The cis-regulatory dynamics of the Drosophila CNS determinant castor are controlled by multiple sub-pattern enhancers

In the developing CNS, unique functional identities among neurons and glia are, in part, established as a result of successive transitions in gene expression programs within neural precursor cells. One of the temporal-identity windows within Drosophila CNS neural precursor cells or neuroblasts (NBs) is marked by the expression of a zinc-finger transcription factor (TF) gene, castor. Analysis of cis-regulatory DNA within a cas loss-of-function rescue fragment has identified seven enhancers that independently activate reporter transgene expression in specific sub-patterns of the wild-type embryonic cas gene expression domain. Most of these enhancers also regulate different aspects of cas expression within the larval and adult CNS. Phylogenetic footprinting reveals that each enhancer is made up of clusters of highly conserved DNA sequence blocks that are flanked by less-conserved inter-cluster spacer sequences. Comparative analysis of the conserved DNA also reveals that cas enhancers share different combinations of sequence elements and many of these shared elements contain core DNA-binding recognition motifs for characterized temporal-identity TFs. Intra-species alignments show that two of the sub-pattern enhancers originated from an inverted duplication and that this repeat is unique to the cas locus in all sequenced Drosophila species. Finally it was shown that three of the enhancers differentially require cas function for their wild-type regulatory behavior. Cas limits the expression of one enhancer while two others require cas function for full expression. These studies represent a starting point for the further analysis of cas gene expression and the TFs that regulate it (Kuzin, 2012).

Using the EvoPrinter repeat finder search program and composite eBLAT intra-genomic alignments, an inverted repeat was identified that partially overlaps two of the upstream conserved sequence clusters (CSCs) and is present once in each of the 12 Drosophila genomes examined. In D. melanogaster, the proximal half of the inverted repeat is located 680 bp upstream of the predicted start of transcription and is separated from the distal half by an 1,881 bp spacer that spans the cas-6 CSC. Both distal and proximal halves contain CSBs that are part of the cas-5 and cas-7 CSCs respectively, and both of these CSCs contain unique CSBs that flank the repeat halves. Analysis of the inverted duplication in different Drosophila species revealed that all sequenced species have the inverted repeat, but the size, extent of repeat sequence identity and the orientation of its intervening region vary among different species. For example, the sequence identity between proximal and distal repeat halves is not complete in D. melanogaster, as there are base-pair differences within the central region of each repeat and larger identity gaps in their outer ends. In addition, the repeats within the D. virilis and D. mojavensis species both have differences when compared to D. melanogaster. In contrast to D. melanogaster, the D. virilis repeat is larger (distal half 1,587 bp vs. 1,188 bp) and the central regions of the repeat halves are nearly identical with only a single base-pair difference between them, while the D. mojavensis repeat sequence identity is significantly lower than that observed in the repeat of other species. Comparison of the D. mojavensis repeat halves reveals that the central portion of the repeats has not been conserved (Kuzin, 2012).

The inter-species alignments also revealed that the cas-6 CSC (located within the intervening spacer between the repeat halves) had flipped its orientation in five of the Drosophila species (D. persimilis, D. willistoni, D. virilis, D. mojavensis and D. grimshawi) relative to the repeat-spacer-repeat arrangement present in D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae and D. pseudoobscura. The intra-species alignments also revealed that, unlike any of the other species examined, the cas locus within D. grimshawi is duplicated, and one of the duplicates does not include the adjacent pollux gene. cis-Decoder analysis of the conserved sequences (including both intra- and inter-CSC alignments) reveals that many of the CSCs share different combinations of conserved sequence elements, and many of these elements are repeated within the individual CSCs. As discussed above, cas-5 and cas-7 are structurally related to one-another. The shared repeat and unique elements within the cas-5 and -7 CSCs have been used to discover other late temporal network Drosophila NB enhancers (Brody, 2012). In addition, the inter-CSC alignments also revealed that the cas-3 CSC shares an 18 bp sequence (ATTTGCATAATTTTGGCA) with both the cas-5 and -7 CSCs with all but the first base conserved in the cas-3 CSC. The shared sequence contains a POU-homeodomain TF DNA-binding octamer sequence (ATTTGCAT) and the core DNA-binding motif for Antennapedia class homeodomain TFs (TAAT). Unlike cas-5 and cas-7, which both activate expression in the embryo, cas-3 drives expression in a set of larval brain neurons. Future experiments are required to delineate function of the 18 bp sequence (Kuzin, 2012).

The presence of shared conserved sequence elements also suggests that cas-1 and -6 CSCs may belong to a structurally related family of enhancers. Alignments revealed that the two CSCs share the following sequences GCAAGGGTT, TTGGGTTG and TCAAAGGGT. These elements all contain Kr consensus DNA-binding sites. Although it is beyond the scope of this paper to examine the role of Kr in the regulation of these enhancers, this result is suggestive of a role for this temporal-identity TF in the regulation of cas expression. Previous work of others has shown that other CSCs in the cis-Decoder database contain clusters of conserved Kr binding sites including a number of segmentation and neural enhancers that are either known or putative targets of Kr regulation, including the hb HZ enhancer driving expression in the embryonic ectoderm (Berman, 2004), the pdm-2 CE8012 neuroblast enhancer and a hairy stripe 1 enhancer (Kuzin, 2012).

The comparative analysis also highlighted conserved amino acid codons within the ORF. Invariant codon nucleotide positions for the poly-glutamine (first exon) and the Zn-fingers (exons 2 and 3) domains were the most prominent while the 3' end of the ORF and the 3' UTR lacked sequence conservation. Unlike the Zn-finger domain codons, that show evolutionary divergence in many of their wobble positions, most of the codons for the conserved poly-glutamine tracks within the first exon showed significantly fewer substitutions in their wobble positions, most likely reflecting the fewer nucleotide options for the glutamine codon wobble position. To test if any of the ORF CSCs may also function as enhancers, their potential to activate transgene reporter expression during different phases of development was investigated, and no cis-regulatory activity was detected (Kuzin, 2012).

To determine the cis-regulatory behavior(s) of cas enhancers, each of the CSCs contained in the complete rescue fragment were tested using gypsy-insulated enhancer/reporter transgenes. To further control for chromosomal integration-specific events that could possibly affect reporter expression, the phiC31 integrase mediated site-specific integration system was used to insure that all reporter transgenes were inserted into the same chromosomal environment. Multiple independent transformant lines were tested for each CSC reporter transgene, and no significant variability was detected among the independent lines for each construct. Mutational analysis of enhancers has revealed that when CSBs are removed or altered, the enhancer often becomes unstable, triggering a high degree of cis-regulatory variability. For example, functional analysis of conserved sequences within the nerfin-1 NB enhancer reveal that its bilateral expression symmetry within ventral cord NBs that flank the midline is dependent on maintaining its CSBs and their individual sequences (Kuzin, 2012).

Whole-mount embryo in situ mRNA localization of transgene reporter expression revealed that all but four of the tested CSCs activate expression in different or overlapping regions of the cas gene embryonic expression window. Reporter expression was not detected in tissues outside of the CNS for any of the CSCs tested nor was expression observed outside the cas late temporal window within the embryonic CNS. One of the four CSCs that did not activate embryo reporter expression, the cas-3 CSC, was found to direct transgene expression in the larval CNS while the other three fragments that span the ORF, introns and 3'UTR (fragments 9-12) were not active during any of the developmental windows examined in this study (Kuzin, 2012).

During embryonic stage 9, cas gene expression is first detected in segmental clusters of ventral midline mesectodermal cells and in cells that line the anterior midgut primordium. At this stage, no expression is detected in either cephalic lobe or ventral cord NBs. The expression pattern observed with the cas-2 CSC/reporter transgene matches the early onset of the endogenous cas expression. Located 10.3 kb 5' to the predicted transcription start site, the cas-2 enhancer contains 10 CSBs of 6 bp or greater in length that span 557 bp of genomic DNA. cis-Decoder intra-CSC alignments of the CSBs identified both repeat and palindromic conserved sequences, including two copies of a 6 bp repeat (CCCTTT) and the palindromes (TTATAA and CAATTG). Core DNA-binding motifs for bHLH and Tramtrack TFs are present in three of the CSBs. Also present, but only partially conserved in the 12 species that were included in the EvoPrint (not present in D. virilis, D. mojavensis and D. grimshawi) is a docking site for the Single-minded (Sim) and Tango (Tgo) dimer (CACGTG) (Kuzin, 2012).

Expression analysis of cas-1, -4, -5, -7 and -8 CSC/reporter transgenes suggest that these enhancers participate in regulating different or overlapping aspects of the spatial and temporal cas embryonic NB expression dynamics. Analysis of cas gene expression has shown that subsets of cephalic lobe and ventral cord NBs initiate cas expression during embryonic stage 10. The first ventral cord NBs to express Cas are the late delaminating medial row NB6-1 NBs located on the posterior edge of the gnathal, thoracic and abdominal segments. Soon after this onset, additional medial, intermediate and lateral row NBs initiate expression, and by stage 12 most ventral cord and cephalic lobe NBs have detectable levels of cas mRNA and/or protein. Cas expressing NBs, identified based on their large cell body diameters and their position underlying the ectoderm, and their Cas positive descendants (GMCs, nascent neurons and glia) are positioned on the outer surface of the cephalic lobes and on the ventral/ventral-lateral surfaces of the developing subesophageal ganglion and ventral cord neuromeres. Similar to wild-type cas expression, cas-1, -4, -5, -7 and -8 CSCs activate reporter expression in overlapping patterns of ventral cord and cephalic lobe NBs, while cas-8 directs expression predominantly in GMCs and nascent neurons (Kuzin, 2012).

The less-conserved central regions of the inverted repeat within the cas-5 and -7 CSCs indicate that these relatively large CSCs might span multiple independent enhancers. For example in D. mojavensis, which has the poorest conservation within the inverted repeat, the distal half of the repeat within the cas-5 CSC contains a 600 base-pair gap of less-conserved sequence between CSBs. To test if the different sub-regions of cas-5 and -7 could function independently as enhancers and if the combined expression patterns of the sub-fragments recapitulate the full expression domains of the CSCs, three smaller enhancer/reporter transgenes were generated for the cas-5 CSC (cas-5a, b and c) and two that cover subsets of the cas-7 CSBs (cas-7a and b). Although each sub-region of both the cas-5 and -7 CSCs activated reporter expression within different sub-patterns of their full CSCs expression domain, collectively the sub-fragments of each CSC did not recapitulate the full embryonic expression of the cas-5 or cas-7 enhancers. Both full enhancers activate reporter expression in greater numbers of NBs, with the most significant difference between complete CSCs and their sub-regions being detected in the cephalic lobes and lateral ventral cord NBs. Corresponding sub-regions of the inverted repeat halves exhibited similar cis-regulatory behaviors. For example, both the cas-5c and cas-7a sub-regions activate reporter expression in medial row ventral cord NBs. The sub-fragments that span both repeat and unique flanking CSBs (cas-5b and cas-7b) do not overlap in their expression patterns, suggesting that the unique CSBs flanking the repeats harbor spatial cis-regulatory information for these regions. Interestingly, although both cas-5 and -7 CSCs direct expression within subsets of ventral midline cells and both contain highly conserved Sim/Tgo DNA-binding sites, one of the cas-7 sub-regions, cas-7a, includes one of the conserved Sim/Tgo core docking site flanked by other conserved sequences (TTTCTCACGTT), but does not activate midline expression (Kuzin, 2012).

Near the end of the cas temporal NB expression window (starting at the end of stage 12 and progressing through stage 13), the cas-6 enhancer activates reporter expression in a subset of NBs positioned along the outer edges of the cephalic lobes and along the ventral/ventral-lateral edges of the ventral cord neuromeres. As with endogenous cas expression, cas-6 enhancer/reporter expression diminishes during stage 14 until only a small subset of ventral cord cells have detectable levels of expression (Kuzin, 2012).

As an initial step toward identifying the TFs that are required for cas expression, the reporter expression patterns of the cas embryo enhancers were examimed in H23AD1 casnull mutant embryos. EvoPrint analysis revealed that all but one of the cas CSCs (cas-2) have multiple CSBs that contained core recognition motifs for Cas DNA-binding. Three of the enhancers were found to require cas for wild-type behavior. One of the enhancers, the cas-1 CSC, which activates reporter expression in both cephalic lobe and ventral cord NBs, was hyper-expressed in casnull embryos. Both extra cells and higher levels of expression were observed. cas-4 and cas-6 exhibited diminished expression in a casnull background. For both constructs, only brain expression remained; for cas-4, expression in the brain was not impaired, while for cas-6, a reduced number of cells expressing the reporter was also apparent in the brain cephalic lobes. It is unclear whether the effects of cas mutant background on cas enhancer/reporter expression are direct or indirect, since many regulatory events have been observed to be downstream of cas. It has been suggested that the broad temporal window regulated by cas could be subdivided into multiple feed-forward loops. Absence of cas function had no detectable effect on the expression of cas-2, cas-5 or cas-7. Interesting two of the non-responsive enhancers, cas-5 and cas-7 both contain multiple Cas binding sites. Future studies are required to understand the mechanism of cas self-regulation (Kuzin, 2012).

Previous studies have shown that cas is expressed in the larval ventral nerve cord and in the larval brain. cas enhancer-trap expression studies have also revealed that cas is most likely expressed in a subset of ellipsoid body and fan-shaped body fibers, and in the pars intercerebralis. Given these observations, cas CSC function was examined in larvae or adults (Kuzin, 2012).

In the 3rd instar larvae, cas-1, -2 and -8 CSCs drove reporter expression within ventral nerve cord and brain NB lineages. Many larval NB lineages are made up of coherent clusters of cells and a projecting axonal fascicle. cas-1 enhancer/reporter activity was detected in many NB lineages of the central brain and thorax, and also in a two terminal abdominal neurons whose axonal projections did not cross the midline. The cas-2 enhancer/reporter drove expression in sets of brain and thoracic neurons that cross the midline. Compared to cas-1 fewer cells expressed the reporter, suggesting that cas-2 regulates expression predominantly in neurons. cas-8 (the 5'UTR) drives reporter expression in a large set of central brain lineages, including medullary NBs and their progeny, and many neurons project between the hemispheres. The reporter is also expressed in lamina and medulla neurons of the optic lobe. cas-8 is expressed in lateral thoracic neurons that cross the midline and in many ventral lineages (Kuzin, 2012).

The expression patterns of three other CSCs (cas-3, -5 and -6) are represented by a single z-series representing the full dorsal/ventral extent of the third-instar larva. cas-3 enhancer/reporter, which was silent in the embryo, is expressed in a small set of larval brain neurons. Given their position and size, the anterior cells are probably the median neurosecretory cell group in the pars intercerebralis. cas-4 enhancer/reporter drove expression in a single pair of medial brain neurons that project posterior, again corresponding to a putative neurosecretory cell. cas-5 enhancer/reporter, which includes the distal inverted repeat, expressed in several more posterior central brain neurons. cas-6 enhancer/reporter, consisting of the spacer between the inverted repeats, expressed in fewer lineages, comparable in number to cas-2, and like cas-2 fewer NBs are labeled. Positive thoracic neurons are arrayed laterally and project axons across the midline. cas-7 enhancer/reporter was completely inactive in early 3rd instar larvae. Additional co-labeling studies are required to identify specific NBs and the identities of the post-mitotic cells within their lineages (Kuzin, 2012).

In the adult, both cas-1 and cas-8 CSCs activate expression in the olfactory lobe. The membrane-tagged GFP-mCD8 marks the projection neurons that project to the mushroom body. This expression could be related to a previously documented requirement of cas for axon pathfinding within the mushroom body. The cas-4 CSC regulates reporter expression in an anterior cell from the medial neurosecretory group, while cas-6 drives expression in two anterior medial cells that project axons posteriorly and a pair of large lateral cells that project axons towards the midline, and neurites throughout the adult brain (Kuzin, 2012).

This analysis reveals that the embryonic expression of cas is controlled by multiple enhancers, termed here sub-pattern enhancers, that activate expression in overlapping similar but non-identical subsets of cells in the brain and ventral cord. Even the CSCs that include the inverted repeats regulate expression in non-identical subsets of cells, as double labeling experiments revealed that the patterns overlap but were not identical. Given that the first three distal-most enhancers direct expression to different spatial/temporal windows of cas expression, the absence of these enhancers in the partial rescue fragment most likely results in its inability to completely rescue the casnull mutant (Kuzin, 2012).

The inverted repeat upstream of the cas transcribed sequence partially constitute two different but functionally related enhancers. The selective pressures that maintain the inverted repeat in the different Drosophila species are currently unknown. Models for the evolution and maintenance of the inverted duplication can be built on the following assumptions: First, having repeat copies of a regulatory sequence must be advantageous since both sets are highly conserved; and second, the species-specific nature and extent of inverted sequence identity between the two repeat halves indicates that each may play a role in maintaining repeat identity with its partner. For example, one half of the repeat could possibly serve as a template for rectifying differences between the two. Given that the extent of sequence identity between the two halves, varies between species, sequence corrections (rectification) most likely occurred at different times during species divergence. For example, the relatively high sequence identity between the D. virilis repeat halves suggests that its putative sequence rectification was more recent compared to any corrections that may have occurred in D. mojavensis (Kuzin, 2012).

Analysis of the cas enhancer cis-regulatory activity in larvae and adults shows that many of these enhancers are multi-functional; that is, they direct gene expression in embryos, larvae and/or adults. cis-Decoder analysis reveals that some of the enhancers fall into two structurally related families based on the sharing of conserved sequence elements; one class, represented by the cas-3, cas-5 and cas-7 CSCs are characterized by the presence of multiple POU-homeodomain TF DNA-binding sites. The second class is represented by cas-1 and cas-6, which each share many sequence elements including multiple Kr binding sites but lack POU TF docking sites (Kuzin, 2012).

The existence of cas sub-pattern enhancers can be compared to recent studies of segmentation enhancers in Drosophila. Multiple enhancers function to drive gap gene expression in similar non-identical fashion acting to ensure the full pattern of expression of the regulated gene. Similarly, transgenic rescue experiments suggest that most of the partially redundant enhancers associated with the sloppy-paired locus of Drosophila are required for full gene function in maintaining wingless expression and parasegment boundaries throughout embryogenesis. Another study discovered that multiple enhancers associated with the nerfin-1 gene can drive expression in different subsets of neural precursor cells and neurons. Finally, the presence of sub-pattern enhancers is not confined to Drosophila. In vertebrates, expression of Ngn1 in the midbrain, hindbrain, trigeminal ganglia, and ventral-neural tube appear to be due to sub-pattern enhancers that are located both 5′ and 3′ of the Ngn1 coding sequence. This study and those of others indicate the existence of sub-pattern enhancers is likely a general phenomenon required by the different regulatory environments in different tissues and lineages generated during development. These insights into cas regulation provide the basis for further, more detailed analysis of the molecular events that enable NBs to transition from one temporal gene expression program to the next (Kuzin, 2012).

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 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 focused to 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).

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

The effects were analyzed of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes (Kim 2013).

Transcriptional Regulation

The ventral nerve cord (VNC) of Drosophila exhibits significant segmental-specific characteristics during embryonic development. Homeotic genes are expressed over long periods of time and confer identity to the different segments. castor (cas) is one of the genes which are expressed in neuroblasts along the VNC. However, at late embryonic stages, cas transcripts are found only in head and thoracic segments and terminal abdominal segments, while Cas protein lasts longer in all segments. This study investigated the regulation of temporal and spatial expression of cas by the bithorax complex genes. In the loss-of-function mutants of Ultrabithorax (Ubx) and abdominal-A (abdA), cas transcripts were ectopically expressed in abdominal segments at late embryonic stage. However, unlike in Ubx and abdA mutants, in Abdominal-B (AbdB) loss-of-function mutant embryos, cas disappeared in the terminal region. Ectopic Ubx and abdA suppressed cas expression, but ectopic AbdB activated cas expression in most abdominal segments. Moreover, cas was co-expressed in the cells in which AbdB was normally expressed, and overexpressed in the ectopically expressed AbdB embryos. These results suggest that the expression of cas is segment-specifically regulated negatively by Ubx and abdA genes, but positively by the AbdB gene (Ahn, 2010).

cas is transiently expressed in a subset of neuroblasts in their cell lineage. Its transcripts are present with homologous patterns in thorax and abdomen at early embryonic stages. At late embryonic stages, cas transcripts are found in only a few cells per hemisegment in thoracic and posterior abdominal segments, but not in other abdominal segments. This indicates that cas is expressed in segment-specific mode during late embryonic stages. This study investigated how this regional diversity was produced (Ahn, 2010).

The segment-specific expression of cas was regulated by the homeotic genes. Ubx or abdA mutation caused the homeotic transformation of abdominal cuticle belts to the more anterior ones. These transformation patterns were also observed in cas expression. Mutations in Ubx or abdA genes caused ectopic cas expression in abdominal segments, which was normally observed in the thoracic segments at stage 15 of wild-type embryo, suggesting transformation of the thoracic pattern to an abdominal pattern at that stage. This transformation was synergistically enhanced in the Ubx and abdA double mutants (Ahn, 2010).

cas was ectopically expressed in A1 segment in Ubx mutant embryos. This result is coincident with the function of Ubx to specify the posterior thorax and a portion of A1 segment. cas was also ectopically expressed in A1 to A4 segments in abdA mutant embryos, which coincide with the function of abdA. In Ubx abdA double mutant embryos, cas was expressed in virtually all abdominal segments and in more cells than in each single mutant embryo. The roles of Ubx and abdA on cas expression in the abdominal segments were confirmed in the ectopically expressed Ubx and abdA mutant embryos. For this experiment, proper embryonic stages were very important because cas expression changed dramatically in the abdominal segments between stages 14 and 15. Whether ectopic Ubx or abdA repressed cas expression in the abdominal segments was tested at this stage. The GAL4-mediated induction of Ubx or abdA suppressed cas expression in the abdominal segments (Ahn, 2010).

However, in contrast to the Ubx and abdA mutations, AbdB mutation caused reverse effects on cas expression in the abdominal segments, which have never been reported. Loss-of AbdB function caused lack of cas expression, while ectopic ABDB activated cas expression in the abdominal segments. This phenotype was also observed in Polycomb mutant embryos. Although Polycomb mutation induced ectopic expression of Ubx, abdA and AbdB at the same time, cas was ectopically expressed in the abdominal segments of stage15 embryos, suggesting that ABDB dominated the effects of ectopic UBX or ABDA. The co-localization of cas and ABDB is found in a few cells in the posterior abdominal segments, supporting the positive regulation of cas by ABDB (Ahn, 2010).

This idea was further intensified by the appearance of the ectopic cas mRNA in the numerous abdominal cells with the ectopic AbdB expression. Real-time PCR experiment showed the overexpession of cas mRNA in the ectopically expressed AbdB embryos, also supporting the positive regulation of cas by ABDB. Furthermore, seven AbdB DNA binding sites were found within 5kb upstream from the cas transcription start site enhancing the possibility that ABDB directly regulates the cas expression. ABDB binds preferentially to a sequence with an unusual 5'-TTAT-3' core (Ahn, 2010).

One of questions was why all the cells with the AbdB expression does not show cas mRNA expression. In wild-type embryos, all AbdB-expressing cells does not show cas mRNA. Only a few cells among AbdB-expressing cells could maintain the expression of cas and the other cells lost it. This might be that the homoetic proteins carry out their function by interacting with other cofactors to regulate distinct sets of downstream genes (Ahn, 2010).

Accumulating evidence shows that the bithorax complex genes are involved at different steps in the segment-specific divergence of the CNS. Ubx or abdA activity is required for the abdominal pathway of the NB1-1 lineage. Both ectopic induction of Ubx- or abdA expression until several hours after gastrulation and homeotic de-repression in Polycomb mutants, override thoracic determination of NB1-1. The abdominal NB6-4 lineage is also specified by the abdA and AbdB. abdA is expressed in the NB6-4 lineage of abdominal segments A1-A6, whereas AbdB is expressed in the NB6-4 lineage of segments A7-A8. They specify the abdominal NB6-4 lineage by down-regulating levels of G1 Cyclin (CycE) (Ahn, 2010).

In summary, UBX and ABDA suppress cas expression in abdominal segments, so that mutation in both genes causes ectopic expression of cas in abdominal segments at late embryonic stage. However, ABDB activates cas expression, which is supported by co-localization of cas and ABDB in cells ectopically expressing AbdB, and real-time PCR in ectopically expressed AbdB embryos (Ahn, 2010).

Targets of Activity

CAS/Ming is required for the correct CNS expression of engrailed (Cui, 1992). Ganglion mother cells generated early in development normally express en. Interstripe neurons usually expressing en are absent in cas/ming mutants, and a reduction of en expression by a factor of two is found in later cells (Mellerick, 1992).

To determine if Cas is a pdm repressor, Pdm-1 and Pdm-2 expression was analyzed in cas null embryos. In stage 9 and in younger embryos, no differences were detected between the cas- and wild-type expression patterns of Pdm-1 or -2. However, starting at stage 10, NBs fail to terminate expression of both Pdms. Ectopic Pdm expression is observed in most, if not all, late developing sublineages in all CNS ganglia. The sustained Pdm expression is most likely due to transcriptional derepression, since PDM-1 mRNA in situ hybridizations also reveal that its message persists in cas- late NBs. In vivo analysis of pdm-1 genomic DNA has identified the main cis-regulatory elements controlling its embryonic expression. These control elements lie within a 6.3 kb DNA fragment, flanking the 5' side of its transcribed sequence. The pdm-1 regulatory DNA contains enhancer(s) that can drive the expression of reporter genes in the same cells in which pdm-1 is normally expressed. In a cas- background, transgenes are ectopically expressed in NBs during late sublineage development. This result demonstrates that the enhancer(s) within the 6.3 kb regulatory DNA are negatively regulated by Cas. To explore the possibility that Cas may play a direct role in silencing pdm gene expression, Cas-DNA immunoprecipitation was carried out with pdm-1 promoter fragments to test for potential Cas DNA-binding sites. DNA sequence analysis of bound fragments reveals 32 potential DNA-binding sites, all sharing at least 8 out of the 10 bp with the Hb consensus sites. Cas binds to these sites. Base pair substitutions in one of the sites demonstrate that the A/T rich core sequence is essential for Cas binding. All together, the results suggest that Hb and Cas regulate pdm expression by interacting directly with their cis-regulators to deactivate controlling enhancer(s), with Hb repressing the pdm genes early and Cas silencing late in CNS development. To test if Cas can silence Pdm-1 expression outside of Cas's endogenous sublineage boundaries, the effects of misexpressing Cas were studied early in CNS development. Indicating that Cas can act as a pdm repressor outside of its normal late expression boundaries, the temporally misexpressed Cas significantly reduces Pdm-1 expression when compared to wild-type embryos stained under identical conditions (Kambadur, 1998).

The role of Castor in regulating pdm genes raises the possibility that it may regulate expressions of other POU genes. To test this, the expression domains of Cas and Drifter/Ventral veins lacking were examined. Drf expression was examined in cas- embryos. In addition to its established role in midline glia and tracheal development, Drf is also expressed in a subset of NB progeny in both the developing brain and ventral cord. Many Cas-expressing NB sublineages also express Drf. Thus, it appears that Cas does not repress drf expression: to the contrary, a marked reduction in late-lineage Drf expression is observed in cas- embryos, suggesting Cas either directly or indirectly plays a role in activating and/or sustaining drf expression in these sublineages. Ectopically activated Cas has no effect on Drf expression. In the absence of castor function, I-POU expression is lost in a subset of ventral cord cells, but ectopic Cas has no effect on the I-POU wild-type expression pattern. It is not known if Cas is a direct activator of drf and/or I-POU. However, the data indicate that if Cas is playing a direct activator role, it most likely requires co-factors that are not expressed outside of its normal domain (Kambadur, 1998).

The mammalian NAB proteins have been identified previously as potent co-repressors of the EGR family of zinc finger transcription factors. Drosophila NAB (dNAB: CG15000), like its mammalian counterparts, binds EGR1 and represses EGR1-mediated transcriptional activation from a synthetic promoter. In contrast, dNAB does not bind the Drosophila EGR-related protein Klumpfuss. dnab RNA is expressed exclusively in a subset of neuroblasts in the embryonic and larval central nervous system (CNS), as well as in several larval imaginal disc tissues. Targeted deletion mutations were created in the dnab gene and the identification is described of additional, EMS-induced dnab mutations by genetic complementation analysis. Null alleles in dnab cause larval locomotion defects and early larval lethality (L1-L2). A putative hypomorphic allele in dnab instead causes early adult lethality due to severe locomotion defects. In the dnab -/- CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of Eve+ neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that dnab -/- clones are either very small or absent. Similarly, dNAB overexpression in the eye causes eyes to be very small with few ommatidia. These dramatic eye-specific phenotypes will prove useful for enhancer/suppressor screens to identify dnab-interacting genes (Clements, 2002).

To identify regulators of dnab gene expression, mutants of several NB-expressing transcription factors were screened for loss of dnab RNA expression. Castor is a zinc finger transcription factor expressed in Drosophila NBs and GMCs; castor loss of function causes reductions in CNS axonal density and reductions in the number of neurons expressing the homeodomain protein Engrailed. Furthermore, Castor positively regulates expression of the POU domain transcription factors Drifter and Acj6 and negatively regulates expression of the POU domain transcription factors Pdm-1 and Pdm-2 in the embryonic CNS. dnab expression was examined in cas minus embryos, which contain deletions removing the entire castor gene; dnab expression is affected in these mutants. dnab expression in midline cells at stage 11 appears normally; however, expression fails to spread to NBs during stages 12/13. In the cephalic lobes, only a few cells express dnab. This finding is in contrast to wild-type, where dnab is robustly expressed in many cephalic NB. These results indicate that dnab is either a direct or indirect target gene of Castor. The temporal expression patterns of castor and dnab support these conclusions. Castor expression first appears in NBs at stage 10 and spreads to 9-10 NBs per hemisegment by late stage 11, the stage at which dnab expression is first observed. The Castor protein has been shown to bind the consensus DNA sequence (G/C)C(C/T)(C/T)AAAAA(A/T). A genomic region containing the entire dnab transcription unit, as well as 10 kb upstream and 10 kb downstream of dnab was scanned for Castor binding sites. This analysis revealed that no consensus Castor binding sites occur in the putative dnab promoter region or in dnab introns, although at least two closely related sites occur in the putative promoter region. It may be possible that Castor directly regulates dnab expression through these sites, or through sites in a distal enhancer element. Alternatively, dnab expression might be directly regulated by the transcription factors encoded by the Castor target genes drifter, Acj6, pdm-1, or pdm-2. In these scenarios, drifter and Acj6 might normally function as positive regulators of dnab expression (Clements, 2002).

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, a very highly specific marker was used that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. The data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, it was shown that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, a targeted genetic screen was performed to identify players involved in ABCA specification. It was found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, it was shown that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third s segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification (Gabilondo, 2011).

The findings strongly suggest that the Capaergic abdominal ABCA neuron arises from NB 5-3. This conclusion is based on the expression in ABCA cells of gsb, wg and unpg, and the absence of the markers lbe(K) and hkb. However, even though gsb expression is known to be maintained specifically in the lineage of rows 5 and 6 NBs, whether expression of the other genetic markers used to identify NBs at stage 11 changes late in embryogenesis remains unanswered. Nonetheless, the specific combination of NB markers found in ABCA cells and their position in the hemineuromere are consistent with their birth from NB 5-3. Previous accounts showed that this NB gives rise to a lineage of 9–15 cells. Additionally, observations derived from studies in which PCD was blocked showed that NB 5-3 can potentially produce a large lineage (ranging from 19 to 27 cells), suggesting that it could generate 13 or 14 GMCs. The lack of a NB 5-3 specific-lineage marker prevented resolution of its complete lineage, and thus determining the birth order of the ABCA cell (Gabilondo, 2011).

Recent findings on the NB 5-6 and NB 5-5 demonstrate that cas and grh act together as critical temporal genes to specify peptidergic cell fates at the end of these lineages. cas mutants lack ABCA cells and Cas is expressed in these cells, while the normal pattern of ABCA cells is found in grh mutants, and Grh is not present in ABCA neurons. These data strongly support the birth of ABCA cells in a cas-only temporal window. This is different from the s Capaergic SECA cells, which while also arising from NB 5-3, show a reduction in cell number in both pdm and cas mutants, demonstrating birth at a mixed pdm/cas temporal window. Previous studies in other lineages have shown that when a temporal gene is mis-expressed, all progeny cells posterior to that temporal window can be transformed to the specific fate born at that particular temporal window. However, cas mis-expression failed at inducing ectopic ABCA cells, suggesting that cas in necessary but not sufficient to specify the ABCA fate (Gabilondo, 2011).

Programmed cell death (PCD) is a basic process in normal development. The results suggest that the ABCA and its sibling are equivalent cells committed to achieve the ABCA fate. First, it was shown that the ABCA-sibling cell dies by apoptosis, but produces an ABCA-like Capaergic neuron if PCD is inhibited. Second, when PCD is blocked, NB 5-3 also produces a GMC generating two ABCA-like Capaergic cells in the A5–A7 segments. These data indicate that a segment-specific mechanism prevents death of the ABCA cells in A2–A4 neuromeres. Segment specific cell death has been previously reported for the NB 5-3 lineage, and detailed studies on segment-specific apoptosis of other lineages have shown that this process is under homeotic control. In addition, the results show a different timing in the PCD undergone by the ABCA sibling and the ABCA cells born in A5–A7. This interpretation is based on the differential effect of p35 expression when cas-Gal4 or elav-Gal4 drivers were used. Although elav-Gal4 is transiently express in NBs and GMCs, robust and maintained driver expression commences in differentiating neurons. In contrast, cas expression starts in the NB and is maintained in the GMC and neuronal progeny. Therefore, the finding that death of the ABCA-sibling cell can be prevented by directing p35 with cas-Gal4, but not with elav-Gal4, suggests that the death of the ABCA sibling occurs earlier in development than the death undergone by the ABCA cells in A5–A7 segments (Gabilondo, 2011).

In the ABLK/LK peptidergic fate (derived from the NB 5-5), activation of Notch (N) signaling in the peptidergic cell prevents its death, while its sibling, NOFF cell undergoes apoptosis. On the contrary, in the EW3/Crz peptidergic fate (derived from the NB 7-3), silencing of N signaling is essential for the neuron survival, and therefore for it proper specification. The current results are in accordance with the last scenario, in which the ABCA cell is NOFF, and it sibling, which undergoes apoptosis, is NON. Therefore, Notch signaling must be switch off for the proper specification of the ABCA neuron (Gabilondo, 2011).

To search for genes involved in specification of the ABCA neural fate, a reduced set of mutants was screened of genes that are expressed in the embryonic CNS at stage 11, a time at which distinctly defined sublineages are being generated from all active NBs. Even though this method will certainly overlook important genes, the results reveal that it is in fact a very effective way to find genes involved in specification of a particular neural fate. Indeed, the ratio of success has been very satisfactory: 33.3% of the genes analyzed display a significant phenotype. Moreover, the set of identified genes could be further expanded by, for example, searching in interactome databases, and performing the subsequent screen on those putative interactors (Gabilondo, 2011).

It is assumed that the specification of a concrete cellular fate requires the combination of several transcription factors, namely a genetic combinatorial code. Recently, a detailed combinatorial code has been reported for three neuropeptidergic fates: ap4/FMRFa, ap1/Nplp1 and ABLK/Lk. However, very little is known about the specification of the rest of the 30 peptidergical fates. This study has identified several genes involved in the specification of the ABCA fate, which fit into three categories. First, genes were found whose loss-of-function produces a relevant increase of the number of ABCA cells. Most remarkable are the klu and rn phenotypes, which consist of duplications of the ABCA cells. These phenotypes suggest that these two transcription factors repress the ABCA fate in other neural cells (or/and NBs/GMCs). Interestingly, the normal phenotype of nab mutants indicates that, contrary to its mode of action in the wing, Rn does not work with the transcription cofactor Nab in this context (Gabilondo, 2011).

Second, genes were found whose loss-of-function produces a significant decrease of the number of ABCA neurons. In this category, the zhf2, ftz and grain phenotypes stand out. The effects of mutations on ftz are in agreement with its early role in segmentation: ftz is a pair-rule segmentation gene that defines even-numbered parasegments in the early embryo, and absence of ABCA cells was found in the A3 segment in ftz mutants. However, zfh2 and grain seem to be part of the specific combinatorial code of the ABCA cells. The Drosophila GATA transcription factor Grain has been reported to be involved in the specification of other cell fates, such as the aCC motoneuron fate. Based on its expression, the zinc finger homeodomain protein zfh2 has been proposed to mediate specification of the serotoninergic fate, but this has not been further demonstrated. Interestingly, during wing formation, zfh2 is required for establishing proximo-distal domains in the wing disc, and it does so partly by repressing gene activation by Rn. The opposite phenotypes that was observed in rn and zfh2 mutants suggest that similar interactions occur during ABCA specification. Analyses aimed to test this hypothesis are currently being performed (Gabilondo, 2011).

Third, two genes were found whose loss-of-function abolishes the ABCA fate: Grunge and hh. Grunge encodes a member of the Atrophin family of transcriptional co-repressors that plays multiple roles during Drosophila development. Taken together, studies from C. elegans to mammals suggest that Atrophin proteins function as transcriptional co-repressors that shuttle between nucleus and cytoplasm to transduce extracellular signals, and that they are part of a complex gene regulatory network that governs cell fate in various developmental contexts. Similarly, Hh is an extracellular signaling molecule essential for the proper patterning and development of tissues in metazoan organisms. It is noteworthy that two genes implicated in extracellular signaling pathways, Grunge and hh, are absolutely required for ABCA fate. Further studies will be needed to identify at which step/s they exert their actions, and to unravel possible interactions between them and with other players of the combinatorial code for ABCA specification (Gabilondo, 2011).

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).

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