Interactive Fly, Drosophila



cis-Regulatory Sequences and Functions

Cis-acting elements for the expression of btd head stripe expression are contained in a 1 kb DNA fragment, located about 3 kb upstream of the promoter, The four maternal coordinate systems are necessary for correct btd head stripe expression, most likely by acting through the 1 kb cis-acting control region. Expression of the btd head stripe depends on bicoid. bcd-dependent activation also involves the activity of the morphogens of the posterior and dorsoventral systems, hunchback and dorsal, respectively, which act together to control the spatial limits of the expression domain. Finally, tailless, a torso dependent repressor of btd, takes part in the regulation of btd head stripe expression by enhancing activation at low levels of activity and repression at high levels of activity (Wimmer, 1995). btd expression in the peripheral nervous system anlage is mediated by the btd 5.2 kb upstream region (Wimmer, 1996).

Transcriptional Regulation

Terminal development is disrupted in the dead ringer maternal and zygotic mutant embryos. Both head and tail defects are invariably observed in dri maternal and zygotic mutant embryos. One of the most consistent and striking phenotypes is severe disruption of the cephalo-pharyngeal skeleton. Germline and zygotic dri mutant embryos still have a recognizable dorsal bridge, dorsal and ventral arms and mouth hooks, but the H-piece and lateralgraten are missing or severely malformed. In addition, the atypical anterior position of pharyngeal muscles, visualized using anti-muscle myosin immunostaining, indicates that head involution does not proceed properly (Shandala, 1999).

The appearance of these defects prompted an examination of genes that play a role in the formation of terminal structures. Expression of the terminal gene tailless and the genes buttonhead, empty spiracles, orthodenticle and argos was examined. Of these genes, disruption to only argos (aos) and buttonhead (btd) expression was observed. In wild-type embryos, aos is initially expressed at stage 5 in two terminal domains and a domain that flanks the position of the cephalic furrow. In embryos lacking dri maternal and zygotic product, expression of aos in the terminal domains is almost completely eliminated while expression in the region of the cephalic furrow is maintained, both before and after division into two stripes at the time of cephalic furrow formation. Zygotic aos mutant embryos exhibit head defects that are similar to those observed in maternal and zygotic dri mutant embryos, indicating that the dri mutant head defects are likely to be the result of loss of anterior aos expression in the dri mutant embryos. Analysis of btd expression reveals a regulatory relationship that accounts for another consistent dri mutant phenotype, the appearance of ectopic cephalic furrows. btd expression is found to be partially derepressed in the trunk of dri germline and zygotic mutant embryos. The cephalic furrow arises where expression of the head specific gap gene btd overlaps the first stripe of expression of the primary pair rule gene eve. The repetitive appearance of ectopic cephalic furrows is therefore likely to be the result of the coincident ectopic trunk expression of btd with the more posterior eve stripes. The ectopic furrows do not progress, most probably due to the incomplete derepression of btd in this region (Shandala, 1999).

Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead

The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. This study reports that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btb) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btb is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btb restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity (Komori, 2014).

Maintaining functionally distinct stem cell populations allows higher organisms to generate the requisite number of diverse cell types required for organogenesis. For example, neural stem cells in the subventricular zone and in the outer subventricular zone collectively contribute to the generation of all the cell types required for the development of a human brain. Similarly, heterogeneous stem cell pools have also been reported in other organs including blood and intestine. Although the mechanisms that specify the identity of distinct stem cell types within a given organ have been proposed, the mechanisms that maintain the functional heterogeneity of stem cells have never been reported. This study used the two well-defined yet functionally distinct types of neuroblasts in the fly larval brain to investigate the mechanisms that maintain stem cell functional heterogeneity during neurogenesis. It was discovered that Trx functions uniquely to maintain a type II neuroblast identity through the H3K4 methylation activity of the SET1/MLL complex, thereby contributing to neuroblast heterogeneity during larval brain neurogenesis. The homeodomain transcription factor Btd was identified as a direct downstream target of Trx in the maintenance of a type II neuroblast identity. This Trx-Btd-dependent mechanism provides the first mechanistic insight into the maintenance of stem cell functional heterogeneity within an organ. The homologs of Trx and Btd have been shown to play critical roles in regulating vertebrate neural stem cell functions. The current findings lead to a speculation that the SET1/MLL histone methyltransferase complex might also contribute to the maintenance of stem cell heterogeneity in other higher eukaryotes (Komori, 2014).

The SET1/MLL complex elicits biological responses by maintaining its target genes in an active state through the methylation of H3K4. The core components of the SET1/MLL complex is required for the maintenance of the H3K4 methylation in a type II neuroblast and the maintenance of a type II neuroblast functional identity. Most importantly, over-expression of rbbp5FL, but not rbbp5SG, which encodes a mutant Rbbp5 protein that partially compromises the H3K4 methylation activity of the SET1/MLL complex (Cao, 2010), restored both H3K4 methylation and a type II neuroblast functional identity in rbbp5 null type II neuroblasts. These results indicate that the H3K4 methylation activity of the SET1/MLL complex is required for maintaining the functional identity of a type II neuroblast. In the fly genome, Trx, Trr and dSet1 can each bind to the core components of the SET1/MLL complex. Although the methylation activity of Trx was required for maintaining the type II neuroblast functional identity, removing trx function did not alter the global H3K4 methylation. In contrast, knocking down the function of trr or dset1 did not affect the maintenance of a type II neuroblast functional identity despite resulting in the global loss of H3K4 mono- or tri-methylation. These data strongly suggest that Trx maintains a type II neuroblast functional dentity by regulating H3K4 methylation in specific downstream target loci (Komori, 2014).

The functional identity of a type II neuroblast is defined by the competence of a neuroblast to generate INPs. The data indicate Trx plays a central role in maintaining the functional identity of a type II neuroblast by promoting the expression of a small number of genes. This study identified the btb gene as a critical downstream target of Trx that is both necessary and sufficient for the regulation of the type II neuroblast functional identity. btb encodes a C2H2 zinc finger transcription factor required for required for proper patterning of the head segment during fly embryogenesis and likely functions as a transcription activator. However, the role of Btd in regulating neuroblasts has never been established, and the mechanisms by which Btd elicits biological responses remain unclear. Several possible reasons exist to explain the relatively inefficient nature of eliciting the type II neuroblast functional identity in a type I neuroblast by the mis-expression of btb. First, certain co-factors might be required for Btd to efficiently activate its target gene transcription, and a lower abundance of these co-factors in type I neuroblasts hinders the functional output of mis-expressed Btd. Second, the epigenetic landscape might be vastly different between the two types of neuroblasts such that mis-expressed Btd may not have access to all of its target genes required to elicit the type II neuroblast functional identity in a type I neuroblast. Lastly, additional transcription factors might function in parallel with Btd to regulate the functional identity of a type II neuroblast. Btd is a highly conserved transcription factor. Future studies to elucidate the mechanisms by which Btd regulates the functional identity of a type II neuroblast will provide critical insight in the regulation of neural stem cell heterogeneity during both invertebrate as well as vertebrate neurogenesis (Komori, 2014).

This study has identified the pnt gene as another direct downstream target of Trx. It was initially hypothesized that Pnt might function in parallel with Btd to maintain the functional identity of a type II neuroblast. This hypothesis was extremely appealing in light of a previous study demonstrating mis-expression of PntP1 can transform a type I neuroblast into a type II neuroblast. Unexpectedly, knocking down the function of the pnt gene, which encodes at least three alternatively spliced transcripts, had no effect on the maintenance of the type II neuroblast functional identity, and instead, resulted in the formation of supernumerary type II neuroblasts. This result to a proposal that Pnt functions in the immature INP to specify an INP identity. Consistently, heterozygosity of the pnt locus dominantly enhanced the supernumerary neuroblast in the brat or erm hypomorphic genetic background. These two genetic backgrounds have been used extensively for elucidating the mechanisms that regulate the specification of an INP identity in the immature INP. Furthermore, over-expression of pntP1 failed to restore the functional identity of a type II neuroblast in trx mutant type II neuroblasts. Together, these data strongly suggest that pnt mainly functions to specify an INP identity rather than to maintain the type II neuroblast functional identity. Thus, it is proposed that in addition to maintaining the type II neuroblast functional identity, Trx also functions to promote INP identity specification through pnt (Komori, 2014).

Strategies that uniquely target the functional properties of cancer stem cells will revolutionize cancer treatments. Cancer stem cells generate a hierarchy of progeny that include cell types directly contributing to the exponential expansion of cancer stem cells. Thus, reprogramming their functional identity to bypass the cell types that directly contribute to the exponential expansion of cancer stem cells should halt further tumor growth. In this study, removing trx function efficiently reduced the number of supernumerary type II neuroblasts, which are proposed to serve as cancer stem cells in several Drosophila brain tumor models increased the number of differentiated cells in the brat or erm mutant brain. Similarly, attenuating the competence of type II neuroblasts to generate INPs by removing btb function also efficiently halted the expansion of brat or erm mutant brain tumors/ The results strongly support the hypothesis that reprogramming the functional identity of putative cancer stem cells can significantly alter the course of tumorigenesis. As such, understanding the mechanisms that maintain stem cell heterogeneity during normal development might provide novel insight into designing rational therapies to promote switching of cancer stem cells to an alternative, non-cancerous stem cell type (Komori, 2014).

Targets of Activity

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. (Mohler, 1995).

cap n'collar anterior domain is found to be activated by Bicoid and Torso maternal pathways. The posterior domain (correpsonding to intercalary and mandibular segment primordia) involves activation by BTD and repression by OTD anteriorly and Snail ventrally (Mohler, 1993).

Genetic and molecular analyses of patterning in the Drosophila embryo have shown that the process of head segmentation is fundamentally different from the process of trunk segmentation. The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems. The initial step in CF formation is a change in shape and the apical positioning of a single row of cells. The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped. The position of the furrow coincides with the second row of Even-skipped-expressing cells in stripe 1. Re-examination of the btd and eve phenotypes in live embryos indicates that both genes are required for CF formation. Eve expression in initiator cells is found to be dependent on btd activity. The control of eve expression by btd in these cells is the first indication of a new level of integrated regulation that interfaces the head and trunk segmentation systems. In conjunction with previous data on the btd and eve embryonic phenotypes, these results suggest that interaction between these two genes both controls initiation of a specific morphogenetic movement, which separates the two morphogenetic fields, and contributes to patterning of the hinge region, which demarcates the procephalon from the segmented germ band. The position and size of Eve stripe 1 is determined by repressor elements acting downstream of Bicoid and overriding buttonhead dependent activation. These results strengthen the conclusion that btd might be a 'generic transcriptional activator' required for transcriptional activation of specific target genes, such as eve and collier, an ortholog of mammalian early B-cell factor/Olfactory-1 (Crozatier, 1996). collier's expression is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, anterior to the CF domain, possibly parasegment 0. However, buttonhead's expression is not instructive for head development (Vincent, 1997).

Interestingly, Engrailed expression in PS1 requires btd but does not require eve, contrary to the setuation in parasegments 2 and 14. Despite having no effect on En expression in PS1, eve may still have a role in patterning this parasegement, since its expression and placement in relation to En is conserved between long germ-band and short germ-band insects. Recent data on the activation of collier (Crozatier, 1996) in PS0/mitotic domain 2, suggests a possible mechanism by which btd and eve cooperate to pattern PS1. Activation of col requires btd. Conversely, in the absence of eve, col expression is expanded posteriorly to overlap a region roughly corresponding to PS1, indicating that Eve acts as a repressor of col in this parasegment. Likewise, expression of string in mitotic domain 2, which also requires btd, is expanded posteriorly in eve mutant embryos. The current working model holds that the activation of eve by btd in anterior PS1 cells allows for differential gene expression between PS0 and PS1. In addition to the control of CF formation, the btd/eve interaction may thus assign separate gene expression and mitotic programs to cells on either side of the pro-cephalon/posterior head border (Vincent, 1997 and references).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. col expression is first detected during the interphase of mitotic cycle 14, when expression of head-gap genes has already resolved from initial broad domains into defined stripes. The stripe of col expression is included in that of btd, overlaps that of ems, and is restricted both dorsally and ventrally to neuroectodermal cells. Examination of dorsal (dl) mutant embryos shows that Dl is required for col repression in the mesodermal plate. The ectopic expression of col observed in twist (twi) and snail (sna) mutant embryos suggests that Dl target genes, rather than Dl itself, are involved. Embryos lacking ems function also show a ventral derepression of col expression. Further, at stage 10, ems mutant embryos show an abnormal pattern of col mRNA accumulation, with a mandibular stripe in addition to intercalary stripe of col-expressing cells. This suggests a second role for ems in regulating col. In btd mutant embryos, there is a complete loss of col expression, whereas there is no change in embryos lacking both slp (slp1 and slp2) genes, consistent with previous data establishing that btd but not slp is required for intercalary en and wg expression (Crozatier, 1999).

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning: it determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. Surprisingly, each of these elements are located at positions downstream of the transcription start site. The early-active enhancer element is a direct target of twist, a gene necessary for tinman activation that encodes a basic helix-loop-helix (bHLH) protein. This 180 bp enhancer includes three E-box sequences that bind Twist protein in vitro and are essential for enhancer activity in vivo. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. This repression requires the function of buttonhead, a head-patterning gene. The second expression domain, restricting tin mRNA expression in the dorsal mesoderm, is triggered by Dpp-mediated induction events (Yin, 1997).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Rusch, 2000).

Many of the genes that are members of either the gap, pair rule, or segment polarity genes have some effect on the pattern of pb accumulation. For the most part, mutations in genes of these classes reduce the number of cells expressing pb but do not eliminate Pb entirely from the affected segments. In no case do they cause pb to accumulate ectopically. The most striking results were caused by zygotic mutations in the genes buttonhead (btd), giant (gt), and hunchback (hb). btd is a head gap gene required for formation of the mandibular segment. In btd mutants, no mandibular structures are seen and no pb accumulation occurs anterior of the maxillary segment. pb accumulation is normal in the other gnathal segments. Mutations in both gt and hb disrupt the formation of the labial lobe and result in concomitant loss of pb expression therein. When the pb reporter #7 was tested in a hb mutant background no lacZ expression in the presumptive labial segment was found. In the case of gt, pb expression is not entirely extinguished. Weak pb accumulation can sometimes be seen in the most dorsal and posterior cells of the presumptive labial segment, overlapping with the few remaining cells of the engrailed stripe in the labial segment. For both gt and hb, this reduction or loss of pb expression in the labial lobe cannot be attributed to alterations in the Scr pattern because Scr accumulates in the cells posterior to the maxillary segment (Rusch, 2000).

An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene

Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. This laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. One of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. A consensus binding site has been derived for the Sp1/KLF Drosophila family members; this consensus sequence is shown to be present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila (Brown, 2005).

PREs are complex elements -- no single DNA binding site can act as a PRE. Instead, PREs are made up of binding sites for many different proteins. To date most PREs studied at the molecular level have been shown to contain DNA binding sites (usually multiple copies) for the proteins Pleiohomeotic (Pho) and its partially redundant homolog Pleiohomeotic-like (Phol), Zeste, GAGA Factor (GAF)/Pipsqueak (Psq) and Dorsal Switch Protein 1 (Dsp1). Clustered pairs of GAF/Psq, Zeste and Pho/Phol sites can predict the location of many PREs. However it has recently been shown that a combination of Zeste, Dsp1, GAF/Psq and Pho/Phol sites in the same number, orientation and spacing as in the native PRE is insufficient to restore full PRE activity . This suggests that all of the DNA binding sites necessary for PRE activity are still not known (Brown, 2005).

With the aim of identifying the binding sites and factors necessary for PRE function, this lab has been trying to define the components that constitute a minimal 181 bp PRE at ~576 to ~395 upstream of the Drosophila engrailed gene. This element was originally identified in a pairing-sensitive silencing assay, an assay used to detect the function of many PREs. When PREs are included in the vector pCaSpeR, they have an unusual effect on the eye color marker mini-white. Normally, when transgenic flies are made with pCaSpeR, flies homozygous for the transgene have a darker eye color than flies heterozygous for the transgene. However, when a PRE is included in the pCaSpeR vector, the eye color of homozygous flies is often lighter than that of heterozygotes. This phenomenon is dependent on the chromosomes being able to pair. Fragments of DNA that mediate pairing-sensitive silencing are called pairing-sensitive elements (PSEs). Not all PSEs have been shown to act as PREs and vice versa. The engrailed 181 bp element behaves both as a PRE and a PSE (Brown, 2005).

The 181 bp engrailed PRE contains 3 GAF/Psq sites, 2 Pho/Phol sites, 2 potential Zeste sites and 1 Dsp1 site that almost entirely overlaps the Pho/Phol site that has been studied by mutational analysis. In addition to these known sites, a number of other protein-binding sites required for pairing-sensitive silencing have been identified. This study investigated the role of one of these protein-binding sites, Site 2, in PRE function and shows that the Sp1/KLF family of proteins bind to this site. Sp1/KLF binding sites are present in most well characterized PREs (Brown, 2005).

Site 2 is required for pairing-sensitive silencing of the 181 bp engrailed PRE. This study asked whether Site 2 is also important for the PRE activity of that fragment. The bxd-Ubx-lacZ reporter construct was used to test the effect of mutations in Site 2 on PRE activity. In the absence of a PRE, the bxd-Ubx-lacZ reporter construct expresses lacZ throughout the embryo, in both the ectoderm and the nervous system, late in development. When the 181 bp PRE from the Drosophila engrailed gene is included in this vector, lacZ expression is restricted to parasegment six and posterior segments. Restricted expression patterns were seen in 75% of the lines, a number consistent with what has been seen by other investigators as PREs are not active in all chromosomal insertion sites (Brown, 2005).

Mutations were introduced into either Site 2 or the two Pho/Phol binding sites in the 181 bp fragment in the context of the bxd-Ubx-lacZ vector. The effect on PRE activity, as assayed by embryonic ß-galactosidase expression patterns, was analyzed. Mutation of either the Pho/Phol sites or Site 2 caused a reduction in the percentage of lines that had PRE activity. Both mutated constructs gave results that were significantly different from the expected frequency of 75% PRE activity for the unmutated construct. There were still a small percentage of lines with restricted expression in the Pho/Phol (20%), and the Site 2 mutant transgene (29%) as well as in the vector only control (8%). This probably reflects the fact that the bxd enhancer and the Ubx promoter are poised to work with flanking genomic PREs and may contain weak PRE activity on their own. When the first 350 bp of the bxd sequence that borders the 181 bp element in these constructs were scanned for the known PRE DNA binding sites, 3 Pho/Phol sites, 3 Dsp1 sites, 1 Zeste site and 2 potential Sp1 sites were found. This may explain why mutation of Pho/Phol or Site 2 did not completely eliminate the PRE activity of the 181 bp engrailed fragment in this vector. Nevertheless, the results suggest that both Pho/Phol and Site 2 binding sites contribute to the activity of the engrailed PRE, and that neither alone is sufficient for PRE activity (Brown, 2005).

In order to identify the protein(s) that interact with Site 2, a yeast one-hybrid screen was carried using multimerized Site 2 as bait. Four cDNA clones were isolated that showed specific binding to Site 2 but not to a mutated Site 2. Each of these four clones encoded a member of the Sp1/KLF family of zinc finger proteins [CG5669, CG12029 (two different length clones) and luna]. The consensus binding sequence for the Sp1/KLF family based on mutational analysis of a KLF family member (KLF4) binding site is (G/A)(G/A)GG(C/T)G(C/T). The Site 2 sequence contains a perfect match to this consensus sequence (Brown, 2005).

The Sp1/KLF family is an important group of proteins that in mammals have been shown to be involved in cell morphogenesis, differentiation and cancer. These proteins share a high degree of homology over 3 Cys2/His2 zinc fingers (>65% sequence identity with each other), located at or close to the C-terminal end of the protein. The N-terminal regions are generally unique (Brown, 2005).

Three members of the Sp1/KLF family were identified in the yeast one-hybrid screen, however the screen was not saturating. When the Drosophila genome sequence was searched for homology to the zinc finger region of these proteins a total of 10 members of this class was identified in Drosophila. The zinc finger regions of these members are highly conserved. The mammalian factors can be sub-divided into three classes (Sp1/class I family, class II family and class III family) based on closer homology between members of the subfamily relative to members outside the subfamily. This homology sometimes extends to functional protein domains that lie outside the DNA binding domain. The Drosophila proteins can also be placed into different groups based on homology of the zinc fingers with the zinc fingers of the human SP1/KLF proteins. CG5669, dSp1 and Btd are more closely related to the Sp1/Class I proteins than to the KLF proteins. In fact, dSp1 has 97% amino acid identity with hSp8 zinc fingers and also has homology to hSp8 in the region N-terminal to the zinc fingers. For CG5669 and Btd it is hard to assign a homolog since they have significant identity with a number of Class I proteins. Sequence identities of CG5669 with the zinc fingers of Class I range from 76% to 87%, for Btd the range is 65%–74%. CG12029, Luna and CG9895 have closer amino acid identity to the Class II human proteins. Luna is most closely related to KLF6 and KLF7 and shows conservation of a putative activation domain at the N-terminus. Cabot (also known as CG4427) and Bteb2 seem to belong to the class III subclass showing 72%–85% identity with human members of this class. Hkb and CG3065 are harder to place. CG3065 has 62%–65% identity to members of the Sp1 family and 62%–64% identity with KLF9 and KLF14 members of class III. Hkb is the most diverged member of this class of proteins. The highest degree of homology for Hkb is 53% identity with hSp5 and it is difficult to say whether it should be placed in this family at all. It is noted that Hkb binds only weakly to sequences that match the Sp1/KLF consensus (Brown, 2005).

Each of the zinc finger regions of these 10 proteins were cloned in frame into an in vitro transcription/translation vector and expressed in vitro. The products were tested for binding to Site 2. Nine of the 10 Drosophila Sp1/KLF family member zinc fingers show binding to Site 2 and only CG3065 shows no binding. Binding to each of these factors is specific based on competition experiments. Binding of Luna, Cabot and Hkb to Site 2 is very weak. This result is not due to the production of inactive protein since, with the exception of Hkb, strong binding of these factors is seen to other binding site sequences (Brown, 2005).

Identifying which Sp1/KLF factor acts through Site 2 is not a easy task. Not only are a number of members of this class genetically uncharacterized there is also the possibility that there may be functional redundancy as is seen with Pho and Phol. The existence of a viable and fertile Bteb2 mutant suggests that functional redundancy will be observed with the Sp1/KLF family in Drosophila. Experiments using family member-specific antibodies in chromatin-immunoprecipitation experiments on PREs will help elucidate which Sp1/KLF family members play a role in PRE function in Drosophila (Brown, 2005).

What role the Sp1/KLF family of proteins play in recruiting the PcG complexes to the PRE remains to be elucidated. In fact, for most of the other proteins required for PRE function, their roles are not yet clear. Both GAF and Psq bind the sequence GAGAG, a sequence shown to be important for PRE function. Psq has been shown to be in a complex with PcG proteins isolated from the Drosophila cell line SL2 and psq mutations enhance the mutant phenotypes of the PcG genes polyhomeotic (Ph) and Polycomb (Pc) in larval and adult tissues. This suggests that Psq may be important for PRE function. GAF has also been reported to co-purify with some PcG proteins and has been shown by chromatin-immunoprecipitation experiments to be present at PREs. GAF is a member of the TrxG of genes but may also play a role in PcG repression. The DNA binding protein Pho has been shown to bind in vitro to a chromatinized PRE template only if GAF is present. GAF and Psq can interact through their BTB protein–protein interaction domains and it has been proposed that they may function together in vivo (Brown, 2005).

Zeste has been shown to be important for both PRE and TRE activity. Zeste is a stoichiometric component of the biochemically purified PcG complex, PRC1 suggesting a role in PcG repression, and it has been report that Zeste is required for the PcG-mediated repression of an Ubx transgene. In contrast, experiments with the iab-7 PRE have shown that Zeste binding sites are important for the ability of this DNA to act as a TRE, not as a PRE (Brown, 2005).

Pho and Phol have recently been shown to be required to recruit an Esc-E(z) complex to a PRE . In vitro, Pho interacts directly with E(z) and Esc whereas Phol interacts with Esc. Recruitment of the Esc-E(z) complex leads to methylation of lysine 27 of histone H3 by the SET domain of E(z). The methylated K27 recruits a Pc-containing complex through interaction of the chromo-domain of Pc with the methylated histone tails. Pho has also been shown to interact with Pc in vitro. Pho/Phol double mutants have a very strong PcG phenotype, much stronger than mutations in the genes encoding the other PRE-binding factors suggesting that Pho/Phol play a central role in PRE function. It has been proposed that Dsp1 facilitates the binding of Pho/Phol to the PRE (Brown, 2005).

The role that the Sp1/KLF family may play remains to be elucidated but it is intriguing to note that mammalian Sp1 has been reported to interact directly with YY1 (the mammalian homolog of Pho). This interaction requires the first one and a half zinc fingers of YY1, a region that is 96% identical between the Drosophila and mammalian proteins. The 158 amino acid C-terminal region of Sp1 (includes the three zinc fingers and one of the activation domains, domain D), can mediate the interaction leading to an increase in the level of correctly initiated transcripts. These data raise the possibility that Pho or Phol may interact with Sp1/KLF proteins at PREs (Brown, 2005).

Anterior-posterior positional information in the absence of a strong Bicoid gradient

The Bicoid (Bcd) transcription factor is distributed as a long-range concentration gradient along the anterior posterior (AP) axis of the Drosophila embryo. Bcd is required for the activation of a series of target genes, which are expressed at specific positions within the gradient. This study directly tested whether different concentration thresholds within the Bcd gradient establish the relative positions of its target genes by flattening the gradient and systematically varying expression levels. Genome-wide expression profiles were used to estimate the total number of Bcd target genes, and a general correlation was found between the Bcd concentration required for activation and the positions where target genes are expressed in wild-type embryos. However, concentrations required for target gene activation in embryos with flattened Bcd were consistently lower than those present at each target gene's position in the wild-type gradient, suggesting that Bcd is in excess at every position along the AP axis. Also, several Bcd target genes were positioned in correctly ordered stripes in embryos with flattened Bcd, and it is suggested that these stripes are normally regulated by interactions between Bcd and the terminal patterning system. These findings argue strongly against the strict interpretation of the Bcd morphogen hypothesis, and support the idea that target gene positioning involves combinatorial interactions that are mediated by the binding site architecture of each gene's cis-regulatory elements (Ochoa-Espinosa, 2009).

This study used genetic and transgenic manipulations to create pure populations of embryos with flattened Bcd gradients. These manipulations expanded specific subregions of the body plan, which reduced the complexity of cell fates in the embryo compared with wild type, and increased signal-to-noise ratios in the microarray experiments. The three levels of Bcd generated in these experiments, ≈4%, 11%, and ≈40%, cover the lower half of the full range of the Bcd gradient, and these experiments identified 13 of the 18 known Bcd target genes (Ochoa-Espinosa, 2009).

The 13 known Bcd target genes are included in a set of 242 genes that are differentially activated by increasing levels of Bcd. Ninety-seven of these genes have been tested for expression in the early embryo, and 48 are expressed differentially along the AP axis. Of these, 30 are likely to be direct targets based on known or predicted Bcd-dependent CRMs. If a linear extrapolation of this number is used to take into account the full set of 242 genes, the genome-wide estimate is ≈74 genes, and if the fact that these experiments did not identify five previously known Bcd target genes (27%), the estimate increases to ≈103 genes (Ochoa-Espinosa, 2009).

Six other genes were identified as Bcd targets based on the microarray experiments and the presence of nearby clusters of Bcd sites, but these genes are either expressed ubiquitously or in dorsal-ventral patterns, with no apparent modulation along the AP axis. It is possible that Bcd-dependent activation may partially contribute to these patterns by activating expression in anterior regions, which is consistent with recent studies that showed ChIP-chip binding of DV transcription factors to AP-expressed genes and vice versa. If these are real target genes, they would slightly increase the estimate of the total number of Bcd target genes (Ochoa-Espinosa, 2009).

Bicoid has been considered as one of the best examples of a gradient morphogen. Several lines of evidence suggest that Bcd does indeed function as a morphogen, including the coordinated shifts of morphological features and target gene expression patterns in embryos with different copy numbers of the bcd gene, and the ability of bcd mRNA to establish anterior cell fates when microinjected into ectopic positions. Furthermore, manipulations of the Bcd-binding sites in the hb P2 promoter and synthetic constructs with defined Bcd sites showed that cis-regulatory elements can be designed to be more or less sensitive to Bcd-mediated transcription. These studies led to the hypothesis that differential sensitivity to Bcd binding may control the relative positioning of different target genes (Ochoa-Espinosa, 2009).

The current findings suggest that differential sensitivity to Bcd binding is not the primary mechanism that controls the relative positioning of its target genes. Though some target genes respond in an all-or-none fashion to different levels of flattened Bcd, the levels required for activation are much lower than those present in the wild-type gradient in the regions where those genes are activated. These findings suggest that Bcd concentrations are in excess of those required for activation at every position along the length of the wild-type gradient (Ochoa-Espinosa, 2009).

It was also shown that the head gap genes otd, ems, and btd are expressed in correctly ordered stripes in embryos containing flattened Bcd gradients. This is most dramatically demonstrated by the mirror-image duplication of otd, ems, and btd stripes in the posterior region of 6B (6 copies) vas exu embryos, where the Bcd gradient slopes in the opposite direction to the order of striped expression. It is proposed that these genes are patterned by the terminal system in the absence of a Bcd gradient, and though Bcd function is required for their activation, the Bcd gradient does not play a major role in establishing their relative positions along the AP axis (Ochoa-Espinosa, 2009).

Bcd seems capable of bypassing the terminal system if expressed at high levels. For example, the anterior defects in terminal-system mutants can be partially rescued by increasing bcd copy number. Also, in 6B (6 copies) vas exu embryos, higher levels of Bcd are present throughout the embryo, with a relatively weak gradient along the AP axis. This causes expansions of the anterior otd, ems, and btd expression patterns into central regions of the embryo. The posterior boundaries of these patterns are positioned correctly, suggesting that the Bcd protein gradient is sufficient to position these target genes in regions where the terminal system does not reach. This is consistent with the observation that microinjected bcd mRNA can autonomously specify anterior structures (Ochoa-Espinosa, 2009).

These data are consistent with previous studies that failed to find a strong correlation between the relative positioning of target genes and the Bcd-binding 'strength' of their associated cis-regulatory elements. They further support a model in which Bcd functions as only one component of an integrated patterning system that establishes gene expression patterns along the AP axis. A second major component is maternal Hb, which is expressed in an AP protein gradient. Hb synergizes with Bcd in the activation of several specific target genes. In vas exu embryos, the loss of vas causes ectopic translation of maternal hb in posterior regions, so Hb protein is ubiquitously expressed and available for combinatorial activation with Bcd. This combination is likely sufficient to lead to the near ubiquitous expression of zygotic hb and Kr in 1B vas exu embryos, and gt in 2B vas exu embryos (Ochoa-Espinosa, 2009).

A third major component is the terminal system, which seems to affect the expression patterns of Bcd target genes in two ways. First, it causes a repression of all known Bcd target genes at the anterior pole by a mechanism that is not clearly understood. Second, the data suggest that the terminal system functions with Bcd for the establishment of the posterior boundaries of the head gap genes. This interaction appears to be important for regulating at least two other target genes, gt and slp1, which are expressed in anterior domains that shift toward the anterior pole in terminal system mutants. Both gt and slp1 are also activated in anterior and posterior stripes in embryonic regions containing low levels of flattened Bcd. These findings suggest that interactions with the terminal system may be required for positioning most Bcd target genes. The only known target genes that may not be directly influenced by the terminal system are zygotic hb and Kr, which are expressed in middle embryonic regions, far from the source of the terminal system activity (Ochoa-Espinosa, 2009).

How synergy between Bcd and the terminal system is achieved for each target gene is not clear. One possibility is that the Torso phosphorylation cascade directly modifies the Bcd protein, increasing its potency as a transcriptional activator. Mutations in Bcd's MAP-kinase phosphorylation sites partially reduce the ability of Bcd to activate otd, consistent with this hypothesis. Alternatively, the terminal system has been shown to repress the activities of ubiquitously expressed repressor proteins. Perhaps repression by the terminal system creates posterior to anterior gradients of these proteins, which then compete with Bcd-dependent activation mechanisms to establish posterior boundaries of target gene expression (Ochoa-Espinosa, 2009).

Interactions between Bcd, maternal Hb, and the terminal system may be critical for the initial positioning of target gene expression patterns, but it is clear that other layers of regulation are required for creating the correct order of gene expression boundaries in the anterior part of the early embryo. Almost all known Bcd target genes are transcription factors, and there is evidence that they regulate each other by feed-forward activation and repression mechanisms. Each target gene contains one or more CRMs, each of which is composed of a specific combination and arrangement (code) of transcription factor binding sites. Unraveling the mechanisms that differentially position Bcd target will require the detailed dissections of CRMs that direct spatially distinct expression patterns (Ochoa-Espinosa, 2009).

Multiple regulatory safeguards confine the expression of the GATA factor serpent to the hemocyte primordium within the Drosophila mesoderm

Serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. This study found that srp is sufficient to transform trunk mesoderm into hemocytes. Two disjoint cis-regulatory modules were identified that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium (Spahn, 2013).

The evolutionary conserved transcription factor Sp1 controls appendage growth through Notch signaling

The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms in which they participate to control limb growth are mostly unknown. This study analyzed the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. Sp1 plays a more prominent role controlling leg growth than btd. A regulatory function of Sp1 in Notch signaling was identified, and a genome wide transcriptome analysis was performed to identify other potential Sp1 target genes contributing to leg growth. The data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling (Cordoba, 2016).

Understanding the molecular mechanisms that control the specification and acquisition of the characteristic size and shape of organs is a fundamental question in biology. Of particular interest is the development of the appendages of vertebrates and arthropods, i.e., non-homologous structures that share a similar underlying genetic program to build them, a similarity that has been referred to as 'deep homology.' Some of the conserved genes include the Dll/Dlx genes, Hth/Meis and the family of Sp transcription factors. The Sp family is characterized by the presence of three highly conserved Cys2-His2-type zinc fingers and the presence of the Buttonhead (BTD) box just N-terminal of the zinc fingers (Cordoba, 2016).

Members of the Sp family have important functions during limb outgrowth in a range of species from beetles to mice. In vertebrates, Sp6, Sp8 and Sp9 are expressed in the limb bud and are necessary for Fgf8 expression and, therefore, for apical ectodermal ridge (AER) maintenance. Moreover, Sp6/Sp8 phenotypes have been related to the split-hand/foot malformation phenotype (SHFM) and, in the most severe cases, to amelia (the complete loss of the limb) (Cordoba, 2016).

In Drosophila, two members of this family, buttonhead (btd) and Sp1, are located next to each other on the chromosome and share similar expression patterns throughout development. Recently, another member of the family, Spps (Sp1-like factor for pairing sensitive-silencing) has been identified with no apparent specific function in appendage development. The phenotypic analysis of a btd loss-of-function allele and of a deletion that removes both btd and Sp1 led to the proposal that these genes have partially redundant roles during appendage development. However, the lack of a mutant for Sp1 has prevented the analysis of the specific contribution of this gene during development (Cordoba, 2016).

In Drosophila, leg development is initiated in the early embryo by the expression of the homeobox gene Distal-less (Dll) in a group of cells in each thoracic segment. Later on, Dll expression depends on the activity of the Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways, which, together with btd and Sp1, restrict Dll expression to the presumptive leg territory. Therefore, the early elimination of btd and Sp1 completely abolishes leg formation and, in some cases, causes a leg-to-wing homeotic transformation (Estella, 2010). As the leg imaginal disc grows, a proximo-distal (PD) axis is formed by the differential expression of three leg gap genes, Dll, dachshund (dac) and homothorax (hth), which divides the leg into distal, medial and proximal domains, respectively. Once these genes have been activated, their expression is maintained, in part through an autoregulatory mechanism, and no longer relies on Wg and Dpp. Meanwhile, the distal domain of the leg is further subdivided along the PD axis by the activity of the epidermal growth factor receptor (EGFR) signaling pathway through the activation of secondary PD targets such as aristaless (al), BarH1 (B-H1) or bric-a-brac (bab). During these stages, btd and Sp1 control the growth of the leg but are no longer required for Dll expression (Estella, 2010). How btd and Sp1 contribute to the shape and size of the leg and the identity of their downstream effector targets is unknown (Cordoba, 2016).

One important consequence of the PD territorial specification is the generation of developmental borders that activate organizing molecules to control the growth and pattern of the appendage. In the leg, PD subdivision is necessary to localize the expression of the Notch ligands Delta (Dl) and Serrate (Ser), which in turn activate the Notch pathway in concentric rings at the borders between presumptive leg segments. However, it is still unknown how Notch controls leg growth and how the localization of its ligands is regulated. The present study generated a specific Sp1 null mutant, which, in combination with the btd mutant and a deletion that removes both btd and Sp1, allow analysis of the individual contributions of these genes to leg development. This study finds that Sp1 plays a fundamental role during patterning and growth of the leg disc, and that this function is not compensated by btd. The growth-promoting function of Sp1 depends in part on the regulation of the expression of Ser and, therefore, on Notch activity. In addition, other candidate targets of Sp1 affecting leg growth and morphogenesis were identified. Intriguingly, some of these Sp1 potential downstream targets are ecdysone-responding genes. These results highlight a mechanism by which btd and Sp1 control the size and shape of the leg, in part through regulation of the Notch pathway (Cordoba, 2016).

The two Sp family members in Drosophila, Sp1 and btd, display a similar spatial and temporal expression pattern during embryonic and imaginal development. Previous work suggested that btd and Sp1 have partially redundant functions during development. However, the lack of an Sp1 mutant has prevented the detailed analysis of the individual contributions of each gene. This study has generated an Sp1 null mutant that allowed elucidation unambiguously of the individual contributions of each of these genes to leg development (Cordoba, 2016).

Appendage formation starts in early embryos by the activation of Dll (through its early enhancer, Dll-304), btd and Sp1 by Wg, and their expression is repressed posteriorly by the abdominal Hox genes. Some hours later, there is a molecular switch from the early Dll enhancer (Dll-304) to the late enhancer (Dll-LT) to keep Dll expression throughout the embryo-larvae transition restricted to the cells that will form the leg. At this developmental stage, Sp1 and btd play redundant roles in Dll activation, as only the elimination of both genes suppresses Dll expression and Dll-LT activity in the leg primordia. Once Dll expression is activated in the leg disc by the combined action of Wg, Dpp and Btd/Sp1, its expression is maintained in part through an autoregulatory mechanism. At this time point, during second instar, btd and Sp1 are co-opted to control the growth of the leg. The leg phenotype of Sp1 and btd single mutants demonstrates the divergent contributions of each gene to leg growth. Removing btd from the entire leg only slightly affects the growth of proximo-medial segments, whereas loss of Sp1 causes dramatic growth defects along the entire leg. The different phenotypes of Sp1 and btd mutant legs could be a consequence of their distinct expression pattern along the leg PD axis, with btd being expressed more proximally than Sp1 (Cordoba, 2016).

The growth defects observed in Sp1 mutant legs are not due to gross defects in the localization of the different transcription factors that subdivide the leg along the PD axis, nor to defects in the expression of the EGFR ligand vn. By contrast, the results suggest a role for Sp1 in the regulation of the Notch ligand Ser. Notch pathway activation is necessary for the formation of the joints and the growth of the leg, and defects in these two processes were observed in Sp1 mutant legs. Moreover, the results demonstrate that Sp1 is necessary and sufficient for Ser expression at least in the distal domain of the leg and is therefore required for the correct activation of the Notch pathway. These results are consistent with the proposed role of Sp8 in allometric growth of the limbs in the beetle where the number of Ser-expressing rings is reduced in Sp8 knockdown animals (Cordoba, 2016).

The regulation of Ser expression is controlled by multiple CREs that direct its transcription in different developmental territories. Interestingly, although the wing and leg are morphologically different appendages and express a diverse combination of master regulators (e.g. Sp1 selects for leg identity whereas Vg determines wing fate), the same set of enhancers are accessible in both appendages, with the exception of the ones that control the expression of the master regulators themselves. These results imply that appendage-specific master regulators differentially interact with the same enhancers to generate a specific expression pattern in each appendage. The current analysis of Ser CREs identified a specific sequence that is active in the wing and in the leg. In the leg, this CRE reproduced Ser expression in the fourth tarsal segment and require the combined inputs of Sp1 and Ap. It is proposed that Sp1, in coordination with the other leg PD transcription factors, interacts with different Ser CREs to activate Ser expression in concentric rings in the leg. Meanwhile, given the same set of Ser CREs in the wing, the presence of a different combination of transcription factors regulate Ser expression in the characteristic 'wing pattern' (Cordoba, 2016).

Transcriptome analysis identified additional candidate Sp1 target genes that contribute to control the size and shape of the leg. Appendage elongation depends on the steroid hormone ecdysone through several of its effectors, such as Sb. Sb, as well as other genes related to the ecdysone pathway, were misregulated in Sp1 mutant discs. The characteristic change in cell shape that normally occurs during leg eversion does not happen correctly in these mutants. Other genes identified in this study are the Notch pathway targets dys and Poxn, which are both required for the correct development of the tarsal joints. dys and Poxn downregulation is consistent with Sp1 regulation of the Notch ligand Ser. Interestingly, the upregulation of the antenna-specific gene danr in Sp1 mutants might explain the partial transformation of the distal leg to antennal-like structures observed when two copies of Sp1 and one of btd are mutated. Interestingly, btd and Sp1 are only expressed in the antenna disc in a single ring corresponding to the second antennal segment whereas in the leg both genes are more broadly expressed. Consistent with this, misexpression of Sp1 in the antenna transforms the distal domain to leg-like structures, suggesting that different levels or expression domains of Sp1 helps distinguish between these two homologous appendages (Cordoba, 2016).

A considerable group of Hsp-related genes were downregulated in Sp1 mutant legs. Although their contribution to Drosophila leg development is unknown, downregulation of DnaJ-1, the Drosophila ortholog of the human HSP40, affects joint development and leg size, suggesting a potential role of these genes during leg morphogenesis (Cordoba, 2016).

An ancient common mechanism for the formation of outgrowths from the body wall has been suggested. Members of the Sp family are expressed and required for appendage growth in a range of species from Tribolium to mice. Consistent with the current results, knockdown of Sp8/Sp9 in the milkweed bug or the beetle generated dwarfed legs with fused segments that maintain the correct PD positional values. As is the case for Drosophila Sp1 mutants, mouse Sp8-deficient embryos develop with truncated limbs. By contrast, loss of function of Sp6 results in milder phenotypes of limb syndactyly. A progressive reduction of the dose of Sp6 and Sp8 lead to increased severity of limb phenotypes from syndactyly to amelia, suggesting that these genes play partially redundant roles. This phenotypic analysis of Sp1 and btd are consistent with this model, in which Sp1 plays the predominant role in appendage growth and the complete elimination of btd and Sp1 together abolish leg formation. Therefore, Drosophila Sp1 mutants are phenotypically equivalent to vertebrate Sp8 mutants. In vertebrate Sp8 mutant limbs, Fgf8 expression is not maintained and a functional AER fails to form. In Drosophila, FGF signaling does not seem to be involved in appendage development. Nevertheless, another receptor tyrosine kinase, EGFR, is activated at the tip of the leg and act as an organizer to regulate the PD patterning of the tarsus. The current results suggest that Sp1 acts in parallel with the EGFR pathway, as the ligand vn and EGFR target genes maintain their PD positional information in Sp1 mutant legs. However, a potential relationship between Sp1 and the EGFR pathway in later stages of leg development cannot be ruled out (Cordoba, 2016).

The results suggest that the Notch ligand Ser is a target of Sp1, and mediates in part the growth-promoting function of Sp1. Interestingly, members of the Notch pathway in vertebrates, including the Ser ortholog jagged 2 and notch 1 are expressed in the AER and regulate the size of the limb. It would be interesting to investigate further the possible relationship between Sp transcription factors and the Notch pathway in vertebrates, and test whether the functional relationship described in this work is also maintained throughout evolution (Cordoba, 2016).

Protein Interactions

Unlike the trunk segments, the anterior head segments of Drosophila are formed in the absence of pair-rule and HOX-cluster gene expression, by the activities of the gap-like genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd). The products of these genes are transcription factors but only Ems has a HOX-like homeodomain. Indeed, ems can confer identity to trunk segments when other HOX-cluster gene activities are absent. In trunk segments of wild-type embryos, however, ems activity is prevented by phenotypic suppression, in which more posterior HOX-cluster genes inactivate the more anterior without affecting transcription or translation. ems is suppressed by all other Hox-cluster genes and so is placed at the bottom of their hierarchy. Misexpression of Ems in the head transforms segment identity in a btd-dependent manner; misexpression of Btd in the trunk causes ems-dependent structures to develop; and Ems and Btd physically interact in vitro. The data indicate that this interaction may allow ems to escape from the bottom of the HOX-cluster gene hierarchy and cause a dominant switch of homeotic prevalence in the anterior-posterior direction (Schock, 2000).

Combined activities of otd, ems and btd generate and specify Drosophila head segments in the absence of pair-rule and homeotic gene activities. btd alone is required for development of the mandibular segment, btd plus ems for the intercalary segment, and btd, ems plus otd for the antennal segment. Misexpression of btd or otd in the prospective head region fails to cause homeotic transformations showing that neither of the two genes carries the proposed homeotic function in head segmentation. To explore the untested homeotic role of ems and to address a possible cooperation with btd, the ems protein (Ems) was misexpressed in the btd domain of otherwise wild-type embryos. Ems expression is achieved by an ems complementary DNA transgene under control of the btd cis-acting promoter region (Schock, 2000).

Ems expression in the btd domain of wild-type embryos causes a second intercalary-like engrailed expression domain in place of the mandibular segment. Furthermore, these embryos develop a duplicate set of intercalary cuticle elements in place of mandibular structures. The same results are observed in response to Ems expression in the anterior third of blastoderm embryos mediated by a Gal4/UAS system. However, misexpression of Otd in the btd domain has no effect on head formation. Thus, among the three head gap-like genes only ems carries both early segmentation and homeotic selector gene function. However, ems misexpression in several head segments causes only the mandibular into intercalary segment transformation. Since the intercalary segment also depends on btd, and ems does not have transforming activity in btd mutant embryos, it is concluded that ems activity is able to specify head segment identity only when acting in concert with btd. Notably, the direction of the ems-dependent transformation is from a posterior into a more anterior segment identity. This is the opposite of the direction of transformation in response to ectopically expressed HOX-cluster genes in the trunk (Schock, 2000).

Btd is a transcriptional activator with in vitro properties that are indistinguishable from those of human Sp1. However, whereas transgene-derived btd activity causes a full rescue of all head segments that are deleted in btd mutant embryos, Sp1 activity can rescue only mandibular segment development. Similarly, expression of the fusion protein VP16Btdzf , which contains the VP16 transactivator region fused to Btd's zinc finger domain, only rescues mandibular development. Conversely, expression of BtdSp1zf, in which the Btd zinc finger domain is replaced by the zinc finger domain of human Sp1, mediates a complete rescue of btd mutant embryos. Thus, Btd must contain specific features outside its zinc finger domain that are needed for intercalary segment development (Schock, 2000).

To identify the Btd region necessary for the ems-dependent intercalary development, it was asked whether Btd can physically interact with Ems in vitro and which parts of Btd are involved. Btd is able to bind [ 35S]methionine-labelled Ems in vitro. This interaction involves the amino-terminal region of the protein. A specific domain could not be found because several parts of the N-terminal region interact with Ems, excluding the zinc finger domain. Sp1, which has the same biochemical features as Btd, does not interact with Ems. The yeast two-hybrid system also shows a direct interaction between Ems and Btd's N-terminal region that does not involve the homeodomain of Ems (Schock, 2000).

The next question to be examined was whether the Btd mutants that interact with Ems are sufficient to allow homeotic Ems activity in vivo. Transgene-dependent rescue experiments were performed in which Btd deletion mutants were expressed in btd mutant embryos. N-Btd, a protein composed of the combined DNA-binding and N-terminal region, rescues all head segments of btd mutant embryos, whereas C-Btd, a protein lacking the N terminus, causes mandibular segment development in all btd mutant embryos but restores only partial intercalary development in rare cases. Furthermore, Btd variants that lack various portions of the N terminus are able to restore intercalary segment development fully, indicating that Btd-dependent intercalary development depends on the parts of its N-terminal region that also allow physical interaction with Ems (Schock, 2000).

To investigate whether the Btd and Ems interaction causes homeotic transformations in other parts of the embryo, use was made of the observation that ems is also expressed in the trunk region of the embryo from stage 9 onward. Lack of ems activity causes no alteration in trunk segments except that the 'filzkörper', a morphologically distinct structure of the last abdominal segment, fails to develop. In the absence of all HOX-cluster gene activities, however, the trunk segments alter identity and develop ems-dependent sclerotic head plates. Their formation can be phenotypically suppressed by the co-expression of any gene of the HOX-cluster including labial, Deformed or Sex combs reduced, all of which are normally expressed and required in the cephalic region of the embryo. Therefore, ems may be a disconnected member of the ancient HOX-cluster, acting at the bottom of the functional HOX-cluster hierarchy (Schock, 2000).

The ems-dependent homeotic transformation in the head region may be because of a requirement of Ems to cooperate with Btd to escape from phenotypic suppression. To test this in the trunk region, Btd was expressed from a heat-shock-inducible transgene at various early embryonic stages. Ectopic Btd expression up to and during blastoderm stage has no effect on trunk segmentation. However, Btd expression during stage 7-9 of embryogenesis, when ems is initially expressed in the prospective trunk region, causes a range of phenotypes. These included the development of sclerotic head plates reminiscent of the ems-dependent structures observed in embryos that lack the HOX-cluster genes (Schock, 2000).

Most Btd misexpressing embryos develop fusions of trunk segments to varying degrees (149 cases out of 218 heat-shocked embryos examined). However, such segment fusions are also observed at a similar frequency in Btd-expressing homozygous ems mutant embryos. Thus, ectopic Btd activity causes metamerization defects independent of ems activity. However, Btd-expressing wild-type embryos also develop sclerotic plates. In a few cases (11 embryos), segmentation is completely abolished, and sclerotic plates are found. Sclerotic plates are never observed in embryos lacking ems activity. Thus, their formation in the trunk region of embryos depends on combined Btd and Ems activities. ems escapes phenotypic suppression by the HOX-cluster genes without Btd affecting the HOX-cluster gene transcription or translation (Schock, 2000).

The results provide evidence that combined Btd and Ems activities specify the intercalary head segment identity. The gnatho-cephalic homeotic genes labial and Deformed are normally expressed in intercalary and mandibular head segments, respectively, and their products cause phenotypic suppression of Ems. Since the intercalary segment development is dependent on the regions of Btd that can associate with Ems in vitro, it is probable that the Ems-Btd interaction releases the phenotypic suppression. Ems can also overcome phenotypic suppression by the HOX-cluster genes in the trunk when co-expressed with ectopic Btd. It is proposed that the interaction with Btd allows Ems to relocate from the bottom to the top of the HOX-cluster gene hierarchy. Ems then functions in an anterior-prevalent manner, that is, in the direction opposite that of other HOX-cluster genes. The unique homeotic feature of ems among the head gap-like genes is therefore consistent with the proposed origin of the gene from the HOX-cluster. By adopting Btd as a partner, Ems could escape phenotypic suppression by gnatho-cephalic HOX gene activities and specify the intercalary head segment identity (Schock, 2000).

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

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