA and act as an activator. The former is used during normal development of the epidermal barrier and the latter is used to overcome a semi-dormant state during wound response. Another study showed the tyrosine kinase Stitcher activates Grh during epidermal wound healing. In the early embryo Grh may be phosphorylated by Egfr signaling to support activation of ind through the A-box binding site. It is suggested that phosphorylation of both Grh as well as Cic by Egfr signaling can act as a switch to help fine-tune the expression of ind (Garcia, 2011).

Whether a relationship between Grh activation and Cic repression was used in regulation of other genes containing A-box or Cic binding sites was investigated. One other Cic target gene, hkb, was unaffected in Grh mutants. As the A-box site (WTTCATTCATRA) is larger than the Cic consensus binding sequence [T(G/C)AATGAA, complement TTCATT(G/C)A] defined by Ajuria (2011) it is possible that Grh needs the full A-box site to bind. The full A-box sequence is not present in the hkb CRM, but Cic binding may be facilitated by a partial sequence (i.e., TGAATGAA). Alternatively, it is possible that a role for Grh and/or Cic at the A-box is context dependent. For instance, Grh-mediated activation may be a necessary input to support ind expression but not for the support of hkb, which also receives activation input from Bicoid and Hunchback transcriptional activators and is expressed in the pre-cellularized embryo (Garcia, 2011).

Other studies have suggested that Grh acts to repress transcription of fushi tarazu (ftz), dpp, and tll in the Drosophila embryo, but this study is the first to identify a role for Grh-mediated gene activation in the early embryo, in support of dorsoventral patterning. Previous studies had shown that Grh can function as an activator at later embryonic stages. One analysis identified Grh (also called NTF-1 or Efl-1) biochemically using an element from the dpp early embryonic CRM, however the dpp expression domain was unchanged in the grh mutants (Garcia, 2011).

Another recent study also showed Grh binds to sites that are similar to Zelda binding sites (Harrison, 2010). Zelda and Grh each showed stronger affinity for different variations of the shared consensus sequence, but in vitro studies showed they also competed for binding. Harrison (2010) proposed that as levels of Zelda increase it is able to compete against Grh for binding sites and cause activation of the first zygotic genes. Competition at the same binding sites results in a cascading effect in which ubiquitous activators regulate genes in a temporally related manner. It was proposed that Grh functions first to silence gene expression; while, alternatively, the current data is more consistent with a model in which Grh mediated activation follows that of Zelda. ind is considered a 'late' response gene as it appears at mid stage 5 (nc 14), at the onset of cellularization, whereas Zelda was shown to support gene expression earlier at nc 10 (Garcia, 2011).

It is possible that Grh competes for binding to a variety of sites (not only those recognized by Zelda), and that this competition influences gene activation/repression. At the A-box sequence, Cic and Grh may compete to help establish a sharp boundary; unfortunately, the Cic binding to the A-box sequence demonstrated previously in vitro was quite weak (Ajuria1, 2010), so this competition is best examined in vivo in future studies (Garcia, 2011).

This study found there is yet another tier of repression activity that is independent of the A-box mediated repression. Analysis of the eve.stripe3/7-ind-mutant-A-box reporter construct revealed that, while dorsal-lateral repression was lost, there was still repression in the dorsal-most part of the embryo. This led to the idea that other binding sites in the ind CRM, independent of the A-box binding site, mediate repression. Previous research showed ectopic TGF-beta/Dpp signaling can repress ind expression, and therefore it is hypothesized that the repression activity observed in dorsal-most regions of the embryo may be regulated by Dpp signaling (Garcia, 2011).

The results suggested that the Dpp dependent repression supports repression in the dorsal most part of the embryo and not in dorsal lateral regions of the embryo. An expansion of the ind domain in the mutants affecting only this dorsal-most repressor would not be expected, thus the SMM site was mutated in the context of two mutant A-boxes and it was found that the expression pattern was expanded into dorsal regions of the embryo. However, when the A-box sites were mutated, expansion of ind more dorsally into dorsal-lateral regions was seen, but expression was absent in dorsal-most regions. It is possible the embryo can tolerate a slight expansion of ind into dorsal lateral regions of the embryo but expansion of ind into the non-neurogenic ectoderm is detrimental. Thus, two tiers of repression have developed to insure that expression of ind is limited to the neurogenic ectoderm. It is suggested that partially redundant repressor mechanisms are more common than appreciated, because in contrast to activation it is difficult to track repression activity (Garcia, 2011).

Epigenetic changes to DNA and chromatin remodeling have been shown to be vital in repression and activation of genes that define structures in late stages of Drosophila development. For example, Polycomb group genes silence the homeotic genes of the Bithorax complex, which control differentiation of the abdominal segments. To date, little is known regarding how/if chromatin factors play a role in early development of Drosophila embryos. This study has presented evidence that several chromatin-related factors bound an A-box affinity column but did not bind a column containing the mutant A-box element. Although several of these factors did not bind to the A-box element alone when tested by EMSA, it is possible that they bind indirectly via a larger complex. One of these factors Psq has been implicated in both silencing and activation via the Polycomb/Trithorax response elements. Independently, Psq was recently found to positively regulate the Torso/RTK signaling pathway in the germline, while being epistatic to cic a negative regulator of the Torso signaling. It is possible that some of these factors play a role in regulating ind via the A-box element, which would suggest a role for chromatin remodeling early in development - an avenue which is worth pursuing in future studies (Garcia, 2011).

Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference

Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, this study performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, direct TF-DNA interactions were derived using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, network motifs, cis-regulatory codes, and regulators of eye development were found. The predicted target regions of Grainyhead were validated by ChIP-seq and this factor was identified as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions (Potier, 2014).

The development of the Drosophila eye is a classical model system to study neuronal differentiation and patterning. The TFs that represent the core of the retinal determination network are Eyeless (Ey), Twin of Eyeless (Toy), Dachsund (Dac), Sine Oculis (So), and Eyes Absent (Eya). Although many regulatory interactions are known between these TFs, as they intensively cross-regulate each other, knowledge about interactions with downstream target genes and of other TFs involved in the eye-antennal gene regulatory network (GRN) is sparse. This study aimed at combining classical reverse genetics-starting from a mutant allele and analyze its (molecular) phenotype-with genomics. Doing so, attempts were made to unveil genetic regulatory interactions in an unbiased way, and many regulators of the eye and antennal developmental programs were identified; most of these did not require or use any mutation or direct perturbation (Potier, 2014).

The mapping approach began by systematically perturbing the developmental system. Attempts were made to include multiple perturbations into one data matrix to obtain a broad spectrum of expression profile changes. These perturbations included TF mutants, TF overexpression, TF knockdown, and cell sorting (Potier, 2014).

Eye-antennal discs were dissected at the stage where in the WT discs about half of the eye disc contains pluripotent cells that are dividing asynchronously, while the other half contains differentiating PR neurons, in consecutive stages of differentiation. Simultaneously, the antennal disc contains neuronal precursors that are undergoing specification. The expression changes induced by the perturbations often result from a shift in proportion of cell types. This is trivial for the cell-sorting experiments; for example, the GMR>GFP-positive cells show, as expected, a very strong enrichment of genes related to PR differentiation. TF mutants and TF perturbations can also result in cell type shifts; for example, overexpression of Atonal yields more R8 PRs, and the glass mutant results in fewer differentiated PRs. Other TF perturbations cause changes in gene expression downstream of the TF without changing the cell type composition, such as Retained, which disturbs axonal projection. The key technique that was applied, however, was not to compare each TF perturbation with WT discs to identify differentially expressed genes. Rather, linear and nonlinear correlations of gene expression profiles were used across the entire vector of 72 gene expression measurements. This TF-gene coexpression network contains both direct and indirect edges, and although this network is informative, a second layer of predicted TF-DNA interactions was added, thus making this a direct GRN. To increase the sensitivity, a very large collection was used of TF motifs, also including position weight matrices derived for yeast and vertebrate TFs and including computationally derived motifs (e.g., highly conserved words). Using motif-motif similarity measures and TF-TF orthology relationships, each motif was linked to a candidate binding factor. This yielded a large network with 335 TFs and their predicted direct targets. The only functional network of comparable size and comparable directedness to this in vivo network is the TH17 GRN that was derived in vitro in a recent study (Yosef, 2013). That study used a microarray time course of naive CD4+T cells differentiating into TH17. From these gene expression data, they derived TF-gene interactions by clustering and filtered those with TF-DNA interactions obtained by ChIP-seq data, TF perturbations, and cis-regulatory sequence analysis (Potier, 2014).

The predicted direct and functional eye-antennal GRN includes many previously reported interactions, such as known target genes for Eyeless and Sine Oculis. Target genes in the network were also found for late factors (e.g., Glass, Onecut) and very late factors (e.g., Pph13). The fact that information was captured at different time points during development is because several cell populations were sorted that are loosely correlated with the temporal axis of development, consisting of undifferentiated pluripotent cells anterior to the furrow, all PR cells undergoing differentiation posterior to the MF, R8 PR cells, and late populations of chp-positive cells. However, the temporal information encoded in the network is limited to these broad domains, and a more detailed reconstruction of the time axis would require higher resolution cell sorting or microdissection experiments. Although the perturbed TFs were mainly chosen for their development of the retina, master regulators of antennal development, such as aristaless were also identified (Potier, 2014).

Interestingly, general factors like Grainyhead were found that were ubiquitously expressed. Grh was found as one of the TFs with the largest number of target enhancers and its binding correlates with open chromatin. Previous studies have shown that Grainyhead may interact with Polycomb and Trithorax proteins to regulate (both activate and repress) target gene expression. It is speculated that this observed correlation can be explained by the fact that Grh is present ubiquitously in the eye disc, thus yielding many sequence fragments from bound and nucleosome-free enhancers by FAIRE-seq (Potier, 2014).

It is well known that network motifs such as FFLs play an important role in biological networks. One such network motif was examined in more detail, namely the TF pair Glass-Lozenge, and their common targets. These TFs constitute a double-feedback loop (Glass regulates Lozenge, Lozenge regulates Glass, and they together regulate 36 targets). For this network motif, it was found that Glass and Lozenge motifs co-occur at the same enhancer, where they furthermore overlap; this may indicate competition for binding between Glass and Lozenge. Given that Lozenge, an important regulator of cone cell differentiation, could be a repressor and Glass, an important regulator of PR differentiation, could be an activator, such a competition at the CRM level could indeed be a plausible mechanism for their regulatory action (Potier, 2014).

Another interesting feature that can be derived from a GRN is the proportion of autoregulatory TFs (108 autoregulatory TFs in the eye network) and the proportion of activating versus repressive TFs. A recent large-scale study in yeast found a small majority of yeast TFs to have a repressive role. In that study, each individual TF was perturbed, thereby providing information on positive versus negative edges from the TF to its direct predicted targets, whereby TF-DNA information was used from ChIP-chip data. Since the eye GRN was started from a coexpression TF-gene network, the correlations between TFs and their candidate targets were revisited, and 151 TFs were found that have their motif enriched in the positively correlated target genes, but not in the negatively correlated targets, and 127 TFs showing the opposite; 62 TFs show enrichment in both. This finding agrees, to some extent, with the results in yeast concerning the high amounts of gene-specific repressors. On the other hand, the eye network suggests relative more TFs with a dual activator/repressor function, while the yeast study found only a few such cases (Potier, 2014).

In conclusion, starting from an expression matrix derived from large-scale perturbations and combining TF-gene coexpression with TF-DNA interactions based on motif inference enabled drawing an extensive eye-antennal GRN. All predicted regulatory interactions, target genes, and candidate regulatory regions are stored in a Neo4J database and can be queried from a laboratory website. The database can also be accessed directly from Cytoscape using the CyNeo4j plugin or can be queried programmatically using the Neo4j query language Cypher. Although many known regulators and cis-regulatory elements were uncovered and several other ones were revealed, a large part of the predicted network, including how the dynamics of the developmental program are encoded in the cis-regulatory regions and in the topology of the network, remains to be explored (Potier, 2014).

Protein Interactions

GRH functions as a heterodimer. GRH mutant proteins lacking the novel activation domain act as trans-dominant inhibitors of GRH-directed gene activation in tissue culture cells (Attardi, 1993). GRH cannot activate Ubx from the upstream promoter by direct interaction with TATA binding protein, but requires six tightly bound TBP-associated factors (Dynlacht, 1991).

The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. The human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor Grainyhead binding to dring, the fly homolog of dinG. Electrophoretic mobility shift assays demonstrate that the Grh-dring complex forms on regulatory elements of genes whose expression is repressed by Grh but not on elements where Grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes (Tuckfield, 2002).

Strong evolutionary conservation of amino acid sequence exists between the mammalian and Drosophila members of the Grainyhead-like family. The likelihood of a similar conservation of function led the idea of the existence of a Drosophila homolog of dinG. Database searches identified a sequence that has been termed dring (FlyBase term: Sex combs extra), which has 44% identity and 61% similarity to the dinG amino acid sequence and 50% identity and 68% similarity in the domain of the dinG protein which interacts with the GRH-like family. To determine whether the Drosophila factor Dring could interact with Grh, radiolabeled in vitro-transcribed and translated Grh was generated for GST chromatography assays. Grh was shown to be specifically retained on a GST-Dring matrix but not on GST alone, confirming the evolutionary conservation of this interaction (Tuckfield, 2002).

DinG can interact with CP2 and repress transcription from a CP2-dependent promoter. These data were generated in the context of a concatemerized consensus CP2 binding site. No physiological target genes of CP2-mediated repression have been identified in mammalian systems. In contrast, the regulatory regions in the dpp and tll genes involved in Grh-mediated repression have been clearly defined in vivo. In view of the significant homology between Grh and CP2 in the DNA binding domain, whether the CP2-dinG complex could form on the Grh-responsive element in the dpp promoter was examined. A probe containing the DRE-B region of the dpp promoter was studied in an EMSA in the presence of nuclear extract from the mammalian cell line JEG-3. Addition of this extract to the DRE-B probe resulted in the formation of a DNA-protein complex. This complex was ablated by the addition of either anti-CP2 or anti-dinG antiserum. To extend this observation, whether the GRH-DRING complex could assemble on the regulatory regions in the dpp and tll genes that are critical for GRH-mediated repression was examined. Probes containing the DRE-B region of the dpp promoter and the tor-RE element in the tll promoter were studied in an EMSA with Drosophila embryo extract in the presence and absence of anti-Grh antiserum or anti-dinG antiserum (which cross-reacts with the Drosophila DRING protein). The be2 element of the Ddc promoter (where Grh functions as a transcriptional activator) was also studied. A complex consisting of at least Grh and Dring formed on both the dpp and tll elements. In both settings, the complex was ablated (or shifted out of the gel) by anti-Grh and anti-dinG antisera. In contrast, the complex formed on the Ddc promoter was ablated by the addition of anti-Grh antiserum but remained unchanged in the presence of anti-dinG antiserum (Tuckfield, 2002).

Efficient and specific targeting of Polycomb group proteins requires cooperative interaction between Grainyhead and Pleiohomeotic

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. The grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, it is proposed that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity (Blastyak, 2006).

The iab-7 PRE lies next to the Fab-7 boundary, a chromatin domain insulator element between the neighboring iab-6 and iab-7 cis-regulatory domains of BX-C. Fab-7 ensures the functional autonomy of these cis-regulatory domains; iab-7 is inactive in the sixth abdominal segment (A6), where iab-6 is active, while iab-7 is activated in segment A7. A large set of internal BX-C deficiencies is available, making this region ideal for genetic studies (Blastyak, 2006).

Class II deletions, which remove only the boundary region, fuse the otherwise intact cis-regulatory elements iab-6 and iab-7. The consequence of this fusion is that in some A6 cells iab-6 is inactivated by iab-7, while in some other A6 cells iab-6 ectopically activates iab-7. As a result, A6 will become a mixture of cell clones with either A5 or A7 identity. Due to the fact that the Abd-B gene, the expression of which is controlled by these cis regulators, is haploinsufficient, such transformations are evident even under heterozygous conditions. Class I deletions, which remove both the Fab-7 boundary and the adjacent iab-7 PRE, transform A6 into a perfect copy of A7, suggesting that in the case of class II deletions it is the iab-7 PRE that mediates the inactivation of iab-6 in A6; thus, the inactivation may depend on Pc-G-mediated silencing. Indeed, if a class II deletion is combined with some, but not all, Pc-G mutations, the resulting phenotype is indistinguishable from that of class I deletions. Based on this result, it should be possible to identify mutations in factors that specifically interact with the iab-7 PRE as enhancers of the phenotype of class II deletions (Blastyak, 2006).

Accordingly, several X-ray mutagenesis screenings were performed with the class II allele Fab-72. Among the enhancer mutants, one complementation group, represented by five alleles in the collection, is described here. Two alleles are associated with a cytologically visible breakpoint in 54F, and deficiency mapping placed the locus between the proximal breakpoints of the Pcl11b and Pcl7b deletions. Previously, four complementation groups were isolated within this interval. Noncomplementation with alleles of one of the four complementation groups showed that new mutant alleles were isolated of the previously described gene grainyhead (grh). The previously isolated grh alleles, including the molecularly characterized amorphic allele B37, are also strong Fab-72 enhancers, indicating that loss-of-function grh mutations affect the function of the iab-7 PRE (Blastyak, 2006).

Genome-wide prediction has indicated that the occurrence of the same limited set of consensus motifs can fairly accurately predict the PRE function of a DNA sequence (Ringrose, 2003). This observation suggests that many, if not all, PREs use the same set of DNA binding proteins. One of the frequently occurring consensus sequences within PREs is a poly-T motif. Many, although not all, GRH binding sites are T rich, and the current studies indicate that at least in some cases the poly-T consensus sequence may be a binding site for this protein. However, like other DNA binding proteins involved in PRE function, GRH alone cannot explain the specificity of targeting, since its function is not limited to PREs. In other contexts, GRH acts as a transcriptional activator. The fact that an array of distinct sequence motifs is required to accurately predict PREs probably means that there is no single major targeting activity. Indeed, in the case of the engrailed PRE it was demonstrated that all binding sites of DNA binding proteins are equally important for silencing activity. Identification of GRH as a PRE-related DNA binding protein and, in particular, its cooperative interaction with another member of this group both in vivo and in vitro may help in understanding the targeting of PC-G to PREs during development (Blastyak, 2006).

A cooperative interaction between GAF (Trithorax-like) and PHO has been demonstrated (Mahmoudi, 2003). In contrast to the case of GRH and PHO, cooperation between GAF and PHO is independent of the physical interaction between the two proteins and requires a nucleosomal context. Although the physical basis of this cooperative interaction is not understood, it also suggests that cooperativity may be an important principle in the organization of nucleoprotein assembly at PREs (Blastyak, 2006).

What could be the impact of cooperativity on PC-G targeting? Theoretically, one of the most significant problems encountered by a DNA binding protein is the huge excess of potential binding sites in the genome, including both functional sites and pseudosites. It can be assumed that if any of the DNA binding proteins involved in targeting are present in limited amounts in the nucleus, then their binding occurs only at the highest-affinity sites, where a combination of certain binding sites facilitates their cooperative binding. Several observations contradict this simple model. First, if the amount of these DNA binding proteins were limited, their mutations would be expected to result in strong haploinsufficient phenotypes, which is not the case. Second, studies on the DNA binding proteins EVE, FTZ, and GAF demonstrated that in vivo they also bind to genes that are not controlled by them. These functionally irrelevant sequences may represent pseudosites, and the relatively low level of binding at these sites may indicate a low binding affinity. Thus, it appears that restricted binding site occupancy of DNA binding proteins is not necessary for specificity in gene regulation. Likewise, even though the DNA binding proteins present on PREs may bind to nonfunctional sites, it is likely that the functionally relevant high-affinity sites are distinguished from pseudosites in vivo by the unique arrangement of distinct, stably bound cooperative partners. However, although in this model of targeting of PRC1 to the iab-7 PRE, cooperativity at the level of the DNA binding proteins is critically required for binding stability, by itself it is insufficient to provide the required specificity of the targeting process (Blastyak, 2006).

In contrast to the DNA binding components, other constituents of the silencing complex appear to be limiting factors. This is suggested by the fact that most Pc-G genes were identified either on the basis of their characteristic haploinsufficient phenotypes or on the basis of their dominant genetic interaction with other known Pc-G members. The number of potential PRE sequences is also relatively small, as a genome-wide survey estimated it to be not more than a few hundred in Drosophila. This brings us to the question of how the abundant DNA binding proteins link the limited amount of PC-G complexes to the low-frequency target sites with high specificity (Blastyak, 2006).

The first clue comes from studies showing that all of the PRE DNA binding proteins have the ability to interact with various PC-G proteins that are all subunits of the same preformed protein complex, PRC1. These interactions appear to be weak by themselves, as illustrated by the fact that although the occurrence of these interactions can be demonstrated by using short protocols like immunoprecipitation, the resulting complexes do not survive nonequilibrium methods used for traditional biochemical purification of protein complexes. The consequence of the cooperativity at the level of DNA binding proteins is that the otherwise weak interaction surfaces are integrated into a stable composite surface that can serve as a high-affinity docking site for the limited amount of PRC1 complex. In the model, this second level of cooperativity would provide targeting specificity (Blastyak, 2006).

Notably, the same DNA binding proteins involved in PC-G targeting can separately participate in weak interactions with various other protein complexes involved in processes unrelated to, or the opposite of, Pc-G-dependent silencing, such as TFIID-dependent transcription or chromatin remodeling by SWI/SNF. Based on the available data, interaction surfaces of any such complex are not shared by these DNA binding proteins, and according to this model, their concerted recruitment to PREs is unlikely. Also, in agreement with the experimental data, this model predicts that in the absence of DNA none of the DNA binding proteins will be able to interact stably with the complex to be recruited. The integration of several weak protein-protein interaction modules into a single entity is a prerequisite for the complex to dock on chromatin (Blastyak, 2006).

It has been shown that transcription through the iab-7 PRE displaces PC-G proteins and results in concomitant recruitment of the TRX and BRM proteins. Thus, iab-7 PRE appears to be a switchable element and the potential, for example, of PHO to interact with protein partners having a function that is the opposite of PC-G silencing might be realized under certain circumstances. There is insufficient data to explain the mechanism underlying this switch. One possibility is that binding of some DNA binding proteins to DNA or to their interacting partners is modified by posttranslational modifications, as it was shown in the case of the human homologue of Grh. According to the model, even the modification of a single actor (e.g., GRH) can radically influence the overall assembly configuration of the targeting complex and might be responsible for the dynamic nature of the iab-7 PRE (Blastyak, 2006).

This model shows remarkable similarity to the functional and structural organization of enhanceosomes. For example, multimerization of the binding sites of any of the DNA binding proteins involved in beta interferon (IFN-ß) enhanceosome formation does not reproduce faithfully the virus inducibility of the intact enhancer. Instead, these synthetic enhancers respond promiscuously to inducers that are normally not involved in regulation of the IFN-ß gene. The molecular basis of the selective inducer response of the enhanceosome is established by the following cooperative interactions. First, in their original context, the mutually cooperative interactions at the level of DNA binding proteins promote binding stability. Second, on the resulting spatially arranged protein surface, each DNA binding protein contributes to the recruitment of a protein complex through interactions with one of its subunits. It is concluded that the integration of different, hierarchical levels of cooperativity could be a general principle in the targeting of protein complexes to chromatin (Blastyak, 2006).

The validity of the enhanceosome model has already been demonstrated by in vitro reassembly of the IFN-ß enhanceosome with well-defined recombinant components. In vitro studies with a nucleosomal template have provided valuable insights into the role of PRC1 in regulation of the chromatin structure. However, in this experimental system the excess of PRC1 and nonspecific DNA binding of PRC1 complex members overcomes the problem of targeting. An initial attempt to reconstitute cooperativity at the level of DNA binding proteins failed, possibly because the simultaneous presence of several other DNA binding proteins is required for cooperative assembly. Until these components of PREs are identified, it is likely that PC-G targeting cannot be faithfully reconstituted in vitro. Hopefully, the identification of as-yet-unknown DNA binding protein components of PREs, together with the conceptual framework presented here, will facilitate these studies (Blastyak, 2006).

Recent results showed that in vivo stable recruitment of PC to the Ubx PRE critically depends on the presence of the E(Z) protein. E(Z) is a member of a PC-G complex, which is distinct from PRC1, and possesses histone methyltransferase activity. These findings led to a model wherein, upon binding of the EZ complex, its enzymatic activity could provide the mark for the specific targeting of PRC1. Hence, recruiting of PRC1 would only indirectly depend on sequence-specific DNA binding proteins, as they primarily act as recruiters of the E(Z) complex, but not PRC1. Contrary to the predictions of this model, it was found that although mutations in PRC1 complex members are similarly strong dominant enhancers of the Fab-72 phenotype as grh and pho, amorphic E(z) alleles in heterozygous condition are not. Thus, the current results indicate a rather intimate link between these DNA binding proteins and PRC1 complex members. However, it is still possible that in a nucleosomal context the histone mark could provide an additional constituent for binding whose presence can be critical in vivo in certain tissues. Certain PC-G group members have a tissue-specific phenotype, and GRH is also not ubiquitously expressed, which supports this notion (Blastyak, 2006).

Phosphorylation of Grainy head by ERK is essential for wound-dependent regeneration but not for development of an epidermal barrier

Grainy head (GRH) is a key transcription factor responsible for epidermal barrier formation and repair, whose function is highly conserved across diverse animal species. However, it is not known how GRH function is reactivated to repair differentiated epidermal barriers after wounding. This study shows that GRH is directly regulated by extracellular signal-regulated kinase (ERK) phosphorylation, which is required for wound-dependent expression of GRH target genes in epidermal cells. Serine 91 is the principal residue in GRH that is phosphorylated by ERK. Although mutations of the ERK phosphorylation sites in GRH do not impair its DNA binding function, the ERK sites in GRH are required to activate Dopa decarboxylase (Ddc) and misshapen (msn) epidermal wound enhancers as well as functional regeneration of an epidermal barrier upon wounding. This result indicates that the phosphorylation sites are essential for damaged epidermal barrier repair. However, GRH with mutant ERK phosphorylation sites can still promote barrier formation during embryonic epidermal development, suggesting that ERK sites are dispensable for the GRH function in establishing epidermal barrier integrity. These results provide mechanistic insight into how tissue repair can be initiated by posttranslational modification of a key transcription factor that normally mediates the developmental generation of that tissue (Kim, 2011).

Interestingly, putative ERK phosphorylation sites are also found in the N-terminal domain of Grhl3, a mammalian homolog of GRH. Given that grhl3 mutant mice display defects in both developmental skin barrier formation and wound-induced repair, and that ERK is required for mammalian wound repair, it is plausible that ERK phosphorylation of GRH might be an evolutionarily conserved event in animal epidermal wound repair. Drosophila GRH and mammalian Grhl proteins do not share extended blocks of amino acid sequence homology that include S-P or T-P motifs that are characteristic of ERK consensus sites, but functional phosphorylation sites can show rapid sequence drift during evolution (Kim, 2011).

Mutations of ERK phosphorylation site residues in GRH do not detectably influence its affinity to DNA binding sites in vitro, but the phosphorylation sites are required for GRH functional activity on epidermal wound enhancers in late embryonic epidermal cells. The wound-specific activity of GRH does not appear to involve phosphorylation-dependent nuclear localization, because the GRH protein is constitutively nuclear, either when expressed by endogenous promoters or epidermal GAL4 drivers. In addition, as measured by the strength of overexpressed ERK-site mutants of GRH in activating Ddc expression, the developmental transcriptional activation function of GRH was not significantly altered by the mutation of ERK phosphorylation sites. However, ERK phosphorylation of GRH-binding affinity might enhance its binding to a coactivator or prevent its binding to a corepressor that is specific during wound response. Because phosphomimetic GRH 2E expression did not constitutively activate transcription of GRH target genes in both developmental and wound response contexts, the phosphorylation of GRH alone is not likely to be sufficient for triggering activation of the wound response genes. Given that both GRH and FOS-D are required for the induction of Ddc and msn, and that FOS function can be also activated by ERK phosphorylation, it is believed that wound-induced signaling input through FOS or other transcription factors is also necessary for the transcription of these wound response genes along with the phosphorylation of GRH. Therefore, the ERK-dependent phosphorylation of both GRH and FOS upon injury may activate both transcription factors to synergistically induce many target genes that facilitate epidermal barrier regeneration (Kim, 2011).

Although GRH function in Drosophila embryonic epidermis is critical for both developmental generation and wound-triggered regeneration of epidermal barriers, it was not known whether it is required for the process of reepithelialization (epidermal wound closure) after wounding. Mammalian Grhl3 has been shown to be required for the keratinocyte wound closure in tissue culture, mainly through its activation of RhoGEF19. However, in Drosophila, it was found that the wound closure phenotypes between wild-type and grhIM homozygous embryos were indistinguishable, indicating that Drosophila GRH is not critical for reepithelialization after wounding (Kim, 2011).

Given the importance of the ERK-GRH axis in transcriptional activation of epidermal wound response genes, the signals and receptors upstream of ERK are of great interest. Mammalian cell culture studies suggest that receptor tyrosine kinases (RTK) are responsible for the wound-dependent activation of ERK. In Drosophila, stitcher (stit) encodes a Ret-family tyrosine kinase that contributes to transmission of an epidermal wound signal, because null mutations in stit result in a partial inhibition of wound-induced ERK phosphorylation, and reduced activation of wound enhancers in embryonic epidermal cells (Wang, 2009). It is very probable that another RTK(s) are responsible for the activation of ERK after epidermal wounding observed in stit null mutant embryos. The PVR RTK is a good candidate because its function has been shown to be required for wound healing in larval epidermal tissue (Kim, 2011).

The RTK-mediated activation of ERK-GRH axis appears to mediate other biological roles in addition to its role in embryonic epidermal barrier repair. GRH has been reported to mediate Torso RTK-dependent repression of the tailless gene in early embryogenesis. Therefore, although the ERK-GRH axis is dispensable for late embryonic epidermal barrier development, it appears to function in other developmental contexts. These distinct functions presumably depend on the context of different transcriptional enhancers with different transcription factor codes (Kim, 2011).

More importantly, the current findings suggest an important mechanism that may underlie injury-induced tissue regeneration. After wounding or amputation, developmental programming must be reinitiated to recover the original structure of the damaged tissue. In the context of the epidermis, GRH is a key transcription factor for generating a normal epidermal barrier during development in a manner independent of ERK phosphorylation. However, GRH is also persistently expressed in terminally differentiated epidermal cells of the embryo, larvae, and adult, and its function in barrier repair must be rapidly and robustly reactivated after wounding. In the current model, a semidormant state of GRH can be overcome by ERK phosphorylation to regain its ability to transcriptionally activate target genes like Ddc, msn, and many others that regenerate epidermal barriers. This model may also apply to c-Jun in mammals, which does not require JNK-dependent phosphorylation sites for developmental eyelid and neural tube closure, but does require those sites for closure of epidermal wounds. Thus, some regeneration processes in tissues or organs of diverse animal species after injury may be initiated through a similar molecular mechanism-posttranslational reactivation of essential transcription factors that are normally involved in developmental morphogenesis (Kim, 2011).

Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function, an inducible version of the biotinylation tagging approach was established to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. Many TrxG related proteins were found, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, this analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function (Strübbe, 2011).

Combinations of one-step capture with streptavidin, low stringency washes, specific elution, and detection of peptides using a highly sensitive LTQ-FT-ICR mass spectrometer enabled the identification of even labile and transient interactions. It has been well recognized that PcG and TrxG proteins exert their counteracting activities at the level of chromatin by employing various biochemical activities directed against histones, like methylation, acetylation, and chromatin remodeling. Indeed, this study reveals a substantial number of Pc-interacting proteins implicated in TrxG action. The genes encoding for Rdx, Ebi, CG1845, Rad21, and Fs(1)h have been shown genetically to belong to the TrxG suppressing PcG mutant phenotypes and activating HOX gene expression, for example. Additionally, Pp1-87B has been found to interact with Trx or its homologue MLL. These data indicate that Pc and specific members of the TrxG may physically cooperate to maintain the on/off state of genes (Strübbe, 2011).

So far, the DNA-binding proteins Zeste, Gaf, Pho, Dsp1, Sp1/Klf family members, Psq, and Grh have been connected to PcG function on the basis of genetic interactions, biochemical copurification, functional assays, and/or colocalization on PREs. This study found direct biochemical interactions of Grh and Pho with Pc. Moreover, a Pc-interacting protein called Fs(1)h was identified that might, as well, contribute to recruitment of PRCs to chromatin. Fs(1)h interacts strongly with Ubx, trx, and ash1 mutations and leads to homeotic phenotypes when overexpressed. Fs(1)h is essential for development and conserved in mammals. Whether Pc is recruited by Fs(1)h or opposes its function in gene activation needs to be established. Beside the aforementioned DNA-binding proteins, Enok is a Pc interactor with a putative DNA-binding domain. Enok forms part of the MYST domain family of histone acetyl transferases (HATs), and mutants with defects in the HAT domain show retarded development and pupal lethality (Strübbe, 2011).

Enok's HAT domain is conserved in the vertebrate Moz/Morf proteins. They typically form complexes comprising one protein per BRPF-, ING-, and EAF family member. In Drosophila, a Moz/Morf like complex may consist of Enok, CG1845 (homologue of Brpf1-3), and Eaf6 as all these proteins copurified with Pc and were detected with high confidence. Moz and Brpf1 are TrxG proteins required for HOX gene expression in vertebrates. Although MYST-domain-containing HATs have generally been associated with transcriptional activation, there are also examples with a link to HOX gene repression in Drosophila (Strübbe, 2011).

This work uncovered a connection between Pc and the cohesin complex. Cohesin has been described in detail for its roles in mitosis and meiosis, embracing sister chromatids in mitotic cells. Interestingly, mutations in Ph-p, Psc, and Pc have been reported to result in chromosome missegregation phenotypes in embryos. Besides its traditional role in sister chromatid cohesion, cohesin has also been implicated in both activation and repression of transcription. Furthermore, mutations in the Rad21 subunit of the cohesin complex strongly enhance TrxG and suppress PcG loss of function phenotypes. Pc and cohesins are not colocalized on salivary gland chromatin, and removal of cohesin does not affect Pc binding. It cannot be ruled out that Pc is needed for recruitment of cohesin, however. For example, chromatin binding of cohesin in S. pombe depends on formation of heterochromatin, requiring another chromo domain protein, HP1 (Strübbe, 2011).

A hallmark of PcG repression in flies is pairing-sensitive silencing (PSS), depending on pairing of homologous chromosomes in interphase chromatin. It is known that multiple copies of a transgenic PRE interact with each other if inserted on the same or even on different chromosomes. Because cohesin plays a role in pairing of homologous chromosomes in meiosis and has been suggested to facilitate long-range DNA interactions, it may also facilitate PRE pairing. The transgenic reporter for PSS used in this study only showed PRE-dependent silencing upon PRE pairing. The observation that cohesin mutant alleles reduce PSS supports a model in which cohesins contribute to PRE pairing in interphase chromatin. The identification of Pc-interacting proteins was made possible by employing the in vivo biotinylation system combined with highly sensitive mass spectrometric analysis, thereby preserving near physiological conditions for protein purification. The identification of substoichiometric levels of interacting proteins shows that in vivo biotinylation was effective in capturing even weakly or underrepresented associated proteins. Inducible biotinylation tagging is currently limited to the use of Gal4 drivers that trigger biotinylation well above the background levels. Generation of libraries of different UAS-BirA transgenic lines with less leaky expression and flies carrying BirA under direct control of tissue-specific promoters will further improve and expand this tool, making it a versatile system for proteomic and genomic studies in specialized cell types. As a major advantage over tissue-specific expression of tagged bait proteins, biotin tagging allows to express the bait protein under control of endogenous promoter sequences, whereas the induction of the BirA ligase can be independently induced via the Gal4/UAS system avoiding bait protein misexpression artifacts. This work opens the perspective for tissue-specific applications, potentially enabling a systems analysis on how protein networks can control subsets of genes in specialized cells (Strübbe, 2011).


grainy head: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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