InteractiveFly: GeneBrief

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

Gene name - Beadex

Synonyms - LIM-only (Lmo) and dLMO

Cytological map position - 17C2

Function - Transcription factor

Keywords - Wing

Symbol - Bx

FlyBase ID:FBgn0265598

Genetic map position - 1-59-4

Classification - LIM-only protein

Cellular location - nuclear

NCBI link: Entrez Gene
Bx orthologs: Biolitmine
Recent literature
Han, H., Fan, J., Xiong, Y., Wu, W., Lu, Y., Zhang, L. and Zhao, Y. (2016). Chi and dLMO function antagonistically on Notch signaling through directly regulation of fng transcription. Sci Rep 6: 18937. PubMed ID: 26738424
Genes apterous (ap), chip (chi) and beadex (bx) play important roles in the dorsal-ventral compartmentalization in Drosophila wing discs. Meanwhile, Notch signaling is essential to the same process. It has been reported that Ap and Chi function as a tetramer to regulate Notch signaling. At the same time, dLMO (the protein product of gene bx) regulates the activity of Ap by competing its binding with Chi. However, the detailed functions of Chi and dLMO on Notch signaling and the relevant mechanisms remain largely unknown. This study reports the detailed functions of Chi and dLMO on Notch signaling. It was found that different Chi protein levels in adjacent cells activate Notch signaling mainly in the cells with higher level of Chi. Also, dLMO induces antagonistical phenotypes on Notch signaling compared to that induced by Chi. These processes depend on their direct regulation of fringe (fng) transcription. 
Kairamkonda, S. and Nongthomba, U. (2018). Beadex, a Drosophila LIM domain only protein, function in follicle cells is essential for egg development and fertility. Exp Cell Res 367(1): 97-103. PubMed ID: 29580687
LIM domain, constituted by two tandem C2H2 zinc finger motif, proteins regulate several biological processes. They are usually found associated with various functional domains like Homeodomain, kinase domain and other protein binding domains. LIM proteins that are devoid of other domains are called LIM only proteins (LMO). LMO proteins were first identified in humans and are implicated in development and oncogenesis. They regulate various cell specifications by regulating the activity of respective transcriptional complexes. The Drosophila LMO protein (dLMO), Beadex (Bx), regulates various developmental processes like wing margin development and bristle development. It also regulates Drosophila behavior in response to cocaine and ethanol. Analysis of Bx null flies has shown Bx essential function in neurons for multiple aspects of female reproduction. However, it was not known whether Bx affects reproduction through its independent function in ovaries. This paper shows that female flies null for Bx lay eggs with multiple defects. Further, through knock down studies the function of Bx in follicle cells was shown to be required for normal egg development. Function of Bx is particularly required in border cells for Drosophila fertility.
Nian, X., Chen, W., Bai, W., Zhao, Z. and Zhang, Y. (2019). miR-263b controls circadian behavior and the structural plasticity of pacemaker neurons by regulating the LIM-only protein Beadex. Cells 8(8). PubMed ID: 31426557
Circadian clocks drive rhythmic physiology and behavior to allow adaption to daily environmental changes. In Drosophila, the small ventral lateral neurons (sLNvs) are primary pacemakers that control circadian rhythms. Circadian changes are observed in the dorsal axonal projections of the sLNvs, but their physiological importance and the underlying mechanism are unclear. This study identified miR-263b as an important regulator of circadian rhythms and structural plasticity of sLNvs in Drosophila. Depletion of miR-263b (miR-263b(KO)) in flies dramatically impaired locomotor rhythms under constant darkness. Indeed, miR-263b is required for the structural plasticity of sLNvs. miR-263b regulates circadian rhythms through inhibition of expression of the LIM-only protein Beadex (Bx). Consistently, overexpression of Bx or loss-of-function mutation (BxhdpR26) phenocopied miR-263b(KO) and miR-263b overexpression in behavior and molecular characteristics. In addition, mutating the miR-263b binding sites in the Bx 3' UTR using CRISPR/Cas9 recapitulated the circadian phenotypes of miR-263b(KO) flies. Together, these results establish miR-263b as an important regulator of circadian locomotor behavior and structural plasticity.
Chatterjee, A., Aavula, K. and Nongthomba, U. (2019). Beadex, a homologue of the vertebrate LIM domain only protein, is a novel regulator of crystal cell development in Drosophila melanogaster. J Genet 98. PubMed ID: 31819023
Haematopoiesis is a complex process in which the regulatory mechanisms of several implicated transcription factors remain uncertain. Drosophila melanogaster is an excellent model to resolve the unanswered questions about the blood cell development. This study describes the role of Beadex, a Drosophila homologue of LIM domain only 2 (LMO2), in haematopoiesis. Mutants of Beadex were analysed for blood cell abnormalities. Crystal cells, a subset of haemocytes, were significantly more in Beadex hypermorphic flies. Similarly, Beadex misexpression in prohemocytes altered the crystal cell numbers. Stage-specific misexpression analyses demonstrated that Beadex functions after the prohemocytes enter the crystal cell lineage. It was also discovered that Pannier-U-shaped complex is a negative regulator of the crystal cell differentiation and is possibly negatively regulated by Beadex through its interaction with Pannier. This study therefore suggests the mechanism of two novel regulators of crystal cell specification-Beadex and Pannier-during Drosophila haematopoiesis.

Beadex is one of the classic Drosophila mutations, discovered by Bridges in 1923 and described by Morgan, Bridges and Sturtevant in 1925. The gene is X-linked; therefore, only mutant males or homozygous females exhibit beaded-like wings, described as long, narrow and excised along both margins. The mutation is non-lethal. Lifschytz and Green (1979) proposed, with great insight, that Beadex is a dominant mutation in a negatively acting cis-regulatory region. These authors suggested that an overproducing mutation, one producing too much protein product, is expected if a dominant mutation is readily reverted by gene deletion, or, if gene deletion suppresses the expression of the original dominant mutation in flies heterozygous for the deletion. Beadex was shown to meet these criteria. Before Beadex was identified as coding for the LIM-only protein (Lmo), LIM-only was cloned on the basis of homology to genes coding for vertebrate LIM-only proteins. Only in 1998 did three papers appear, almost simultaneously, identifying Lmo and Beadex as being identical.

Beadex has been cloned by three different procedures: homology based cloning (Boehm, 1990, Zhu, 1995 and Shoresh, 1998), using polymerase chain reaction (PCR); P-element insertion (Zeng, 1998), and by a misexpression system (Milan, 1998). The latter method will be described because of its novelty. The modular-misexpression system developed by Rorth (1996) was used to carry out a large-scale screen for genes that perturb wing development (Rorth, 1998). A special P element carrying an EP (enhancer and a basal promoter), oriented to direct expression of adjacent genomic sequences, is randomly inserted into the genome. The EP is regulatable by manipulation of the transcription factor GAL4. When combined with a source of GAL4, the EP element will direct expression of any gene that happens to lie next to its insertion site. Thus the EP element directs a conditional misexpression of genes into which it inserts. The screen identified EP insertions at hh, patched (ptc), and dpp, genes with known roles in limb patterning (Rorth, 1998), as well as a number of new loci implicated in wing patterning by virtue of their overexpression phenotypes. One of the genes identified by the EP screen, Beadex, is involved in dorsal-ventral (DV) patterning of the wing (Milan, 1998).

The LIM-homeodomain protein Apterous (Ap) serves as the selector gene for the dorsal compartment wing imaginal disc. The juxtaposition of Ap-expressing dorsal cells and nonexpressing ventral cells establishes a D-V organizing center at the boundary between these cell types. In the late third instar disc, wingless and cut expression is induced in a three- to six-cell-wide stripe that straddles the D-V boundary; this is the location of the future wing margin. It is known that ap expression needs to be maintained continuously in the dorsal compartment, because loss of Ap function in mutant clones as late as third instar causes cell-autonomous fate transformation from dorsal to ventral. apterous mutant clones in the interior of the dorsal compartment create a new boundary for dorsal versus ventral cells, thereby initiating the genetic program normally reserved for the endogenous D-V boundary. wingless and cut are expressed in stripes of cells flanking the ectopic boundary, signifying the formation of an ectopic margin. Outgrowth organized by this ectopic margin eventually leads to the outgrowth of an ectopic wing from the dorsal surface of the wing blade. An ectopic wing outgrowth phenotype similar to that produced by ap mutant clones is obtained by overexpression of Beadex in the wing disc. When Beadex expression is driven by a patched promoter, an ectopic margin is formed that, as with ap mutant clones, expresses Wingless and Cut in the dorsal compartment of third instar wing discs. These similarities, along with the common LIM domain structure between Beadex and Ap, make it likely that misexpressed Beadex exerts its effect through interference with Ap function (Zeng, 1998 and references).

Beadex has been shown to regulate Apterous activity levels. The classic dominant gain-of-function Beadex mutants reduce Apterous activity. Conversely, loss-of-function Bx mutants appear to increase Ap activity. A LIM-binding protein called Chip has been identified as a possible cofactor for Ap. Bx gain of function mutants overexpress Beadex, which is shown to bind Chip and thereby interfere with formation of a functional complex between Ap and Chip. Ap is shown to induce expression of its antagonist, Beadex, which suggests that Ap and Beadex constitute a feedback mechanism and that the relative levels of Beadex, Chip, and Ap determine Ap activity levels in vivo. Beadex normally is expressed in the wing pouch of the third instar wing imaginal disc during patterning. A mammalian homolog of Beadex is expressed in the developing limb bud of the mouse. This indicates that Lim-only proteins might function in an evolutionarily conserved mechanism involved in patterning the appendages. The dominant interference mechanism of LMO action may serve as a model for the mechanism by which LMO oncogenes cause cancer when misexpressed in T cells (Milan, 1998; Shoresh, 1998; Zeng, 1998).

Selector genes such as ap are often thought of as simple binary switches. The interaction between Chip and Beadex suggest that the situation is more complex and that Ap activity levels are modulated during wing development. The balance in the levels of Ap, Chip, and Beadex proteins is important for determining the level of Ap activity in vivo. This regulation occurs at the level of protein activity, not at the level of gene expression, and it has been suggested that this may reflect a requirement to fine-tune activity levels regionally as the wing develops. Given that Beadex is expressed in regions where Ap is not known to function, it is likely that Beadex may have other functions as well. Chip and Beadex may regulate the activity of LIM-homeodomain proteins in other developmental contexts and in postembryonic homeostatic processes (Milan, 1998).

Drosophila dLMO-PA isoform acts as an early activator of achaete/scute proneural expression

The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). This study investigates, through new genetic tools, the function of dLMO (Beadex), the Drosophila LIM only transcription factor that was already known to control wing development. dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression (Asmar, 2008).

The lack of dLMO protein leads to very distinctive phenotypes. The mutant animals are not able to fly, they have a short life span and show an abnormal gait behaviour. In addition, they show a discreet bristle phenotype. In Drosophila, there are two paralogous LMO factors, dLMO and CG5708. These genes are expressed in the CNS where redundancy is not excluded. However CG5708 is not expressed in the wing discs and presumptive SOPs. Therefore it is concluded that the mild phenotype observed for the adult PNS in dLMO mutants, is not attributed to gene redundancy. dLMO encodes two distinct isoforms, dLMO-PA and -PB, which only differ from their N-terminus. Only dLMO-RA is broadly expressed in the notum, and contributes to the PNS phenotype. dLMO function is also critical in the developing central nervous system for the activity of the ventral lateral neurons, LNvs . It is highly probable that dLMO-RB has some subtle biological activities in the brain, where it has a specific pattern (Asmar, 2008).

In vertebrate, multiproteic complexes composed by GATA-1, LMO2, Ldb-1 and the bHLHs E47 and SCL, are required for normal differentiation of haematopoietic cells. The results of this paper highlight several evidences in favour of dLMO as a GATA coactivator in Drosophila. (1) A genetic synergism exists between pnr and dLMO null alleles. (2) dLMO modulates the activity of a DC:ac-lacZ reporter, the model target of Pnr, in vivo. Loss of function dLMO mutants show reduced level of the DC:ac-lacZ expression, whereas in gain-of-function dLMO mutants the DC:ac-lacZ expression is increased. (3) dLMO-PA isoform directly interact with Pnr in GST pull down assay. Therefore it is concluded that dLMO might enhance the proneural activity of Pnr through direct interaction with the GATA factor. Consistently, dLMO expression overlaps with the dorsal-most domain of Pnr during third instar larval stages. Though Pnr controls the development of both DC and SC bristles, dLMO null alleles affect only DC bristles. dLMO expression, that overlaps both SC and DC proneural clusters in the notum, is significantly weaker in the SC region, suggesting that regulation of proneural ac/sc expression is differentially sensitive and responds to local combinations of transcription factors. These data support studies demonstrating that the proneural activity of Pnr is prominently repressed in the SC region by the LIM-HD transcription factor Isl (Asmar, 2008).

At later stages, dLMO expression is excluded from the corresponding SOP and its derivative cells. In contrast, the proneural factor Sens, that plays an important role for sensory organ specification, is first broadly expressed in proneural clusters at low levels where it functions as a repressor of ac/sc, and then later, is expressed at high levels in the presumptive SOPs, where it acts as a transcriptional activator that directly interacts and synergizes with the proneural proteins, Ac and Sc. It has been shown recently that both Gfi-1 and GATA-1, the mammalian ortholog of Sens and Pnr respectively, are essential for development of the closed related erythroid and megakaryocytic lineages. This study demonstrated that the Sens/Pnr interaction is evolutionary conserved in Drosophila neurogenesis. It is suggested that Pnr could participate to the Sens-dependent positive autoregulation of Ac/Sc in late SOPs where dLMO is not expressed. The synergism between Pnr and Sens would need more detailed investigations. Taken together, the present studies have shown dLMO-PA as a co-activator for Pnr during the establishment of proneural fields and revealed another level of proneural ac/sc regulation during late neurogenesis in the Drosophila PNS (Asmar, 2008).

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs (Hirano, 2016).

This study has identified Bx and Smr as LTM maintenance genes and has characterize a shift in transcription between CREB/CRTC-dependent maintenance (1-4 days) to Bx-dependent maintenance (4-7 days). In addition, a biological consequence of this shift was identified in defining a time window during which LTM can be modified, β-Spec was identified as being required for memory extinction (Hirano, 2016).

LTM maintenance mechanisms change dynamically during storage. In particular, CRTC, which is not required during memory formation, becomes necessary during 4-day LTM maintenance and then becomes dispensable again. Consistent with this, CRTC translocates from the cytoplasm to the nucleus of MB neurons during 4-day LTM maintenance and returns to the cytoplasm within 7 days. On the other hand, Bx expression is increased at both phases, suggesting that transcriptional regulation of memory maintenance genes may change between these two phases. Supporting this idea, it was found that Bx expression requires CRTC during 4-day LTM maintenance but becomes independent of CRTC 7 days after training. It is proposed that CREB/CRTC activity induces Bx expression, which subsequently activates a feedback loop where Bx maintains its own expression and that of other memory maintenance genes (Hirano, 2016).

Although it is proposed that the shifts in transcriptional regulation that were observed occur temporally in the same cells, the possibility cannot be discounted that LTM lasting 7 days is maintained in different cells from LTM lasting 4 days. MB Kenyon cells can be separated into different cell types, which exert differential effects on learning, short-term memory and LTM. Thus, it is possible that LTM itself consists of different types of memory that can be separated anatomically. In this case, CRTC in one cell type may exert non-direct effects on another cell type to activate downstream genes including Bx and Smr. However, as that CRTC binds to the Bx gene locus to promote Bx expression and both CRTC and Bx are required in the same α/β subtype of Kenyon cells, it is likely that the shift from CRTC-dependent to Bx-dependent transcription occurs within the α/β neurons (Hirano, 2016).

Currently, it is proposed that the alterations in histone acetylation and transcription that were uncovered are required for memory maintenance. However, it is noted that decreases in memory after formation could be caused by defects in retrieval and maintenance. Thus, it remains formally possible that the epigenetic and transcriptional changes reported in this study are required for recall, but not maintenance. However, this is unlikely, as inhibition of CRTC from 4 to 7 days after memory formation does not affect 7 day memory, whereas inhibition from 1 to 4 days does. This suggests that at least one function of CRTC is to maintain memory for later recall (Hirano, 2016).

Consistent with a previous study in mice, which suggests distinct transcriptional regulations in LTM formation and maintenance (Halder, 2016), the data indicate that memory formation and maintenance are distinct processes. Although the HAT, CBP, is required for formation but dispensable for maintenance, other HATs, GCN5 and Tip60, are required for maintenance but dispensable for formation. Through ChIP-seq analyses, those downstream genes, Smr and Bx, were identified as LTM maintenance genes and these are not required for LTM formation. Collectively, these results suggest differential requirements of histone modifications between LTM formation and maintenance. Although other histone modifiers besides GCN5 and Tip60 were identified in the screen, knockdown of these histone modifiers did not affect LTM maintenance. There are ~50 histone modifiers encoded in the fly genome, raising the possibility that the lack of phenotype in some knockdown lines is due to compensation by other modifiers (Hirano, 2016).

The results indicate some correlation of increase in CRTC binding with histone acetylation and gene expression. Interestingly, DNA methylation shows higher correlation to gene expression in comparison with histone acetylation in mice. Notably, flies lack several key DNA methylases and lack detectable DNA methylation patterns. Hence, histone acetylation rather than DNA methylation may have a higher correlation with transcription in flies. Reduction in histone acetylation was detected, overlapping with increase in CRTC binding. Those reductions could be due to CRTC interacting with a repressor isoform of CREB, CREB2b or other transcriptional repressor that binds near CREB/CRTC sites. These interactions would decrease histone acetylation and gene expression, and may be related to LTM maintenance. Although this study focused on the upregulation of gene expression through CREB/CRTC, downregulation of gene expression by transcriptional repressors may also be important in understanding the transcriptional regulation in LTM maintenance. The results demonstrate the importance of HATs for LTM maintenance; however, the data do not conclude that histone acetylation is a determinant for gene expression, but rather it might be a passive mark of gene expression. HATs also target non-histone proteins and also interact with various proteins, both of which could support gene expression in LTM maintenance (Hirano, 2016).

Similar to traumatic fear memory in rodents, this study found that aversive LTM in flies can be extinguished by exposing them to an extinction protocol specifically during 4-day LTM maintenance. These observations suggest the time-limited activation of molecules that allows LTM extinction only during the early storage. Supporting this concept, it was found that CRTC is activated during the extinguishable phase of LTM maintenance and prolonging CRTC activity extends the time window for extinction. Thus, CRTC is the time-limited activated factor determining the time window for LTM extinction in flies. In cultured rodent hippocampal neurons, CRTC nuclear translocation is not sustained, suggesting that other transcription factors may function in mammals to restrict LTM extinction (Hirano, 2016).

This work demonstrates that LTM formation and maintenance are distinct, and involve a shifting array of transcription factors, coactivators and HATs. A key factor in this shift is CRTC, which shows a sustained but time-limited translocation to the nucleus after spaced training. Thus, MB neurons recruit different transcriptional programmes that enable LTM to be formed, maintained and extinguished (Hirano, 2016).


Transcriptional Regulation

In wild-type discs dLMO protein is nuclear and is expressed at higher levels in the dorsal compartment of the mature third-instar disc than in the ventral compartment. This expression pattern mirrors that of the MS1096 GAL4 enhancer trap line. In early- to mid-third-instar discs both MS1096 and dLMO protein are restricted to the dorsal compartment. The observation that dLMO is initially expressed in dorsal cells and maintained at elevated levels in the dorsal compartment suggests that dLMO might be regulated by Apterous (Ap). To test this possibility ectopic Ap expression was forced using dpp-gal4 to direct UAS-Ap in a stripe of cells along the anterior-posterior boundary of the wing disc. dLMO is induced in Ap-expressing ventral cells to the same elevated level typical of cells in the dorsal compartment. Thus, Ap induces expression of dLMO in dorsal cells. The transition from exclusively dorsal expression to dorsal and ventral expression suggests that dLMO expression is initiated by Ap but comes under an additional control mechanism as the disc matures (Milan, 1998).

Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso-ventral (D/V) compartment boundary is defined by the expression of the apterous selector gene in the dorsal compartment. Ap activity is under control of dLMO (Beadex) which destabilizes the formation of the Ap-Chip complex. D/V boundary formation in the wing disc also depends on early expression of vestigial. These data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, over-expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early Vg product regulates Ap activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSalpha1 and PSalpha2 integrins. It is concluded that normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose-dependent way with ap (Delanoue, 2002).

Vnd/NK-2 homeodomain affinity column chromatography was used to purify Drosophila DNA fragments bound by the Vnd/NK-2 homeodomain. Sequencing the selected genomic DNA fragments led to the identification of 77 Drosophila DNA fragments that were grouped into 42 Vnd/NK-2 homeodomain-binding loci. Most loci were within upstream or intronic regions, especially first introns. Nineteen of the Drosophila DNA fragments cloned correspond to one locus, termed Clone A, which is 312 bp in length and contains five Vnd/NK-2 homeodomain core consensus binding sites, 5'-AAGTG, and is part of the first intron of the Beadex gene. The interactions between Clone A and Vnd/NK-2 homeodomain protein were further analyzed by mobility-shift assay, DNase I footprinting, methylation interference, and ethylation interference. The DNase I footprinting analysis of Clone A with Vnd/NK-2 homeodomain protein revealed three strong binding sites and one weak binding site between 15 and 130 bp of Clone A. Binding of the Vnd/NK-2 homeodomain to the 5'-flanking sequence of vnd/NK-2 genomic DNA was also analyzed. The DNase I footprinting result showed that there are two strong binding sites and five weak binding sites in the fragment between -385 and -675 bp from the transcription start site of the vnd/NK-2 gene (Wang, 2005).

Targets of Activity

The Drosophila wing primordium is subdivided into a dorsal (D) and a ventral (V) compartment by the activity of the LIM-homeodomain protein Apterous in D cells. Cell interactions between D and V cells induce the activation of Notch at the DV boundary. Notch is required for the maintenance of the compartment boundary and the growth of the wing primordium. Beadex, a gain-of-function allele of dLMO, results in increased levels of dLMO protein, which interferes with the activity of Apterous and results in defects in DV axis formation. A gain-of-function enhancer-promoter (EP) screen was performed to search for suppressors of Beadex when overexpressed in D cells. 53 lines were identified corresponding to 35 genes. Loci encoding for micro-RNAs and proteins involved in chromatin organization, transcriptional control, and vesicle trafficking were characterized in the context of dLMO activity and DV boundary formation. The results indicate that a gain-of-function genetic screen in a sensitized background, as opposed to classical loss-of-function-based screenings, is a very efficient way to identify redundant genes involved in a developmental process (Bejarano, 2008).

A gain-of-function EP-based screen in a Bx1-sensitized background to search for suppressors of the wing-margin phenotype is efficient in identifying known and new genes involved in DV boundary formation as well as in the regulation of Beadex/dLMO gene activity. Dominant genetic interactions of Bx1 with loss-of-function alleles of the suppressor genes identified have demonstrated that the vast majority are involved in wing development. This is in contrast with classic EP screens based on the gain-of-function phenotype of candidate genes, in which the number of genes not participating in the developmental context of interest is relatively higher. Many of the Bx1 suppressors involved in DV boundary formation are not essential during wing development (i.e., taranis, nmd, nuf, draper, and cabut). This observation suggests that these suppressors share redundant activities with other gene products. The EP gain-of-function approach has also been shown to be extremely efficient in unraveling new roles for the recently identified micro-RNAs (miRs). Loss-of-function-based forward genetic screenings have not been as productive in this respect, probably because of the reduced size of these miRs or their redundant activities. Taken together, a suppressor EP-based gain-of-function screen in a sensitized background provides a suitable combination to identify new genes, including miRs and redundant genes, involved in a given process (Bejarano, 2008).

Redundancy and regulatory feedback loops contribute to the robustness of gene regulatory networks. Classical loss-of-function-based forward genetic screenings have been highly productive in identifying genes that behave as hubs in these networks. However, forward genetic screenings are not as effective in identifying redundant genes or regulators of these feedback loops, whose loss of function might not show any overt phenotype. More quantitative in vivo genetic screenings, such as the one done recently in Drosophila for bristle number, or, alternatively, cell culture-based RNAi quantitative screenings have been more efficient in this regard. The current results indicate that an EP-based gain-of-function in vivo genetic screen in a sensitized background is a strong alternative for the identification of redundant genes or regulators of feedback loops involved in developmental gene regulatory networks (Bejarano, 2008).

This study identified, characterized, and discussed four classes of genes in the context of DV boundary formation or dLMO activity: chromatin organization genes, transcription factors, miRs, and proteins involved in vesicle trafficking and membrane fusion. Several conclusions can be drawn. Among the genes involved in chromatin organization, Osa binds Chip and modulates the expression of Ap target genes. Several transcription factors involved in other signaling pathways during wing development have also been shown to act as Bx1 suppressors, suggesting that Notch and these pathways share common elements or that these pathways collaborate with Notch in boundary formation. The finding of genes encoding for proteins that participate in distinct aspects of vesicle trafficking and membrane fusion indicates that the sorting of sufficient levels of certain molecules, including Notch and its ligand Delta, toward the plasma membrane is especially critical to reach appropriate levels of Notch activity at the DV boundary. Consistent with this, it is interesting to note that overexpression of these genes in an otherwise wild-type background does not show any overt wing phenotype, suggesting that the activity of the Notch pathway is finely regulated and buffered during boundary formation (Bejarano, 2008).

The screen was designed and performed to find new genes involved in Ap and/or dLMO activity, as well as new Ap target genes involved in DV boundary formation. Although genes known to participate in DV boundary formation, like fringe or osa, were scored several times, no new transmembrane proteins or cell adhesion molecules were identified involved in the generation of an affinity difference between D and V cells. P elements are known for their preferential insertion in certain regions of the genome called hot spots. The gene or genes involved in this process might be located in the so-called cold spots, thus suggesting that a distinct transposable element, like the lepidopteran piggyBac, with a different profile of hot spots and cold spots, is a good candidate to search, on a similar suppressor gain-of-function basis, for these kinds of genes (Bejarano, 2008).

Transcriptional regulation by CHIP/LDB complexes

It is increasingly clear that transcription factors play versatile roles in turning genes 'on' or 'off' depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. This study used the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, 189 down-stream targets of CHIP/LDB were identified; these genes are enriched for the binding sites of Apterous (AP) and Pannier (PNR), two well studied transcription factors associated with CHIP/LDB complexes. Extensive genetic screens were performed and target genes were identified that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing, novel roles were uncovered for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, these results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development (Bronstein, 2011).

Drosophila SSDP was identified on the basis of its ability to bind the nuclear adaptor protein CHIP/LDB (van Meyel, 2003; Chen, 2002). Both nuclear localization of SSDP and its ability to modulate the transcription activity of the CHIP-AP complex during wing development depend on its interaction with CHIP/LDB. This study implemented a combination of molecular, bioinformatic and genetic approaches that allowed has led to insight into the effect of SSDP on the transcriptional activity of CHIP/LDB complexes and their role in development. A genome wide screen was conducted for SSDP target genes in Drosophila using expression microarrays with mRNA isolated from larvae bearing hypomorphic alleles of ssdp. Analysis of transcription factor binding site enrichment served as an orthogonal assay that validates and extends the microarray results and thus contributes to understanding of the relation between the CHIP-AP and CHIP-PNR transcription complexes in specific tissues (e.g. wing and thorax) (Bronstein, 2011).

SSDP proteins directly bind DNA and mouse SSDP1 activates the expression of a reporter gene in both yeast and mammalian cells indicating that it is capable of regulating transcription activity. Enrichment was found for SSDP binding sites upstream of the genes identified in the microarray experiments on flies lacking SSDP. Moreover, in agreement with the positive transcriptional role of SSDP, enrichment for SSDP binding sites was restricted to the genes showing decreased expression in mutants. This strongly suggests that a significant number of these genes are bona fide SSDP target genes (Bronstein, 2011).

Consistent with the involvement of SSDP with the CHIP-AP complex, it was found that upstream regulatory regions of the SSDP putative target genes are also enriched for the AP binding site and the SSDP binding site. These sites are likely to be functionally significant, since loss of ssdp enhances the wing notching phenotype of a dominant allele of ap. Additionally, over-expression of Dlmo, whose product negatively regulates the CHIP-AP complex, also interacts with mutants of SSDP target genes, demonstrating that SSDP target genes are involved in the CHIP-AP pathway. The efficiency of finding genetic interactions among the genes differentially expressed in the microarray experiments, demonstrated the power of this approach. Specifically, 72% of the loci tested with DlmoBx2 is more than an order of magnitude higher than an EP insertion screen (1.3% interacting) in a DlmoBx1 sensitized background. Combined microarray and genetic loss of function screen allowed the identification of a similar number of Dlmo-interacting genes by screening a much smaller group of putative target genes (Bejarano, 2008). Of the 35 genes identified by Bejarano only CG1943 was found in the 189 genes identified in the current microarray screen. This study specifically identified down-stream targets of SSDP, while Bejarano searched for any modifiers of the Dlmo wing notching phenotype and thus uncovered genes that function in other regulatory pathways or genes that are upstream of the CHIP-AP complexes. This may explain the limited overlap between the current results and those of Bejarano (Bronstein, 2011).

In contrast to the enrichment of SSDP binding sites in the genes down-regulated in ssdp mutants, the PNR binding site was enriched specifically in the genes up-regulated in the ssdp mutants. A model is therefore presented in which loss of SSDP disrupts the balance between the CHIP-AP and CHIP-PNR complexes. Mammalian SSDP proteins protect LDB, LHX and LMO proteins from ubiquitination and subsequent proteasome-mediated degradation by interfering with the interaction between LDB and the E3 ubiquitin ligase, RLIM. It is therefore possible that in the absence of SSDP proteins, CHIP/LDB and LMO can escape degradation by interacting with GATA and beta-HLH proteins that are not subjected to proteasome-mediated regulation. The N-terminus of CHIP/LDB proteins is responsible for interaction with both PNR and RLIM. Thus, PNR/GATA proteins may partially interfere with the interaction between CHIP/LDB and RLIM making the CHIP/LDB-PNR/GATA complex more resistant to proteasome regulation and less dependant on the levels of SSDP proteins then the CHIP/LDB-LHX/AP complex (Bronstein, 2011).

According to the current model, in cells where both the CHIP-AP and CHIP-PNR complexes are active, loss of SSDP should result in the same phenotype as over-expression of PNR. Indeed, it was found that ssdpL7/+ flies display duplications of scutellar sensory bristles, similar to gain of function mutations in pnr. In addition, lowered levels of pnr in ssdpL7/+; pnrVX6/+ flies suppresses scutellar bristle duplication. This indicates that the duplicated scutellar bristle phenotype of ssdpL7/+ flies depends on the presence of PNR. As predicted by the model, since both AP and PNR regulate bristle formation, the functional interactions between SSDP target genes and ssdpL7 and/or Chipe5.5 resulted in either suppression or enhancement of the duplicated scutellar bristle phenotype (Bronstein, 2011).

These results in flies indicate that SSDP contributes differentially to CHIP/LDB complexes containing AP versus PNR. By contrast, mouse SSDP proteins positively contribute to the transcription activity and assembly of both LDB-GATA and LDB-LHX complexes, but the relative contribution of mammalian SSDP proteins to LDB complexes containing LHX proteins versus GATA proteins has not been specifically examined. It is possible that SSDP alters the balance of LIM-based CHIP/LDB complexes and GATA-containing CHIP/LDB complexes in the development of mice, as occurs in flies (Bronstein, 2011).

The search for enrichment of transcription factor binding sites upstream of the putative SSDP target genes identified additional transcription factors that may warrant future study. Some of these factors are associated with SSDP and CHIP/LDB complexes. For example, the binding sites for PNR and ZESTE (Z) were both enriched in the up-regulated putative SSDP target genes. This is in agreement with previous studies showing that Z can recruit the BRAHMA (BRM, the Drosophila homolog of the yeast SWI2/SNF2 gene) complex via its member OSA, which together negatively regulate the CHIP-PNR complex during sensory bristle formation through direct and simultaneous binding of OSA to both CHIP and PNR (Bronstein, 2011).

Some of the additional regulatory inputs at SSDP target genes may be evolutionarily conserved. For example, enrichment of STAT92E and SSDP binding sites was found in the down-regulated SSDP target genes. This may be significant, as a known role of ssdp is regulation of the JAK/STAT pathway during Drosophila eye development. Interestingly, mammalian STAT1 confers an anti-proliferative response to IFN-γ signaling by inhibition of c-myc expression. Similarly, expression of mammalian SSDP2 in human acute myelogenous leukemia cells and prostate cancer cells leads to cell cycle arrest and inhibits proliferation accompanied by down-regulation of C-MYC. These findings indicate that both in Drosophila and in mammals SSDP and STAT proteins have similar functions and may share common target genes (Bronstein, 2011).

While the transcription factor binding site analysis utilized all of the 189 putative SSDP target genes, genetic screens were conducted on a subset of them due to the availability of mutants. This suggests that more genetic interactions will be found among the untested genes. Even among this more limited subset, there are interesting new stories that suggest future experimental directions. For example, an insertion mutation in the Xbp1 gene suppressed the duplicated scutellar bristle phenotype characteristic of ssdpL7/+ and Chipe5.5/+ flies, indicating that XBP1 contributes positively to bristle formation. In contrast, when Xbp1 was silenced in ap-expressing cells both the wings and the scutum displayed a marked excess of sensory bristles while the scutellum was not affected. These results suggest that in the wing and scutum XBP1 acts as a negative regulator of bristle formation. Silencing of Xbp1 in pnr-expressing cells caused a similar excess of bristle on the scutum, accompanied by a reduced number of scutellar bristles, further emphasizing the opposing effects of XBP1 in these two distinct parts of the thorax. Such contrasting phenotypes have been previously documented for several pnr mutants as well. In flies and mammals XBP1 regulates the ER stress response, also termed the unfolded protein response (UPR). Since one of the functions of the ER is the production of secreted proteins, UPR-related pathways are widely utilized during the normal differentiation of many specialized secretory cells. In this respect it would be interesting to examine whether SSDP and CHIP/LDB complexes affect the production of secreted morphogens, such as Wingless (WG), the secreted ligands of the EGFR receptor, Spitz (SPI) and Argos (AOS), or the secreted Notch binding protein Scabrous (SCA) via XBP1 during wing and sensory bristle formation. Alternatively, the transcription factor XBP1 may directly regulate the expression of genes required for differentiation of the wing and sensory bristles. Indeed, carbohydrate ingestion induces XBP1 in the liver of mice, which in turn directly regulates the expression of genes involved in fatty acid synthesis. This role of XBP1 is independent of UPR activation and is not due to altered protein secretory function. Curiously, the two GO function categories 'cellular carbohydrate metabolism' and 'cellular lipid metabolism' which are enriched among Xbp1 target genes in mouse skeletal muscle and secretory cells were also enriched in the list of putative SSDP target genes. Whether this reflects a secondary effect due to the down-regulation of Xbp1 in ssdp mutants or a direct regulation of these processes by SSDP is yet to be determined (Bronstein, 2011).

Additional novel functions for CHIP/LDB complexes are implied by the results regarding the Gs-alpha60A (a.k.a. CG2835) gene. G protein coupled receptors are important regulators of development by for example, signaling via the protein kinase A (PKA) pathway. Activation or inhibition of PKA signaling during pupal wing maturation perturb proper adhesion of dorso-ventral wing surfaces resulting in wing blistering. This phenotype may be due to miss-regulation of wing epithelial cell death in ap-expressing cells. Interestingly, similar wing blisters occur in the wing of DlmoBx2 flies. Moreover, it was found that mutant alleles of Gs-alpha60A enhanced the wing blistering phenotype of DlmoBx2. Silencing of G-salpha60A in ap-expressing cells caused a curled wing phenotype. Such a phenotype can result from differences in the size of the dorsal and ventral wing blade surfaces. In addition, silencing of this gene in pnr-expressing cells caused the posterior pair of scutellar bristles to form in reversed orientation. Bristle orientation have been proposed to be regulated by planar cell polarity genes. Taken together these results point to novel aspects of regulation of wing and sensory bristle development by SSDP and CHIP/LDB complexes mediated by G-alpha proteins (Bronstein, 2011).

This genome-wide expression profiling and bioinformatics analysis of ssdp mutant larvae, combined with genetic screens resulted in gained insight into the intricate context-dependent transcriptional regulation by CHIP/LDB complexes. It was possible to identify 28 putative SSDP target genes that are involved in wing development and 23 putative SSDP target genes that play a role in scutellar bristle formation. Examination of two of these, xbp1 and Gs-alpha60A, suggests novel aspects of developmental regulation such as the involvement of SSDP and CHIP/LDB complexes in ER function and PKA signaling. Furthermore, it was shown that SSDP proteins contribute differentially to transcription activity, and probably to the balance in formation of CHIP-AP and CHIP-PNR complexes. Furthermore potential novel partners of SSDP in regulating transcription of downstream genes during fly development were. It stands to reason that an extension of the genetic analysis to mammals and other vertebrates will reveal a host of additional functions of SSDP and CHIP/LDB during the multifaceted process of transcriptional regulation that underlies the development of multicellular organisms (Bronstein, 2011).

Post-transcriptional Regulation

Drosophila mir-9a regulates wing development via fine-tuning expression of the LIM only factor, dLMO

MicroRNAs are short non-coding endogenous RNAs that are implicated in regulating various aspects of plants and animal development, however their functions in organogenesis are largely unknown. This study reports that mir-9a belonging to the mir-9 family, regulates Drosophila wing development through a functional target site in the 3' untranslated region of the Drosophila LIM only protein, dLMO. dLMO is a transcription cofactor, that directly inhibits the activity of Apterous, the LIM-HD factor required for the proper dorsal identity of the wings. Deletions of the 3' untranslated region, including the mir-9a site, generate gain-of-function dLMO mutants (Beadex) associated with high levels of dLMO mRNA and protein. Beadex mutants lack wing margins, a phenotype also observed in null mir-9a mutants. mir-9a and dLMO are co-expressed in wing discs and interact genetically for controlling wing development. Lack of mir-9a results in overexpression of dLMO, while gain-of-function mir-9a mutant suppresses dLMO expression. These data indicate that a function of mir-9a is to ensure the appropriate stoichiometry of dLMO during Drosophila wing development. The mir-9a binding site is conserved in the human counterpart LMO2, the T-cell acute leukemia oncogene, suggesting that mir-9 might apply a similar strategy to maintain LMO2 expression under a detrimental threshold (Biryukova, 2009).

The dorsoventral (DV) axis of wings is specified, at the second-instar larval stage, by the activity of Apterous (Ap) in the presumptive dorsal cells of the wing primordium. Ap induces Fringe and Serrate within the whole dorsal territory. Fringe, a crucial effector of the Notch receptor, leads to a differential affinity between the ligands Delta and Serrate. Thus, Notch activity is restricted to the DV boundary, where intense signaling by Notch leads to expression of Wingless (Wg). Levels of Ap and the subsequent Ap-dependent expression of Serrate and Fringe are regulated by Chip (the Drosophila Ldb factor) and dLMO during second and early third-instar stages. Ap activity depends strictly on its cofactor Chip and requires the formation of an Ap-Chip-Chip-Ap tetramer. Ap induces expression of its repressor dLMO, which competes with Ap for binding Chip. As a consequence, within the dorsal domain, dLMO downregulates Ap and limits levels of Serrate and Fringe, that are critical for wing cell survival. In the analysed Bx mutants, Ap expression was observed to be strongly misregulated. In addition, no proper DV boundary formation was found in the wing pouch. Wg is expressed along the DV boundary in wing pouch, where it acts locally to induce nearby cells to adopt a wing margin identity. Wg expression was found to be severely affected in the analysed Bx mutants. The dLMO-dependent repression of Serrate and Fringe in early third-instar, is necessary to allow secondary Delta and Serrate expression domains in an Ap-independent mode of regulation. This Ap-independent induction of Serrate maintains Wg and Cut expression in a Notch-dependent positive feed back loop. However, since Ap activity continues to be required for dorsal cell fate specification, it is important to limit Ap downregulation by dLMO. It is suggested that mir-9a could have a critical role in this process (Biryukova, 2009).

The stoichiometry of dLMO is relevant for proper wing development. This study analyzed series of new Bx, the gain-of-function alleles of dLMO that are associated with lack of miRNA sites in the 3' UTR. Among several putative candidates, the investigation focussed on the function of a very ancient miRNA mir-9a, that is conserved from Drosophila to human. dLMO was shown to be a mir-9a target gene that is involved in the control of the Drosophila wing development. Both loss and gain-of-function mir-9a mutants exhibit wing margin defects. Analysis of dLMO expression in loss-of-function mir-9a mutants revealed increased levels of dLMO in the wing discs. Reciprocally, in mir-9a gain-of-function mutants, a reduced expression of dLMO was detected. Strong genetic interactions were observed between dLMO and mir-9a during wing development. Loss of wing margin of mir-9a null flies is fully rescued by removing its target gene, dLMO. Moreover, the wing margin defects in gain-of-function Bx mutants can be rescued by overexpression of mir-9a. These data provide evidence that dLMO is the main target gene of mir-9a for wing development. Loss of wing margin phenotype of mir-9a null flies has been previously attributed to an excess of another mir-9a target gene, senseless (sens). However, this study showed that the wing shape is less sensitive to the dosage of Sens than to the dosage of dLMO. Flies carrying two extra copies of sens+ do not have a modified wing shape, whereas a fly carrying only one extra copy of sens+, shows a gap in the posterior wing margin, a phenotype observed in weak Bx mutants. According to current estimation, a medium to strong Bx mutant, overexpresses 2- to 4-fold the normal level of dLMO transcripts. Loss of wing margin in mir-9a null mutants is more likely a consequence of the overexpression of dLMO or a synergism between overexpressed dLMO and Sens. Besides, no genetic interaction was found between dLMO and Sens during wing development. In this context, it is suggested that mir-9a might maintain the required threshold of dLMO protein levels that are critical for wing cell survival and fine tune the appropriate level of Ap (Biryukova, 2009).

mir-9a was recently described to restrict the generation of sensory organs by downregulating Sens expression within the proneural fields and assuming the robustness of the Notch lateral signaling. Interestingly, dLMO behaves as an early proneural activator in the thorax. dLMO is expressed within the proneural clusters, and later after the SOP selection, it is excluded from the mature neurons, like mir-9a. Thus, when dLMO is downregulated by mir-9a, its proneural activity no longer interferes with the Notch lateral signaling that represses proneural fate within the same cells. It is suggested that regulation of both dLMO and Sens by mir-9a represent further level of complexity for gene regulation that contributes to sensory organ development (Biryukova, 2009).

dLMO plays an important role in multiple biological processes in Drosophila. The gene encodes two functional isoforms dLMO-PA and dLMO-PB that are differentially expressed during development. It has been demonstrated that the major isoform, dLMO-PA participates with Chip in the assembly of a proneural transcriptional complex that includes the GATA factor Pannier and the bHLHs Ac/Sc and Daughterless (Da). Therefore, dLMO acts as an early coactivator of the proneural genes ac/sc. dLMO mutants have consistent pleiotropic phenotypes, for instance, amorphic dLMO mutants show a hypoplasia of neurons. Loss-of-function dLMO mutants exhibit strong locomotion defects and changes in cocaine responsiveness, hence dLMO function is required for circadian pacemaker neurons in the brain. For these aspects of neurogenesis and neurological responsiveness, the gain-of-function Bx mutants show opposite phenotypes with regards to the loss-of-function ones. Since Bx alleles encode more stable truncated mRNAs, the stoichiometry of dLMO might be relevant for diverse dLMO functions. dLMO 3' UTR contains multiple motifs involved in negative post-transcriptional regulation, including Brd-boxes, AU-rich elements (AREs) and miRNA sites. Interestingly, the non-canonical Brd boxes in the dLMO 3' UTR carry the same wobble at position 6 in the seed, that might decrease a tuning efficiency by Brd box related miRNAs, mir-79 and mir-4. Both ARE-binding proteins and specific miRNAs can bind the AREs sequences, modulating the translational regulation of the transcripts. The diversity of miRNA sites might reflect specificities, redundancies and cooperations with the mir-9a site for adjusting dLMO transcripts under a detrimental level in regulating both neurogenesis and wing development, or controlling other functions of dLMO. For instance, dLMO was identified recently as a potential mir-14 target gene. Although mir-14 is expressed during larval stages, loss of mir-14 did not show any overt wing phenotype nor did it enhance the wing margin defects of Bx1/+ wings. Therefore, mir-14 might directly regulate the dLMO protein levels that are required in other developmental contexts in which dLMO activity is involved (Biryukova, 2009).

The mammalian homolog of dLMO, LMO2, is expressed in the mesenchyme of the developing mouse limb bud, suggesting a conserved function of LMO2 between insects and mammals. Lmx-1, a LIM-HD protein like Ap, is expressed in the mesenchyme of the dorsal limb bud during development. Loss of Lmx-1b function causes a biventral phenotype, implicating Lmx-1b as a primary dorsalizing activity in the mouse limb. Like dLMO, LMO2 is expressed in both the dorsal and ventral compartments during limb patterning. Lhx-2, the mammalian ortholog of Ap, is able to rescue ap mutant phenotypes as efficiently as the fly Ap protein. LMO2 or other LMO proteins could interact with Lmx-1 or Lhx-2, in a manner similar to the proposed interaction between Ap and dLMO in Drosophila wings. LMO2 is also expressed at the somite boundaries. Many genes required for formation of the D-V boundary in the developing limb, such as members of the fringe, Wnt, and Notch gene families, also play an important role during somitogenesis. The presence of LMO2 RNA at the somite boundaries might indicate a conserved role of LMO gene family members in the context of boundary formation, including the limb bud, somite, and insect wing disc (Biryukova, 2009).

LMO2 is known as a master regulator of haematopoiesis in mouse and human. Its stoichiometry is critical for proper T-cell differentiation during early haematopoiesis. LMO2 is activated via chromosomal translocation in T-cell acute leukemia (T-ALL). In Drosophila neurogenesis, dLMO is a member of a transcriptional complex similar to the one that controls Tal-1 expression during human haematopoiesis. Interestingly, the human LMO2 3' UTR contains multiple miRNA target sites, including hsa-mir-9 and hsa-mir-9*, that are orthologs of the Drosophila dme-mir-9a and dme-mir-79, respectively. Furthermore, hsa-mir-9 is expressed within the mammalian haematopoietic system. In human, the enforced expression of LMO2 in a significant fraction of T-ALL results from loss of the upstream transcriptional mechanisms that normally downregulate the expression of this oncogene during T-cell development. Therefore, it is suggested that a similar mechanism of LMO2 regulation by mir-9 may operate during normal human haematopoiesis and can be disrupted in pathological conditions, like T-cell acute leukemias and large B-cell non-Hodgkin's lymphomas (Biryukova, 2009).

miR-9a prevents apoptosis during wing development by repressing Drosophila LIM-only

Loss of Drosophila mir-9a induces a subtle increase in sensory bristles, but a substantial loss of wing tissue. This study established that the latter phenotype is largely due to ectopic apoptosis in the dorsal wing primordium, and the wing development could be rescued in the absence of this microRNA by dorsal-specific inhibition of apoptosis. Such apoptosis was a consequence of de-repressing Drosophila LIM-only (dLMO), which encodes a transcriptional regulator of wing and neural development. Cell-autonomous elevation of endogenous dLMO and a GFP-dLMO 3'UTR sensor was observed in mir-9a mutant wing clones, and heterozygosity for dLMO rescued the apoptosis and wing defects of mir-9a mutants. Evidence is provided that dLMO, in addition to senseless, contributes to the bristle defects of the mir-9a mutant. Unexpectedly, the upregulation of dLMO, loss of Cut, and adult wing margin defects seen with mir-9a mutant clones were not recapitulated by clonal loss of the miRNA biogenesis factors Dicer-1 or Pasha, even though these mutant conditions similarly de-repressed miR-9a and dLMO sensor transgenes. Therefore, the failure to observe a phenotype upon conditional knockout of a miRNA processing factor does not reliably indicate the lack of critical roles of miRNAs in a given setting (Bejarano, 2010).

Because of their relative ease of detection, dominant alleles and X-linked mutants constituted a high proportion of the classical spontaneous mutants isolated by Morgan and colleagues. Bridges isolated Bx[1] in 1923, and genetic tests by Green in the early 1950s established that Bx was due to overactivity of the locus. In fact, the recessive allele Bx[r] was associated with a duplication of the region, indicating that as little as a two-fold increase in Bx activity could interfere with wing development. In 1979, Lifschytz and Green further proposed that Bx might be due to a mutation in a cis-acting repressor site in the heldup locus. Indeed, the cloning of Bx by the Cohen, Jan, and Segal labs in 1998 finally revealed that Bx and heldup were gain- and loss-of-function alleles of the dLMO gene, respectively. Moreover, most spontaneous Bx alleles proved to be transposable element insertions in the dLMO 3' UTR, and new Bx mutants were easily obtained by imprecise excisions of a downstream transposable element, so as to delete dLMO 3' UTR sequence. Collectively, these 85 years of research indicated that 3'UTR-mediated post-transcriptional repression of dLMO is critical for normal development (Bejarano, 2010).

A small number of other gain-of-function mutants in Drosophila and C. elegans result from the loss of 3' UTR regulatory elements, and many of these are now appreciated to be key genetic switch targets of miRNAs. The current studies, together with concurrent work from Heitzler and colleagues (Biryukova, 2009), establish dLMO as one of a handful of genes whose loss of miRNA-mediated repression leads to a severe morphological defect. This study found that the lack of mir-9a results in upregulation of dLMO, aberrant apoptosis in the wing pouch, and failure to completely specify and develop the wing margin. Importantly, dorsal-specific expression of miR-9a, dorsal-specific inhibition of cell death (using p35 or Diap1), or heterozygosity for dLmo, all strongly reduced ectopic apoptosis and restored adult wing development in mir-9a null animals (Bejarano, 2010).

Ectopic apoptosis in the wing pouch has previously been reported to result in loss of wing margin. However in other cases, the disc is able to compensate for cell loss in the face of ectopic apoptosis, so that no loss of margin is observed in the adult wing. In the mir-9a mutant, this study shows that excess apoptosis is coupled with a margin specification defect. Despite an ability to rescue the mutant by inhibiting apoptosis, it cannot be ruled out that ectopic apoptosis by itself might be insufficient to induce adult margin loss; perhaps it requires the sensitized background evidenced by the demonstrable failure to fully activate Cut in the third instar. It is also noted that Bx was recently reported not to be suppressed by inhibiting apoptosis, which might be at odds with the current conclusions. However, that study examined Bx/Y hemizygotes, which are substantially stronger in phenotype than Bx/X heterozygotes. It is clear that elevation of dLMO yields a variety of patterning defects that are not seen in mir-9a mutants. It is inferred that loss of miR-9a results in apoptosis and wing margin defects that are attributable to de-repression of dLMO, but that elevation of dLMO can clearly generate developmental phenotypes that are not simply due to excess apoptosis (Bejarano, 2010).

A minor, but quantifiable, consequence of lacking mir-9a is the development of a small number of ectopic sensory organs. This is demonstrably due to the de-repression of the proneural factors Sens and dLMO. Therefore, even though computational approaches provide evidence for hundreds of conserved miR-9a targets, including compelling 'anti-target' relationships with a large number of neural genes, the bulk of its morphologically evident phenotypes can be accounted for by the failure to repress only two target genes, sens and dLmo. In addition to miR-9a, Drosophilid species encode miR-9b and miR-9c, as well as the ancestrally related miR-79. The function of these miRNAs remains to be studied, but conventional knowledge of miRNA targeting suggests that they may have overlapping target capacity since they have similar seeds. It is conceivable that the analysis of double or triple mir-9 mutants may reveal additional targets that mediate compelling phenotypes. Nevertheless, it is clear that miR-9a serves a function to repress dLmo and sens that cannot be substantially compensated by the remaining miR-9-related genes (Bejarano, 2010).

dLMO and miR-9 are both highly conserved between invertebrates and vertebrates. However, vanishingly few miRNA:target interactions have been preserved over this evolutionary distance, indicating that these post-transcriptional target networks are much more plastic than the genes themselves. Therefore, the existence of a key miR-9a:dLmo regulatory connection in flies does not necessary imply that human miR-9 regulates LMO genes, and human LMO genes lack conserved canonical miR-9 seed sites. Heitzler and colleagues proposed that mammalian LMO2 is a conserved target of miR-9 (Biryukova, 2009). However, the candidate site contains a G:U seedpair, a feature that is detrimental to, although not necessarily incompatible with miRNA targeting. Directed studies are needed to assess whether this site alone confers repression by miR-9 g (Bejarano, 2010).

On the other hand, the necessity of restricting LMO activity might well prove to be a conserved feature of invertebrate and vertebrate biology. As in Drosophila, vertebrate LMO proteins can dominantly interfere with LDB:Islet complexes, indicating that its overactivity is especially 'dangerous'. Indeed, elevation of LMO proteins has myriad consequences for downstream transcriptional networks, and LMO2 is in fact a T-cell oncogene. Intriguingly, LMO2, which normally regulates hematopoetic development, has a highly conserved 8mer seed for miR-223. Recent studies demonstrated that mir-223 mutant mice exhibit hematopoetic defects, and that mir-223 deletion has consequences for the neutrophil transcriptome and proteome. While the depth of peptide sampling was insufficient to report on LMO2 status, the microarray data demonstrated LMO2 to be the 60th most-upregulated mRNA across the transcriptome of mir-223 knockout cells. Indeed, it has been recently reported that suppression of LMO2 by miR-223 regulates erythropoiesis. It is notef that its paralog LMO1 contains a highly conserved canonical site for miR-181, another miRNA with a demonstrated function in the hematopoietic system. These observations suggest that the regulation of vertebrate LMO genes by hematopoietic miRNAs, and its potential relevance to cancer, deserves further study (Bejarano, 2010).

To date, relatively few Drosophila or vertebrate miRNA genes have been analyzed using bona fide mutant alleles. As an approximation, many researchers have taken to analyzing the effects of conditional knockout of miRNA biogenesis factors, such as Dicer. This manipulation is presumed to break all miRNA regulatory links, thereby serving as a plausibility test of whether miRNAs might be required in a given setting. Acknowledged drawbacks of this approach include uncertainty as to whether one or many miRNAs might contribute to a given phenotype, and whether phenotypes are a direct or indirect cause of miRNA loss. However, a caveat that is little considered is the potentially canceling effects of removing 'all' miRNAs, so that loss of one miRNA might be compensated for by the concomitant loss of another miRNA(s). While such an outcome might seem to require highly unlikely coincidences, it may be plausible if it is considered that most biological processes are under both positive and negative control, and that most genes are themselves miRNA targets (Bejarano, 2010).

During development of the Drosophila wing primordium, this study has shown that clones lacking mir-9a upregulate dLMO and induce wing notching, whereas dcr-1 and pasha-mutant clones do not. Additionally, mir-9a mutant clones exhibit a more severe phenotype than dcr-1 mutant clones with respect to loss of wing margin, both in the third instar wing pouch and in the adult wing. Although the cells analyzed were homozygous mutant for substantial periods of time (72-96 hours), perdurance of miRNAs on account of Dcr-1 or Pasha proteins inherited by mutant cells conceivably contributes to the phenotypic disparity. For example, perdurance may explain the incomplete phenocopy of bantam mutant discs by dcr-1 or pasha 'whole disc' mutants. However, potential perdurance is not reconciled with the comparable upregulation of miR-9a and dLMO sensor activity in dcr-1 and mir-9a homozygous mutant cells, which report on similar loss of miR-9a activity in these clones. Together, these data suggest that mir-9a mutant cells exhibit phenotypes that are intrinsically different from those of dcr-1 or pasha mutant cells (Bejarano, 2010).

In summary, the failure to observe a phenotype in cells or tissues that are mutant for a general miRNA biogenesis factor cannot reliably be taken as evidence that miRNAs lack substantial roles in the setting of interest. Reciprocally, the observation that loss of miRNA-mediated regulation from a single target gene (e.g. failure to repress dLmo in mir-9a mutant wings) can be of greater phenotypic impact than loss of 'all' miRNA-mediated regulation (e.g, in dcr-1, pasha double mutant wing clones) highlights the disproportionate consequence of releasing particular miRNA targets from amidst a regulatory web that is inferred to encompass most animal transcripts (Bejarano, 2010).

Protein Interactions

Formation of the dorsal-ventral axis of the Drosophila wing depends on activity of the LIM-homeodomain protein Apterous (Ap). Ap activity levels are modulated by dLMO, the protein encoded by the Beadex (Bx) gene. Overexpression of dLMO in Bx mutants interferes with Apterous function. Conversely, Bx loss-of-function mutants fail to down-regulate Apterous activity at late stages of wing development. Biochemical analysis shows that dLMO protein binds to Chip, thus competing with Apterous binding to Chip. These data suggest that Apterous activity depends on formation of a functional complex with Chip and that the relative levels of dLMO, Apterous, and Chip determine the level of Apterous activity. The dominant interference mechanism of dLMO action may serve as a model for the mechanism by which LMO oncogenes cause cancer when misexpressed in T cells (Milan, 1998).

LIM domains are thought to mediate protein interactions and are found in a variety of different types of proteins, often in combination with other recognized protein domains, as in the LIM-homeodomain proteins. dLMO belongs to a class of LIM domain proteins that have two LIM domains and no other recognizable motifs (hence, the designation LMO, for LIM only). In view of the effects of dLMO on Ap function, experiments were designed to discover whether dLMO can interact with Ap, a LIM homeodomain protein and Chip, an LDB (LIM domain binding) protein. LDB proteins bind to the LIM domains of nuclear LMO proteins of the type encoded by Bx. Genetic interactions between chip and ap suggest that as with Ldb1 and XLim1, Chip binding might activate Ap function. When overexpressed, Bx appears to interfere with Ap function without affecting either Chip or Ap protein expression. This raises the possibility that dLMO might interfere with binding between Ap and Chip. This was tested using a coimmunoprecipitation assay in which the binding between constant amounts of Chip and Ap proteins was challenged by increasing concentrations of Bx protein. Chip protein can be immunoprecipitated with T7-epitope-tagged Ap protein and anti-T7 antibody, showing that Ap and Chip proteins bind in vitro. Binding between Chip and Ap was challenged by adding increasing amounts of in vitro-translated dLMO protein. A dose-dependent decrease in the amount of Chip immunoprecipitating with Ap is observed as the amount of dLMO protein is increased and a corresponding increase in the amount of Chip immunoprecipitating with dLMO is also observed. These observations indicate that dLMO can bind to Chip in vitro and can compete for binding between Chip and Ap in a concentration-dependent manner. As a further test, the LIM domains of Ap were expressed as a GST fusion protein and tested for binding to full-length dLMO, Chip, and Ap proteins. Ap binds to itself and to Chip but not to dLMO in the GST-pull-down assay. This suggests that dLMO interferes with formation of the active Ap-Chip complex by competing with Ap for binding to Chip (Milan, 1998).

A tetramer of dLBD (Chip) and Apterous confers activity and capacity for regulation by dLMO (Beadex). To test whether the active form of Apterous is a complex involving two molecules of Ap and two molecules of Chip, the effects of expressing dominant-negative forms of Chip that differ in their ability to bind Ap were compared. Overexpression of wild-type Chip has dominant-negative activity in vivo. It has been suggested that this could be due to formation of trimeric complexes (Ap:Chip:Chip) that lack a second Ap molecule because the dominant-negative activity of Chip can be suppressed by overexpression of Ap. It was reasoned that a truncated form of Chip lacking the LIM interaction domain (ChipdeltaLID) would also serve as a dominant negative but that its activity should not be suppressed by overexpression of Ap. Before testing the activity of the ChipdeltaLID construct in vivo, it was verified that the molecular interactions between Ap and Chip in vitro are consistent with the expectations based on analysis of the human LDB protein, NLI (Milán, 1999).

Complex formation between Ap, Chip, and ChipdeltaLID was assayed using in vitro translated proteins. Ap was expressed with a T7-epitope tag, incubated with 35S-labeled Chip or ChipdeltaLID, and immunoprecipitated with anti-T7. Full-length Chip coprecipitates with T7-Ap. ChipdeltaLID is not recovered above background levels when incubated with T7-Ap, confirming that Chip needs the LID to bind effectively to Ap. ChipdeltaLID coprecipitates when incubated with T7-Ap and full-length Chip, demonstrating formation of a three part Ap:Chip:ChipdeltaLID complex. To verify that a Chip dimer can bridge two Ap molecules, a tagged version of Ap (Ap-TAP) was used. The biological activity of Ap-TAP is comparable to that of wild-type Ap when ectopically expressed in vivo under GAL4 control. Ap-TAP was in vitro translated and bound to IgG beads. The beads were washed and incubated with labeled Ap with or without unlabeled Chip. Without Chip, only background levels of 35S-Ap are recovered. When Chip is present, Ap-TAP beads bind 35S-Ap, indicating formation of the tetrameric complex in vitro (Milán, 1999).

Overexpression of Chip represses the Ap targets fringe-lacZ and dLMO. Overexpression of ChipdeltaLID using patched GAL4 also represses fringe-lacZ and dLMO, indicating that both forms of Chip suppress Apterous activity when overexpressed. Overexpression of Chip under control of ap-GAL4 interferes with wing formation, producing a phenotype resembling the lack of ap function. This can be suppressed by coexpression of UAS-Chip and UAS-Ap. Overexpression of ChipdeltaLID using ap-GAL4 causes a mild apterous phenotype: distal notching of the wing margin and dorsal-to-ventral transformation of the alula. Although the phenotype suggests only partial reduction of Ap activity, the defects caused by overexpression of ChipdeltaLID cannot be suppressed by coexpression of Ap. These results suggest that ChipdeltaLID acts as a dominant negative for Ap activity in vivo by promoting the formation of a trimeric Ap:Chip:ChipdeltaLID complex that cannot bind another Ap molecule (Milán, 1999).

The LIM domain protein dLMO can compete with Ap for binding to its cofactor Chip. If the active form of Ap in vivo is a LIM-HD dimer bridged by a dimer of cofactor, dLMO might compete for Ap activity by displacing an Ap molecule from the Ap:Chip complex to form an Ap:dLMO heterodimer bridged by the cofactor. This model was tested by preparing a form of Ap that could dimerize but that could not be displaced by dLMO. To do so, a fusion protein consisting of the N-terminal dimerization domain of Chip linked to a C-terminal fragment of Ap containing the homeodomain (aa 270-469) was expressed. The new protein, called ChAp, lacks the LIM interaction domain of Chip and the LIM domains of Ap. Its structure should allow it to form an Ap dimer (Milán, 1999).

A test was performed to see whether ChAp has activity comparable to Ap in vivo. When expressed along the anteroposterior compartment boundary under control of dpp-GAL4, UAS-ap and UAS-ChAp produce essentially identical phenotypes. In both cases, ectopic wing margins are induced on both sides of the dpp-GAL4 stripe in the ventral compartment. The ectopic wing margin is due to the ectopic expression of Wingless in the ventral compartment. This correlates with ectopic induction of the dorsally expressed Ap targets fringe-lacZ and dLMO in ventral cells. These observations show that ChAp can mimic the effects of Ap in ectopic expression assays. A rescue assay was used to ask whether ChAp can functionally substitute for Ap in vivo. The wing defect in apGAL4/aprk568 flies is completely suppressed when wild-type Ap is expressed in dorsal cells using ap-GAL4. Dorsal expression of ChAp produces a comparable rescue. These results show that ChAp behaves like wild-type Ap when ectopically expressed and that ChAp can substitute for Ap in vivo (Milán, 1999).

According to the dimer model, ChAp should be sensitive to the dominant-negative activity of ChipdeltaLID but should not be subject to regulation by dLMO in vivo. dLMO is induced by Ap in the wing disc, and loss-of-function dLMO mutants produce defects that are thought to result from overactivation of Ap. ChAp overexpression in the dorsal compartment of an otherwise wild-type wing disc gives a phenotype that closely resembles the dLMO loss-of-function phenotype. The wings are smaller than wild type and are held in an abnormal position. The dorsal compartment is typically smaller than the ventral compartment, giving the wing a slightly curled appearance. The pattern of veins is also abnormal. Coexpression of the dominant-negative form of the cofactor, ChipdeltaLID, suppresses the ChAp overexpression phenotype. This indicates that dimerization is required for ChAp activity in vivo (Milán, 1999).

To ask whether ChAp is subject to regulation by dLMO, the ability of Ap and ChAp to suppress the effects of dLMO overexpression in vivo were compared. apGAL4/+; UAS-dLMO/+ wings show loss of the normal wing margin and sporadic patches of ectopic wing margin, thus promoting local overgrowth. Overexpression of wild-type Ap does not suppress the dLMO overexpression phenotype. Antibody staining shows that Wg is not expressed at the DV boundary in these discs. The wing pouch is very small, and the normally straight boundary between cells expressing Ap and adjacent nonexpressing cells is uneven. These observations suggest that Ap is nonfunctional in these discs in spite of being overexpressed. In contrast, coexpression of ChAp and dLMO restores Wg expression at the DV boundary even though dLMO is expressed at high levels. The resulting wings have a normal wing margin and resemble the dLMO mutant wing. These results suggest that ChAp overexpression phenocopies the dLMO loss-of-function mutant because ChAp is not sensitive to downregulation by dLMO. Consequently, ChAp remains active in the presence of excess dLMO (Milán, 1999).

The bridged dimer model suggests that removing dLMO activity would result in excess Ap activity. To test this, the properties of Chip and Ap interaction were exploited to regulate Ap activity in a dLMO mutant background. dLMO loss-of-function mutants were generated by excision of a GAL4-P element insertion in the second intron of the dLMO gene. Fortuitously, excision line hdpR590 strongly reduces dLMO expression but leaves GAL4 and the cis-regulatory region unaffected, so that the mutant expresses GAL4 in the normal pattern of dLMO. hdpR590 causes aberrant Serrate expression and a reduced dorsal wing pouch. The dLMO loss-of-function phenotype in this mutant can be suppressed by expression of ChipdeltaLID. The small wing size of hdpR590 is fully rescued, and the abnormal venation is partially suppressed. Likewise, expression of a mutant form of Ap lacking only the homeodomain completely suppresses the hdpR590 phenotype. Both of these constructs have mild dominant-negative effects that reduce Ap activity in vivo. These results confirm that the dLMO loss-of-function phenotype results principally from excess Ap activity at later stages of wing development. Further, they support the proposal that the normal function of dLMO is to regulate Ap activity levels by interfering with formation of an active complex consisting of two Ap molecules bridged by a dimer of Chip molecules (Milán, 1999).

The finding that ChAp can completely replace Apterous function in vivo suggests that the relevant feature of this tetrameric complex is the formation of a dimer of Ap. This molecule is not subject to negative regulation by dLMO and so remains constitutively active. The consequence is a failure to downregulate Ap activity as development proceeds. The phenotypic consequences of excess Apterous activity are comparable to those of the dLMO lack-of-function mutant. These findings support the view that the tetrameric complex between Ap and its cofactor Chip provides a means to generate the requisite bridged dimer of Ap, while allowing the activity of the complex to be regulated by the competitive inhibitor dLMO. It is suggested that this may provide a general model for regulation of LIM-HD activity. LMO family proteins may be generic antagonists of LIM homeodomain proteins through binding to their common LDB cofactors. The active complexes may be cofactor-bridged homodimers (as is the case for Ap in wing development) or heterodimers with other LIM-HD transcription factors or other types of LDB-binding transcription factors. Combinations of different transcription factors bridged by a cofactor dimer might broaden the range of possible target genes (Milán, 1999).

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. A difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo (Weihe, 2001).

Apterous activity levels are spatially and temporally regulated in the wing disc by expression of dLMO. Comparing expression of Ap protein and mRNA in the wing imaginal disc suggested that Ap might be subject to post-transcriptional regulation. ap mRNA is expressed at similar levels in the presumptive wing hinge and wing pouch. By contrast, Ap protein levels are considerably lower in the wing pouch than in the hinge region. The region where Ap levels are low coincides with the region in which dLMO is expressed. This suggests that the difference in Ap protein levels reflects a post-transcriptional consequence of dLMO expression. To ask whether dLMO is responsible for reducing Ap levels where the two proteins are co-expressed, genetic mosaics were produced in which dLMO activity was removed from clones of cells. Ap protein was more abundant in cells homozygous mutant for dLMODelta39. The increased level of Ap protein in the clone was similar to the level detected in the hinge (Weihe, 2001).

These observations suggest that dLMO protein is responsible for the reduced level of Ap protein in the dorsal wing pouch. To further test this possibility, clones of cells overexpressing dLMO were created and Ap protein levels were assessed. As expected from the loss-of-function data, dLMO expression reduced Ap levels in the hinge region, where Ap levels are usually high. The lower level of Ap in the dorsal pouch was further reduced by elevated dLMO expression. It is therefore concluded that dLMO reduces Ap levels in third instar imaginal wing discs. To determine whether Ap protein might be degraded in dLMO-expressing cells by a proteasome-dependent mechanism, wing discs were incubated with the proteasome inhibitor MG-132. Ap protein levels were increased in the wing pouch relative to the levels in the hinge in drug treated. This suggests that Ap protein is more susceptible to proteasome-mediated degradation in cells expressing dLMO (Weihe, 2001).

Since dLMO competes with Ap for binding to Chip, the possibility that Ap protein may be protected when it is in a complex with Chip was examined. To test this, Chipe5.5 mutant clones, which lack Chip protein and therefore lack Ap activity, were created. Ap protein levels were reduced in Chip mutant clones, and increased in the wild-type twin spots which contain a higher level of Chip protein. To verify that reduced Chip activity does not affect ap mRNA levels ap-lacZ reporter gene expression was examined in discs expressing the dominant negative form of Chip, ChipDeltaLID. Ap protein levels were reduced in cells expressing ChipDeltaLID but ap-lacZ levels were unaffected. Thus, loss of Chip leads to reduced levels of Ap protein. It was noted that Chip mutant clones also lack dLMO expression. Thus, loss of Ap protein in Chip mutant clones does not correlate with expression of dLMO, as in wild-type cells. Rather, reduction of Ap levels correlates with the availability of Chip as a binding partner. This suggested that binding to Chip contributes to stabilization of Ap (Weihe, 2001).

If Ap stability decreases when it is unable to bind DNA, it was reasoned that providing additional binding sites might stabilize the protein. To test this possibility a cell culture system was used in which the number of Ap-binding sites could be varied by transfection. It was first verified that co-expression of dLMO would decrease Ap stability in transfected cells. A constant amount of a plasmid directing expression of a Myc-tagged Ap protein was co-transfected with varying amounts of a plasmid directing expression of myc-tagged dLMO. As observed in the wing disc, overexpressed dLMO reduces Ap protein levels in S2 cells. It was noted that high levels of dLMO are required to reduce Ap levels. The relative levels of the two proteins can be directly compared in this assay by virtue of the myc-epitope tag. Comparison of relative levels of the endogenous proteins is not possible (Weihe, 2001).

dLMO has been proposed to act as a competitive inhibitor of Ap in vivo. This model suggests that overexpression of Ap should be able to produce phenotypes similar to those caused by reduced levels of dLMO; however, this has not been observed. Overexpression of Ap in its endogenous domain does not produce alterations in the wing comparable with those caused by loss of dLMO activity. By contrast, expression of fusion proteins between Chip and Ap, which are insensitive to repression by dLMO, produce the expected phenotypes. This suggests that Ap does not compete effectively with dLMO for interaction with Chip, even when overexpressed. These observations could be explained by an intrinsic difference in the affinities of the two LIM domain proteins for Chip. To test this possibility, a fusion protein was constructed that contains the LIM domains of dLMO (100 amino acids) but otherwise consists entirely of Ap sequences. This protein was called dLAp to indicate LIM-domains of dLMO in Ap. To distinguish dLAp from endogenous Ap and dLMO proteins, a C-terminal flag tag was included (Weihe, 2001).

To ask whether overexpression of dLAp in dorsal cells would compete effectively with dLMO to produce a net increase in Ap activity levels, wing development was compared in flies expressing dLAp or Ap under apGal4 control. Ap overexpressing wings are normal. In apGal4/+; uas-dLAp/+ flies defects were observed in wing veins, especially in the posterior crossvein and vein 5, and a held up wing phenotype. These defects resemble the dLMO mutant phenotype, which has been shown to be due to excess Ap activity. Another feature of dLMO mutant wings is overexpression of Serrate in the D compartment. Overexpression of wild-type Ap under apGal4 control does not cause abnormal Serrate expression; however, expression of dLAp in apGal4/+;uas-dLAp/+ wing discs induces ectopic Serrate in the dorsal compartment and causes mild reduction of the D compartment. Similar, though somewhat stronger effects were obtained by overexpression of the Chip/Ap fusion protein ChAp, which is insensitive to competition by dLMO. Thus, dLAp expression can increase Ap activity to levels above normal in the presence of dLMO (Weihe, 2001).

Ap activity can be abolished by overexpression of dLMO under apGal4 control in the wing disc. Providing additional Ap protein by co-expression of Ap does not overcome the inhibitory effects of dLMO. Wingless is not expressed at the interface between D and V cells and the wing pouch is very small. By contrast, co-expression of dLAp restores Wingless expression along the DV boundary and wing pouch growth. This indicates that dLAp is able to restore Ap activity in the presence of dLMO. Taken together, these observations indicate that dLAp competes efficiently with dLMO for binding to Chip, whereas Ap does not. Since the only difference between Ap and dLAp is in the LIM domains, their different behavior is attributed to an intrinsic property of the LIM domains (Weihe, 2001).

This report addresses the problem of asymmetry in the competition between dLMO and Ap. The simplest model for competitive inhibition by dLMO would suggest that Ap should compete effectively with dLMO for binding to Chip when overexpressed. However, overexpression of Ap does not produce an excess of Ap activity. dLMO competes effectively for Ap activity, but the reverse is not true. Swapping the LIM domains of Ap for those of dLMO produces a functional Ap protein that is able to compete effectively with dLMO. This finding may provide an explanation for the non-reciprocal properties of Ap and dLMO. The effectiveness of dLMO as an inhibitor of Ap activity is attributed to an intrinsic difference in the ability of the LIM domains of these two proteins to bind to Chip. It is considered likely that the LIM domains of dLMO bind the LID of Chip with higher affinity than the LIM domains of Ap. However, these proteins have not been produced in soluble form at adequate concentrations and so the affinities of these binding interactions have not been determined directly (Weihe, 2001).

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO (Beadex). Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. A modular P-element based EP (enhancer/promoter) misexpression screen was conducted to look for genes involved in Apterous activity. Osa, a member of the Brahma chromatin-remodeling complex, was found to be a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. It is proposed that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression (Milan, 2004).

This study presents evidence that Osa, a member of a subset of Brahma chromatin remodeling complexes, behaves overall as a general activator of Apterous activity in the Drosophila wing. Overexpression of Osa rescues and loss of Osa enhances the Beadex1 phenotype. It does so by modulating the expression levels of Apterous target genes, some of them being activated (e.g. Serrate and probably other unknown target genes) and some repressed (e.g. Delta, fringe). Chip has been shown to bind Osa. The fact that Osa has different effects on the transcription of Apterous target genes suggests that Chip recruits Osa to the promoters and in combination with other unknown factors mediates either transcriptional repression or activation. Osa mediates repression of both Apterous dependent and independent expression of fringe, suggesting a direct and probably Chip independent effect of Osa on fringe transcription (Milan, 2004).

Apterous activity is regulated during development by dLMO. Osa is required to mediate repression of dLMO expression. Since both early and late expression of dLMO depend on Chip, it is postulated that Chip forms a transcriptional complex with Apterous in the D compartment and an unknown transcription factor expressed in the wing pouch. Osa may interact with Chip thus recruiting the Brahma complex to the dLMO locus and remodeling chromatin in a way that limits dLMO transcriptional activation. High levels of dLMO protein reduce Apterous activity and the Notch dependent organizer is not properly induced along the DV boundary. Osa mediated repression of dLMO expression may ensure moderate levels of expression of dLMO in the wing, thus allowing proper wing development. Gain of function mutations that cause misexpression of vertebrate LMO proteins have been implicated in cancers of the lymphoid system. Truncating mutations in the human SWI-SNF complex, the human homologues of the Brahma complex, cause various types of human cancers. The SWI-SNF complex may be required to mediate repression of LMO expression in lymphoid tissues. Thus, it would be very interesting to analyze if truncating mutations in members of the human SWI-SNF complex cause higher levels of LMO expression and are associated with lymphoid malignancies (Milan, 2004).

It has been shown that the Brahma complex plays a general role in transcription by RNA Polymerase II. Then, is Osa having a general effect on the expression levels of every gene involved in wing patterning? Several observations indicate this is not the case. (1) Osa is a component of a subset of Brahma (Brm) chromatin complexes. (2) Brahma and Polycomb were shown to have non-overlapping binding patterns in polytenic chromosomes. Those genes involved in wing patterning and regulated by Polycomb (i.e. Hedgehog) may not be affected by overexpression of Osa. (3) Overexpression of Osa has different effects on the expression levels of Serrate, Delta and fringe. (4) Osa has been shown to specifically regulate the expression of Wingless target genes and the Achaete-scute complex genes, interestingly by restricting their expression levels (Milan, 2004).


To determine the expression pattern of Beadex, in situ hybridization was carried out using Beadex cDNA as a probe. In third instar wing imaginal discs, Beadex is expressed at high levels in the dorsal compartment and at lower levels in the ventral compartment. Beadex also is expressed in the leg and eye discs. In the embryo, Beadex is expressed in the brain and in a subset of cells in the developing central nervous system. The presence of Beadex in many different structures suggests that perhaps dLMO serves multiple functions during development (Zeng, 1998).


A model system for the identification of presumptive overproducing mutations from among visible dominant mutations in D. melanogaster is described. An overproducing mutation is expected if a dominant mutation is readily reverted by gene deletion and if gene deletions suppress the expression of the original dominant mutation in flies heterozygous for the deletion. The Beadex mutations are shown to satisfy these requirements, since Bx dominant mutations are reverted by an induced deletion, and are also suppressed in trans by such a deletion. In addition, all 13 mutations recovered as Bx reversions or suppressors are associated with recessive held up (hdp) mutations. It is suggested that hdp functions as a Bx deletion, and may therefore represent the structural gene that is cis-regulated by the overproducing Bx mutations (Lifschytz, 1979).

Recombinant lambda phage clones were isolated spanning 49 kilobases of DNA containing the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structures of eight dominant mutant alleles of the Beadex locus that showed increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffer deletions that include at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene (Mattox, 1984).

Dlmo (LIM-like) resides in polytene band 17C1-2, where Beadex (Bx) and heldup-a (hdp-a) mutations map. Bx mutations disrupt the 3'UTR of Dlmo, and thereby abrogate the putative negative control elements. This results in overexpression of Dlmo, which causes the wing scalloping that is typical of Bx mutants. The erect wing phenotype of hdp-a results from disruption of the coding region of Dlmo. This provides molecular grounds for the suppression of the Bx phenotype by hdp-a mutations. Phenotypic interaction is demonstrated between the LMO gene Dlmo, the LIM homeodomain gene apterous, and the Chip gene, which encodes a homolog of the vertebrate LIM-interacting protein NLI/Ldb1. It is proposed that by analogy to their vertebrate counterparts, these proteins form a DNA-binding complex that regulates wing development (Shoresh, 1998).

The process of wing patterning involves precise molecular mechanisms to establish an organizing center at the dorsal-ventral boundary, which functions to direct the development of the Drosophila wing. Misexpression of LIM-like (dLMO or Beadex), a Drosophila LIM-only protein, in specific patterns in the developing wing imaginal disc, disrupts the dorsal-ventral (D-V) boundary and causes errors in wing patterning. When dLMO is misexpressed along the anterior-posterior boundary, extra wing outgrowth occurs, similar to the phenotype seen when mutant clones lacking Apterous, a LIM homeodomain protein known to be essential for normal D-V patterning of the wing, are made in the wing disc. When dLMO is misexpressed along the D-V boundary in third instar larvae, loss of the wing margin is observed. This phenotype is very similar to the phenotype of Beadex, a long-studied dominant mutation that disrupts the dLMO transcript in the 3' untranslated region (Zeng, 1998).

Adult wings of homozygous Bx1 flies show severe scalloping of the wing margin and transformation of dorsal to ventral fate in the alula at the posterior margin of the wing. The abnormalities in Bx wings resemble those produced by reducing ap function. Consistent with the suggestion that Bx mutants reduce Ap function, genetic interaction is observed between Bx and ap, fng, and chip mutants. Flies heterozygous for Bx1 and a wild-type copy of the gene show mild notching. The severity of this weak Bx1 phenotype can be enhanced by simultaneously removing one copy of the ap gene, by removing one copy of fng, or by removing one copy of chip, a gene proposed to act as a cofactor for Ap. The strong Bx1 phenotype can be completely suppressed by increasing the level of ap in dorsal cells. Increasing the level of fng expression also suppresses the wing margin defects but causes other defects in the internal organization of the wing. Taken together, these genetic interactions suggest that the defects caused by overexpressing Bx are due to reduced Ap activity (Milan, 1998).

Bx loss-of-function mutants have been described previously under the name heldup (hdp) because of the abnormal posture of the wings. Unfortunately, the original hdp mutants are no longer available. To study the normal function of dLMO in wing development new mutants were generated by imprecise excision of the MS1096 P element. MS1096 is inserted in the second intron of the Bx-dLMO transcription unit and produces a weak phenotype consisting of venation defects. New Bxhdp mutants were recovered in P-element excision screens on the basis of their adult wing phenotypes. The wings of the new Bxhdp mutants are reduced in size and show abnormalities in vein pattern. In addition, the wing posture is abnormal, as described for the original hdp mutants (Milan, 1998).

hdp mutants behave as dominant suppressors of the Bx gain-of-function phenotype. The MS1096 excision mutants completely suppress the Bx phenotype in heterozygous females, suggesting that they are loss-of-function mutants. This was confirmed by examining dLMO protein, which is expressed at much reduced levels in wing discs of the excision mutant hdpR26 (Milan, 1998).

To ask whether the wing abnormalities caused by reducing dLMO levels might be due to an effect on Ap activity, the effects of the Bxhdp excision mutants were examined on Ap target gene expression. In early third-instar fng-lacZ and Serrate (Ser) are expressed evenly throughout the dorsal compartment of the wing disc and are thought to be regulated by Ap. In Bxhdp mutant discs, the size of the dorsal compartment is considerably reduced, consistent with the small wing phenotype. fng-lacZ expression is not affected in Bxhdp discs. Ser expression is elevated in the dorsal compartment and does not resolve normally into stripes along the DV boundary and wing veins. Ser expression in the ventral compartment appears normal. The stripes of Ser expression along the DV boundary and wing veins are both dorsal and ventral and are under different regulation than the early dorsal-specific domain. The abnormal pattern of Ser in the dorsal compartment of the Bxhdp may be due to superimposition of the early and late expression patterns. It is suggested that this reflects a failure to down-regulate Ap activity as the disc matures. To ask whether elevated Ser levels might contribute to the defects observed in Bxhdp mutant wings, Ser was overexpressed in the dorsal compartment of an otherwise wild-type disc using ap-gal4 to direct UAS-Ser expression. The resulting wings are small and show thickened veins but do not show the abnormalities in vein pattern observed in the Bxhdp mutant wings. Overexpression of fng using ap-gal4 in a wild-type background produces no phenotype. These observations suggest that Ser overexpression contributes to the abnormalities observed in Bxhdp mutant wings but that there are likely to be additional factors. Thus, both gain-of-function and loss-of-function Bx mutant phenotypes can be attributed to abnormal regulation of Ap activity. It is concluded that Ap induces dLMO expression in the wing disc and that dLMO then functions as part of a feedback system to regulate the level of Ap activity (Milan, 1998).

Misexpression of Beadex at the D-V boundary disrupts margin formation and mimics the Beadex phenotype. To test the effect of misexpression of Beadex along the boundary between the dorsal and ventral compartments of the wing disc, Serrate-Gal4 (Ser-G4), which drives expression in the developing wing margin during third instar, was used. Wing margin formation in third instar wing discs was assessed by using wg-lacZ and Cut as markers for the developing wing margin. The margin is discontinuous in discs misexpressing Beadex along the D-V boundary. This disruption of margin formation results in wing scalloping that resembles the Bx phenotype. Misexpression of Beadex along the A-P boundary leads to ectopic margin formation and extra wing outgrowth. ptc-G4 was used to drive misexpression of dLMO along the A-P boundary. A gap in wg-lacZ staining appears in the third instar wing disc where Beadex misexpression intersects the D-V boundary. Additionally, an ectopic wg-lacZ stripe appears along the posterior edge of the ptc-G4 expression domain in the dorsal compartment, perpendicular to the endogenous wing margin. Double-staining for myc-tagged UAS-dLMO and wg-lacZ reveals that wg-lacZ is being expressed in cells both in the Beadex expression domain and in the adjacent cell, similar to wg expression in both the dorsal and ventral cells of the endogenous wing margin. Ct is also expressed along the posterior edge of the Beadex misexpression domain. These markers signify the formation of an ectopic wing margin in the dorsal compartment. Outgrowth organized by this ectopic margin leads to extra wing formation from the dorsal wing blade (Zeng, 1998).

The genetically defined Bx locus corresponds to the 3'UTR of dLMO. Insertion of a P element or a retrotransposon in the 3'UTR of Beadex can result in a dominant wing scalloping phenotype similar to that caused by removal of most or all the AT-rich elements (AREs) and Bearded-like boxes in the 3'UTR. It was surmised that the insertions into the 3'UTR of Bearded (by retrotransposons or P elements or by deletion of parts of the 3'UTR of Bearded) similarly abrogate the negative regulatory effect of the ARE and Brd-like motifs. Consequently, the level of the Beadex transcript in the Bx alleles examined is two- to four-fold higher than that of the wild type, as expected if the ARE and Brd-like boxes have an RNA-destabilizing effect (Shoresh, 1998).

Recessive hdp-a mutations have been genetically mapped to close proximity (0.0045 map units) to the Bx mutations, in the direction opposite from the centromere. Furthermore, hdp-a mutations have been reported to suppress in one dose the dominant wing scalloping of Bx mutations either in cis or in trans. Based on these observations, the hypermorphic nature of Bx mutations has been proposed to result from overexpression of a nearby structural gene, possibly hdp-a. Although all previously existing hdp-a alleles have been lost, new hdp-a mutants were generated in two ways, and both groups recapitulate the two characteristics of the previous alleles, namely erect wings and suppression of the dominant wing scalloping of Bx mutants. Molecular analysis has been carried out on one of them, hdp-a32-4-14, demonstrating that it has a deletion of a major part of the coding region of Dlmo, including part of the second LIM domain, suggesting that hdp-a corresponds to loss-of-function of Dlmo. Comparison to the map of Beadex suggests that the deletion in hdp-aD30r has removed part of the coding region of the gene. Loss of function of Dlmo could be also caused by mutations disrupting the promoter region of the gene (Shoresh, 1998).

Dorsoventral axis formation in the Drosophila wing depends on the activity of the selector gene apterous. Although selector genes are usually thought of as binary developmental switches, Apterous activity is found to be negatively regulated during wing development by its target gene dLMO. Apterous-dependent expression of Serrate and fringe in dorsal cells leads to the restricted activation of Notch along the dorsoventral compartment boundary. Evidence is presented that the ability of cells to participate in this Apterous-dependent cell-interaction is under spatial and temporal control. Apterous-dependent expression of dLMO causes downregulation of Serrate and fringe and allows expression of Delta in dorsal cells. This limits the time window during which dorsoventral cell interactions can lead to localized activation of Notch and induction of the dorsoventral organizer. Overactivation of Apterous in the absence of dLMO leads to overexpression of Serrate, reduced expression of Delta and concomitant defects in differentiation and cell survival in the wing primordium. Thus, downregulation of Apterous activity is needed to allow normal wing development (Milan, 2000).

Removing Apterous activity at different stages of wing development shows that Ap is needed throughout larval stages to confer dorsal cell identity, but its role in Notch activation along the DV boundary is temporally and spatially modulated. This can be explained in terms of changes in Serrate and fringe expression. Some of the changes in Serrate and fringe expression are caused by reducing Ap activity, whereas others are Ap independent. In early second instar wing discs, Ap activity is required in the entire dorsal compartment. Removing Ap activity in mitotic recombination clones at this stage induces Notch activation at the interface between wild-type and mutant cells. This response is independent of the position of the clone within the wing pouch. In early third instar wing discs, Ap-dependent expression of Serrate and fringe is reduced by dLMO. Serrate expression gradually becomes restricted to the region near the DV boundary and, subsequently, by mid-third instar is induced only in cells adjacent to the boundary. The effects of removing Ap activity in clones reflects the gradual retraction of Serrate expression toward the DV boundary. Clones of cells lacking Ap activity induced in early third instar activate the Notch pathway and induce Wg if they are located close to the DV boundary. Clones located more proximally do not show this response. This spatial difference can be overcome by providing Serrate in proximal cells (Milan, 2000).

By mid-third instar, new Ap-independent patterns of Serrate and fringe expression are observed. Serrate is expressed on both sides of the DV boundary by the activity of Wg, and fringe is expressed in four quadrants flanking the DV and AP compartment boundaries. Maintenance of Notch activation along the DV boundary is now under control of a feedback loop between Wg and Serrate and Delta. Ap is no longer required for Notch activation at the DV boundary and removing Ap activity no longer leads to activation of the Notch pathway. In the absence of dLMO, Ap activity remains at high early levels as development proceeds. Serrate and fringe expression remain high throughout the dorsal compartment and fail to undergo normal modulation. In addition, Delta is not expressed in dorsal cells. Ap-dependent repression of Delta at early stages is needed to prevent ectopic activation of Notch in dorsal cells, which are inherently Delta-sensitive due to the activity of Fringe. Some of the defects observed in dLMO mutant wings are correlated with excess Serrate activity and insufficient Delta activity. In addition, abnormally high levels of cell death in the dorsal compartment of the dLMO mutant wing disc are due to excess Ap activity and this leads to overall reduction in the size of the wing. These findings indicate the need to downregulate Ap activity to allow normal wing development. However, Ap activity continues to be required for dorsal cell fate specification and for proper adhesion of D and V wing surfaces. Thus it is proposed that different target genes may be controlled at different levels of Ap activity. Serrate, fringe and Delta may be regulated by a higher level of Ap activity than the target genes involved in surface apposition or fate specification. Temporal changes in the level of Ap activity may be required to modulate activity of different genes at different times to allow normal wing development (Milan, 2000).

Drosophila LIM-only is a positive regulator of transcription during thoracic bristle development

The Drosophila LIM-only (Lmo) protein DLMO functions as a negative regulator of transcription during development of the fly wing. This study reports a novel role of Dlmo as a positive regulator of transcription during the development of thoracic sensory bristles. New dlmo mutants, which lack some thoracic dorsocentral (DC) bristles, were isolated. This phenotype is typical of malfunction of a thoracic multiprotein transcription complex, composed of Chip, Pannier (Pnr), Achaete (Ac), and Daughterless (Da). Genetic interactions reveal that dlmo synergizes with pnr and ac to promote the development of thoracic DC bristles. Moreover, loss-of-function of dlmo reduces the expression of a reporter target gene of this complex in vivo. Using the GAL4-UAS system it was also shown that dlmo is spatially expressed where this complex is known to be active. Glutathione-S-transferase (GST)-pulldown assays showed that Dlmo can physically bind Chip and Pnr through either of the two LIM domains of Dlmo, suggesting that Dlmo might function as part of this transcription complex in vivo. It is proposed that Dlmo exerts its positive effect on DC bristle development by serving as a bridging molecule between components of the thoracic transcription complex (Zenvirt, 2008).

The results presented in this study uncover a novel role of Dlmo in regulation of the development of the thoracic DC bristles. Homozygous, or hemizygous, loss-of-function (dlmohdp) mutants lack the anterior pair of the DC bristles. Moreover, these dlmo mutants displayed genetic interactions with mutants in genes known to regulate DC bristle development, such as pnr and ac, to reduce the number of DC bristles. Consistently, overexpression alleles of dlmo (dlmoBx) also exhibited genetic interactions with these pnr and ac mutants, resulting in an increased number of bristles. In addition, the finding that overexpression of pnr under the regulation of dlmo-GAL4 affects DC bristle development suggests that dlmo is expressed in the region of the wing disc that gives rise to these bristles (Zenvirt, 2008).

These results suggest a role of Dlmo in positive regulation of transcription. The negative role of Dlmo in modulation of transcription during Drosophila wing development has been well documented. The findings indicate that in another context, namely in regulation of DC bristle development by the Chip, Pnr, Ac and Da (CPAD) complex, Dlmo has another role, as a positive regulator of transcription. Lowering the level of Dlmo (in dlmohdp mutants) results in a reduction in the expression of a reporter driven by regulatory sequences of a bona fide target gene of the CPAD transcription complex, suggesting that Dlmo is a positive regulator of CPAD-dependent transcription. While the mechanism by which Dlmo positively regulates transcription in the context of the CPAD complex remains to be elucidated, a first clue to this mechanism may lie in the finding that Dlmo can bind constituent proteins of this complex, including Pnr and Chip, in vitro. Should these interactions also take place in vivo, Dlmo may exert its positive role in transcriptional regulation as a component of the CPAD complex (Zenvirt, 2008).

Insights into the mechanism of positive transcriptional regulation by Dlmo can be gleaned from LMO2, one of the mammalian homologs of Dlmo. LMO2 was demonstrated to participate in a multiprotein transcription complex that contains Ldb1, a GATA factor (GATA-1 or GATA-2), and the bHLH transcription factors TAL1 and E2A, which are homologous, respectively, to the fly components of the CPAD complex, Chip, Pannier, Achaete, and Daughterless. Various lines of evidence indicate that in mammals LMO2 serves as a bridge between components of the complex, and silencing of LMO2 causes disruption of the complex and decreases in the activation of transcription of its target genes, just as does silencing of Ldb1 or Tal1. Similarly to LMO2, Dlmo might serve as a bridge between components of the CPAD complex. LIM domains are protein-interaction modules and could serve Dlmo to bind components of the CPAD complex. This suggestion is supported by the finding that each single LIM domain of Dlmo is capable of binding components of the CPAD complex in vitro, and it agrees with similar reports on other LIM-containing proteins. Notably, a single LIM domain from LMO2 and LMO4 is sufficient to interact with Ldb1 or the related protein CLIM-1a. However, both LIM domains are required for the highest-affinity interactions (Zenvirt, 2008).

This proposed mode of action of Dlmo, as a bridging molecule, which binds a different protein through each one of its LIM domains, predicts that a Dlmo molecule with one defective LIM domain and one intact LIM domain would bind only one protein at a time and not be able to bridge between molecules. Indeed, in the new dlmo mutants, it was found that deletions that span the second zinc finger of the second LIM domain of Dlmo, namely dlmohdp48-1 and dlmohdp185-1, resulted in dlmo loss-of-function mutations. These mutants display partial loss of thoracic DC bristles along with reduced expression of a target gene of the thoracic transcription complex. Interestingly, the wing size of these mutants is normal, unlike the small wings of mutants with lesions in the 5'-UTR of Dlmo, such as dlmohdp58-1, dlmohdp67-2, and dlmohdpR590. This may suggest that the defective Dlmo protein, which has only a single intact LIM domain, is sufficient for its function in the context of the wing, where Dlmo acts as a negative regulator that binds only one protein (CHIP), but is not sufficient when Dlmo acts as a bridging molecule in the thoracic CPAD transcription complex. Finally, the finding that Dlmo can bind other Dlmo molecules to generate homodimers or multimers might provide Dlmo with a greater flexibility of bridging between distant components of the complex. This possibility remains to be examined (Zenvirt, 2008).

In conclusion, Dlmo appears to have a dual role in regulation of transcription, depending on the context. Such a phenomenon has been documented for other transcription cofactors, whose dual function in transcription regulation varies according to their binding partners, the specific tissue, or the developmental stage. Likewise, these results indicate Dlmo has such a dual role, being a negative regulator with respect to the Ap-Chip complex and a positive regulator in the context of the CPAD complex (Zenvirt, 2008).

Beadex function in the motor neurons is essential for female reproduction in Drosophila melanogaster

Drosophila melanogaster has served as an excellent model system for understanding the neuronal circuits and molecular mechanisms regulating complex behaviors. The Drosophila female reproductive circuits, in particular, are well studied and can be used as a tool to understand the role of novel genes in neuronal function in general and female reproduction in particular. In the present study, the role of Beadex, a transcription co-activator, in Drosophila female reproduction was assessed by generation of mutant and knock down studies. Null allele of Beadex was generated by transposase induced excision of P-element present within an intron of Beadex gene. The mutant showed highly compromised reproductive abilities as evaluated by reduced fecundity and fertility, abnormal oviposition and more importantly, the failure of sperm release from storage organs. However, no defect was found in the overall ovariole development. Tissue specific, targeted knock down of Beadex indicated that its function in neurons is important for efficient female reproduction, since its neuronal knock down led to compromised female reproductive abilities, similar to Beadex null females. Further, different neuronal class specific knock down studies revealed that Beadex function is required in motor neurons for normal fecundity and fertility of females. Thus, the present study attributes a novel and essential role for Beadex in female reproduction through neurons (Kairamkonda, 2014: PubMed).


Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL (related protein, Drosophila Helix loop helix protein 3B) and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. The Xenopus homolog of LMO-2 has been cloned and it has been shown to be expressed in a pattern similar to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis (Mead, 2001).

Lmo1, Lmo2, and Lmo3 show individually unique but partially overlapping patterns of expression in several regions of the adult mouse forebrain, including hippocampus, caudate putamen, medial habenula, thalamus, amygdala, olfactory bulb, hypothalamus, and cerebral cortex. In the hippocampal formation, Lmo1, Lmo2, and Lmo3 show different combinatorial patterns of expression levels in CA pyramidal and dentate granule neurons, and Lmo1 is present in topographically restricted subpopulations of astrocytes. Kainic acid-induced limbic seizures differentially regulate Lmo1, Lmo2, and Lmo3 mRNA levels in hippocampal pyramidal and granule neurons, such that Lmo1 mRNA increases, whereas Lmo2 and Lmo3 mRNAs decrease significantly, with maximal changes at 6 hr after seizure onset and a return to baseline by 24 hr. These findings show that Lmo1, Lmo2, and Lmo3 continue to be expressed in the adult mammalian CNS in a cell type-specific manner, are differentially regulated by neuronal activity, and may thus be involved in cell phenotype-specific regulatory functions (Hinks, 1997).

LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors. LMO1, LMO2, and LMO4 have distinct expression patterns in adult tissue, and nuclear retention of LMO proteins is enhanced by the nuclear LIM interactor (NLI). In situ hybridization to early mouse embryos of 8-14.5 days reveals a complex pattern of LMO4 expression spatially overlapping with NLI and LHX genes. LMO4 expression in somites is repressed in mice mutant for the segment polarity gene Mesp2 (related to neurogenic HLH transcription factors) and expanded in Splotch (see Drosophila Paired) mutants. During jaw and limb outgrowth, LMO4 and LMO2 expression defines mesenchyme that is uncommitted to regional fates. Although both LMO2 and LMO4 are activated in thymic blast cells, only LMO4 is expressed in mature T cells. Mesenchymal and thymic blast cell expression patterns of LMO4 and LMO2 are consistent with the suggestion that LMO genes inhibit differentiation (Kenny, 1998).

Many vertebrate homologs of Drosophila genes important for wing patterning have been found to play a role in limb development. To determine whether the LMO genes might also be involved, in situ hybridization was performed using the mouse LMO-2 cDNA on E10.5 mouse embryos. LMO-2 RNA is present in the developing limb bud at E10.5. Expression is seen in a band centered on the D-V boundary of the developing limb bud. In situ hybridization to sections of the limb bud reveals that LMO-2 mRNA is present in a broad field of the mesenchyme underlying the apical ectodermal ridge, a structure known to be an important organizing structure of the limb bud. Expression is also seen at the somite boundaries (Zeng, 1998).

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. A widely expressed protein, Ldb1 (Drosophila homolog: Chip), has been identified whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibits cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibits erythroid differentiation. These studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state (Visvader, 1997).

The product of the scl (also called tal-1 or TCL5) gene is a basic domain, helix-loop-helix (bHLH) transcription factor required for the development of hematopoietic cells. Additionally, scl gene disruption and dysregulation, by either chromosomal translocations or a site-specific interstitial deletion whereby 5' regulatory elements of the sil gene become juxtaposed to the body of the scl gene, is associated with T-cell acute lymphoblastic leukemia (ALL) and T-cell lymphoblastic lymphoma. An inappropriately expressed scl protein, driven by sil regulatory elements, can cause aggressive T-cell malignancies in collaboration with a misexpressed LMO1 protein, thus recapitulating the situation seen in a subset of human T-cell ALL. Inappropriately expressed scl can interfere with the development of other tissues derived from mesoderm. An scl construct lacking the scl transactivation domain collaborates with misexpressed LMO1, demonstrating that the scl transactivation domain is dispensable for oncogenesis, and supporting the hypothesis that the scl gene product exerts its oncogenic action through a dominant-negative mechanism (Aplan, 1997).

The LIM-finger protein Lmo2, which is activated in T cell leukemias by chromosomal translocations, is required for yolk sac erythropoiesis. Because Lmo2 null mutant mice die at embryonic day 9-10, it prevents an assessment of a role in other stages of hematopoiesis. The hematopoietic contribution of homozygous mutant Lmo2 -/- mouse embryonic stem cells has been studied and Lmo2 -/- cells are found not to contribute to any hematopoietic lineage in adult chimeric mice, but reintroduction of an Lmo2-expression vector rescues the ability of Lmo2 null embryonic stem cells to contribute to all lineages tested. This disruption of hematopoiesis probably occurs because interaction of Lmo2 protein with factors such as Tal1/Scl is precluded. Thus, Lmo2 is necessary for early stages of hematopoiesis, and the Lmo2 master gene encodes a protein that has a central and crucial role in the hematopoietic development (Yamada, 1998).

The LIM-only protein LMO2 is expressed aberrantly in acute T-cell leukemias as a result of the chromosomal translocations t(11;14) (p13;q11) or t(7;11) (q35;p13). In a transgenic model of tumorigenesis by Lmo2, T-cell acute leukemias arise after an asymptomatic phase in which an accumulation of immature CD4(-) CD8(-) double negative thymocytes occurs. Possible molecular mechanisms underlying these effects have been investigated in T cells from Lmo2 transgenic mice. Isolation of DNA-binding sites by CASTing and band shift assays demonstrates the presence of an oligomeric complex involving Lmo2 that can bind to a bipartite DNA motif comprising two E-box sequences approximately 10 bp apart, which is distinct from that found in erythroid cells. This complex occurs in T-cell tumors and is restricted to the immature CD4(- )CD8(-) thymocyte subset in asymptomatic transgenic mice. Thus, ectopic expression of Lmo2 by transgenesis, or by chromosomal translocations in humans, may result in the aberrant protein interactions causing abnormal regulation of gene expression, resulting in a blockage of T-cell differentiation and providing precursor cells for overt tumour formation (Grutz, 1998).

The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukemias, is normally expressed in hematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the interaction is unexplained. In erythroid cells Lmo2 forms a novel DNA-binding complex with GATA-1, TAL1, E2A, and the recently identified LIM-binding protein, Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box (CAGGTG), followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex, which may play a role in hematopoiesis (Wadman, 1997).

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, the conserved LIM-interaction domain of Clim coregulators was used to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and a factor was isolated that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in an overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, LMO-4 was used as a bait to screen for such DNA-binding proteins in epidermis. Identified was the mouse homolog of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain (Sugihara, 1998).

It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc) was analysed, with a particular emphasis on intraembryonic hemogenic sites. Both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. Lmo2 mRNA is observed in all the sites endowed with a hemopoietic potential, where its expression is tightly regulated spatiotemporally. The rapid modifications of Lmo2 expression patterns suggest that it allocates specific combinations of transcription factors during key points of development. The overlapping expressions of Lmo2 and GATA-3 suggests combined functions during specific steps of definitive hemopoietic development, namely: (1) endodermal induction leading to the emergence of hemopoietic precursors in the mesoderm; (2) determination of these cells from the mesoderm, and (3) their production in the aortic region from 9 to 12 dpc as well as their release into the blood stream. Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase that determines intraembryonic precursors. A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonization. Furthermore, a strong GATA-3 signal allowed the uncovering of previously unreported mesodermal aggregates beneath the aorta. Combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation (Manaia, 2000).

In the stretch-reflex system, proprioceptive sensory neurons make selective synaptic connections with different subsets of motoneurons, according to the peripheral muscles they supply. To examine the molecular mechanisms that may influence the selection of these synaptic targets, single-cell cDNA libraries were constructed from sensory neurons that innervate antagonist muscles. Differential screening of these libraries identified a transcription regulatory co-factor of the LIM homeodomain proteins, the LIM domain only 4 protein Lmo4, expressed in most adductor but few sartorius sensory neurons. Differential patterns of Lmo4 expression were also seen in sensory neurons supplying three other muscles. A subset of motoneurons also expresses Lmo4 but the pattern of expression is not specific for motor pools. Differential expression of Lmo4 occurs early, as neurons develop their characteristic LIM homeodomain protein expression patterns. Moreover, ablation of limb buds does not block Lmo4 expression, suggesting that an intrinsic program controls the early differential expression of Lmo4. LIM homeodomain proteins are known to regulate several aspects of sensory and motor neuronal development. The results suggest that Lmo4 may participate in this differentiation by regulating the transcriptional activity of LIM homeodomain proteins (Chen, 2002).

Lmo4 can regulate the transcriptional activities of LIM homeodomain factors in several ways. Lmo transcriptional regulatory factors lack a DNA binding domain but contain two protein-protein interaction LIM domains. Lmo proteins can compete for NLI with LIM homeodomain transcription factors, and thereby regulate the formation of LIM homeodomain/NLI complexes and their transcriptional activity. Drosophila Lmo can bind to Chip with higher affinity than the LIM homeodomain of Apterous and thereby regulate Apterous activity levels in vivo. Whether there is a differential affinity to NLI between Lmo4 and other LIM homeodomain proteins is not yet known (Chen, 2002).

Lmo4 may also compete for other co-factors besides NLI that are specific for individual LIM homeodomain proteins and could thus regulate the expression of downstream target genes. For example, data based on the expression of chimeric LIM domains derived from different Islet family members (i.e. Isl1, Isl2 and ISL3) in zebrafish, has led to the conclusion that Isl2 probably forms a transcriptional complex with an Isl2-specific co-factor, in addition to NLI. Interaction with an Isl2-specific co-factor could contribute to the role of Isl2 in the differentiation of primary motoneurons, neuronal positioning, peripheral axonal outgrowth and neuronal transmitter expression in zebrafish (Chen, 2002).

Combinatorial interactions of Lmo4 with other transcription factors might provide additional mechanisms for the regulation of transcription during neuronal development. In enkaphalin-producing neurons, Lmo4 interacts with the transcription factor DEAF1 (deformed epidermal autoregulatory factor 1). DEAF1 has been implicated in opioid production by regulating enkaphalin transcription through a retinoic acid-responsive element. Interestingly, in the fetal and adult mouse brain, Lmo4 expression is region specific: high levels of expression are present in the limbic system and in regions involved in autonomic, motor and neuroendocrine regulation. Recently, studies in breast cancer cell lines have demonstrated that Lmo4 expression is upregulated and forms a multiprotein complex with CtIP and BRCA1. A role for BRCA1 in neurons has not been explored (Chen, 2002).

Different LIM homeodomain proteins are known to activate different downstream target genes. The pattern of neuronal generation in the ventral neural tube is achieved primarily by the spatially restricted expression of transcriptional repressors. By modulating the transcriptional activity of LIM homeodomain proteins, Lmo4 is likely to be involved in the specification of motor neuronal identity. Its restricted expression in subsets of muscle sensory neurons suggests that it contributes to the specification of sensory neurons as well (Chen, 2002).

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activate smooth muscle gene targets. CRP2 is found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocks proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converts pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibits the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation (Chang, 2003).

LMO4 functions as a co-activator of neurogenin 2 in the developing cortex

The proneural protein neurogenin 2 (NGN2) is a key transcription factor in regulating both neurogenesis and neuronal radial migration in the embryonic cerebral cortex. However, the co-factors that support the action of NGN2 in the cortex remain unclear. This study shows that the LIM-only protein LMO4 functions as a novel co-factor of NGN2 in the developing cortex. LMO4 and its binding partner nuclear LIM interactor (NLI/LDB1/CLIM2) interact with NGN2 simultaneously, forming a multi-protein transcription complex. This complex is recruited to the E-box containing enhancers of NGN2-target genes, which regulate various aspects of cortical development, and activates NGN2-mediated transcription. Correspondingly, analysis of Lmo4-null embryos shows that the loss of LMO4 leads to impairments of neuronal differentiation in the cortex. In addition, expression of LMO4 facilitates NGN2-mediated radial migration of cortical neurons in the embryonic cortex. These results indicate that LMO4 promotes the acquisition of cortical neuronal identities by forming a complex with NGN2 and subsequently activating NGN2-dependent gene expression (Asprer, 2011).

The LIM domain protein LMO4 interacts with the cofactor CtIP and the tumor suppressor BRCA1 and inhibits BRCA1 activity

LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators that appear to function as molecular adaptors for protein-protein interactions. Expression of the LMO4 gene is developmentally regulated in the mammary gland and is up-regulated in primary breast cancers. Using LMO4 in a yeast two-hybrid screen, the cofactor CtIP was identified as an LMO4-binding protein. Interaction with CtIP appeared to be specific for the LMO subclass of LIM domain proteins and could be mediated by a single LIM motif of LMO4. The breast tumor suppressor BRCA1 was identified as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediate interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP have no effect on the association of BRCA1 with LMO4. A stable complex comprising LMO4, BRCA1, and CtIP was demonstrated in vivo. The LIM domain binding-protein Ldb1 also participates in this multiprotein complex. In functional assays, LMO4 was shown to repress BRCA1-mediated transcriptional activation in both yeast and mammalian cells. These findings reveal a novel complex between BRCA1, LMO4, and CtIP and indicate a role for LMO4 as a repressor of BRCA1 activity in breast tissue (Sum, 2002).

Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1

The LIM domain protein Lmo2 and the basic helix-loop-helix transcription factor Scl/Tal1 (distantly related to Drosophila Twist) are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-of-function studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. This restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralizing or posteriorizing the embryo. While no ectopic myeloid development from the Scl/Tal1-Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2-induced haemangioblasts differentiate into endothelial cells (Gering, 2003).

Structural basis for the recognition of ldb1 by the N-terminal LIM domains of LMO2 and LMO4

LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1-LID). This study reports the solution structures of two LMO:ldb1 complexes (PDB: 1M3V and 1J2O) and shows that ldb1-LID binds to the N-terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a beta-hairpin in LIM1 domains. These findings constitute the first molecular definition of LIM-mediated protein-protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology (Deane, 2003).

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex

Nuclear LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain-binding protein-1 (Ldb1). A high-resolution X-ray crystal structure of LMO4, a putative breast oncoprotein, has been determined in complex with Ldb1-LID, providing the first example of a tandem LIM:Ldb1-LID complex and the first structure of a type-B LIM domain. The complex possesses a highly modular structure with Ldb1-LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone-backbone hydrogen bonds. A mutagenic screen of Ldb1-LID, assessed by yeast two-hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM-HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains (Deane, 2004).

Identification of the key LMO2-binding determinants on Ldb1

The overexpression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations, retroviral insertion during gene therapy, or in transgenic mice models, leads to the onset of T-cell leukemias. LMO2 comprises two protein-binding LIM domains that allow LMO2 to interact with multiple protein partners, including LIM domain-binding protein 1 (Ldb1, also known as CLIM2 and NLI), an essential cofactor for LMO proteins. Sequestration of Ldb1 by LMO2 in T-cells may prevent it binding other key partners, such as LMO4. This study shows using protein engineering and enzyme-linked immunosorbent assay (ELISA) methodologies that LMO2 binds Ldb1 with a twofold lower affinity than does LMO4. Thus, excess LMO2 rather than an intrinsically higher binding affinity would lead to sequestration of Ldb1. Both LIM domains of LMO2 are required for high-affinity binding to Ldb1. However, the first LIM domain of LMO2 is primarily responsible for binding to Ldb1, whereas the second LIM domain increases binding by an order of magnitude. Mutagenesis was used in combination with yeast two-hybrid analysis, and phage display selection to identify LMO2-binding 'hot spots' within Ldb1 that locate to the LIM1-binding region. The delineation of this region reveals some specific differences when compared to the equivalent LMO4:Ldb1 interaction that hold promise for the development of reagents to specifically bind LMO2 in the treatment of leukemia (Ryan, 2006).

Role of LDB1 in the transition from chromatin looping to transcription activation

Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1; see Drosophila Chip) protein is recruited to the beta-globin locus via LMO2 and is required for looping of the beta-globin locus control region (LCR) to the active beta-globin promoter. This study shows that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2, sufficient to completely restore LCR-promoter looping and transcription in LDB1-depleted cells. The looping function of the DD is unique and irreplaceable by heterologous DDs. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the coregulators FOG1 (U-shaped in Drosophila) and the nucleosome remodeling and deacetylating (NuRD) complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior, as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1 in these processes (Krivega, 2014).

SCL assembles a multifactorial complex that determines glycophorin A expression

SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. This study identified the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and shows that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, these observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression (Lahlil, 2004).

Defective neural tube closure and anteroposterior patterning in mice lacking the LIM protein LMO4 or its interacting partner Deaf-1

LMO4 belongs to a family of transcriptional regulators that comprises two zinc-binding LIM domains. LIM-only (LMO) proteins appear to function as docking sites for other factors, leading to the assembly of multiprotein complexes. The transcription factor Deaf-1/NUDR (see Drosophila Deaf-1) has been identified as one partner protein of LMO4. The Lmo4 and Deaf-1 genes were inactivated in mice to define their biological function in vivo. All Lmo4 mutants died shortly after birth and showed defects within the presphenoid bone, with 50% of mice also exhibiting exencephaly. Homeotic transformations were observed in Lmo4-null embryos and newborn mice, but with incomplete penetrance. These included skeletal defects in cervical vertebrae and the rib cage. Furthermore, fusions of cranial nerves IX and X and defects in cranial nerve V were apparent in some Lmo4(-/-) and Lmo4(+/-) mice. Remarkably, Deaf-1 mutants displayed phenotypic abnormalities similar to those observed in Lmo4 mutants. These included exencephaly, transformation of cervical segments, and rib cage abnormalities. In contrast to Lmo4 nullizygous mice, nonexencephalic Deaf-1 mutants remained healthy. No defects in the sphenoid bone or cranial nerves were apparent. Thus, Lmo4 and Deaf-1 mutant mice exhibit overlapping as well as distinct phenotypes. These data indicate an important role for these two transcriptional regulators in pathways affecting neural tube closure and skeletal patterning, most likely reflecting their presence in a functional complex in vivo (Hahm, 2004).

Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. Gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, was carried out to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, the effect of compound Lmo1/Lmo3 null mutations was assessed. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. These results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways (Tse, 2004).

The LIM domain-only protein LMO4 is required for neural tube closure

Nuclear LIM domain-only proteins (LMOs), which consist of two closely spaced 50 amino acid Zn2+-finger protein interaction modules mediate interactions between several classes of transcription factors important for development. LMO2 is necessary for development of the entire hematopoietic system and overexpression of LMO1 or LMO2 results in human acute T cell leukemia. LMO4 is the most widely expressed LMO but its normal function is unknown. During development, LMO4 is expressed in dividing neuroepithelial cells within the ventricular zone along the entire rostrocaudal axis of the nervous system. In telencephalic and spinal cord regions of the CNS, LMO4 is highly expressed in ventral but is low in dorsal proliferating neuroepithelial cells. To understand the role of LMO4 during mouse development, a homozygous null mutation was generated in the gene. It was found that LMO4 is required for proper closure of the anterior neural tube. In the absence of LMO4, elevation, bending, and proliferation of the ventral neural epithelium and consequent fusion of the prospective dorsal ends of the neural tube do not occur. LMO4 mutant mice die embryonically and exhibit exencephaly, which is associated with abnormal patterns of cell proliferation and with high levels of apoptotic cell death within the neuroepithelium. LMO4 is thus essential for normal patterns of proliferation and for survival of neural epithelial cells in the rostral neural tube. LMO4 is also expressed in Schwann cell progenitors after these contact neurites, a process mediated in part by neuregulin (Lee, 2005).

LMO3 interacts with neuronal transcription factor, HEN2, and acts as an oncogene in neuroblastoma

LIM-only proteins (LMO), which consist of LMO1, LMO2, LMO3, and LMO4, are involved in cell fate determination and differentiation during embryonic development. Accumulating evidence suggests that LMO1 and LMO2 act as oncogenic proteins in T-cell acute lymphoblastic leukemia, whereas LMO4 has recently been implicated in the genesis of breast cancer. However, little is known about the role of LMO3 in either tumorigenesis or development. This study identified LMO3 and HEN2, which encodes a neuronal basic helix-loop-helix protein, as genes whose expression levels were higher in unfavorable neuroblastomas compared with those of favorable tumors. Immunoprecipitation and immunostaining experiments showed that LMO3 was associated with HEN2 in mammalian cell nucleus. Human neuroblastoma SH-SY5Y cells stably overexpressing LMO3 showed a marked increase in cell growth, a promotion of colony formation in soft agar medium, and a rapid tumor growth in nude mice compared with the control transfectants. More importantly, the increased expression of LMO3 and HEN2 was significantly associated with a poor prognosis in 87 primary neuroblastomas. These results suggest that the deregulated expression of neuronal-specific LMO3 and HEN2 contributes to the genesis and progression of human neuroblastoma in a lineage-specific manner (Aoyama, 2005).

The Grainyhead-like epithelial transactivator Get-1/Grhl3 regulates epidermal terminal differentiation and interacts functionally with LMO4

Defective permeability barrier is an important feature of many skin diseases and causes mortality in premature infants. To investigate the control of barrier formation, this study characterized the epidermally expressed Grainyhead-like epithelial transactivator (Get-1)/Grhl3, a conserved mammalian homologue of Grainyhead, which plays important roles in cuticle development in Drosophila. Get-1 interacts with the LIM-only protein LMO4, which is co-expressed in the developing mammalian epidermis. The epidermis of Get-1(-/-) mice showed a severe barrier function defect associated with impaired differentiation of the epidermis, including defects of the stratum corneum, extracellular lipid composition and cell adhesion in the granular layer. The Get-1 mutation affects multiple genes linked to terminal differentiation and barrier function, including most genes of the epidermal differentiation complex. Get-1 therefore directly or indirectly regulates a broad array of epidermal differentiation genes encoding structural proteins, lipid metabolizing enzymes and cell adhesion molecules. Although deletion of the LMO4 gene had no overt consequences for epidermal development, the epidermal terminal differentiation defect in mice deleted for both Get-1 and LMO4 is much more severe than in Get-1(-/-) mice with striking impairment of stratum corneum formation. These findings indicate that the Get-1 and LMO4 genes interact functionally to regulate epidermal terminal differentiation (Yu, 2006).

TAL-1/SCL and its partners E47 and LMO2 up-regulate VE-cadherin expression in endothelial cells

The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. This study investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. It was found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis (Deleuze, 2007).

The Lim-only protein LMO2 acts as a positive regulator of erythroid differentiation

LMO2, a member of the LIM-only protein family, is essential for the regulation of hematopoietic stem cells and formation of erythroid cells. It is found in a transcriptional complex comprising LMO2, TAL1, E47, GATA-1, and LDB1 which regulates erythroid genes. While TAL1 has been shown to induce erythroid differentiation, LMO2 appears to suppress fetal erythropoiesis. In addition to LMO2, the closely related LMO4 gene is expressed in hematopoietic cells, but has unknown functions. This study demonstrates that LMO2 and LMO4 are expressed at the same level in erythroid colonies from mouse bone marrow, implying a function in erythroid differentiation. However, while LMO2 induced erythroid differentiation, LMO4 had no such effect. Interestingly, both LMO2 and TAL1 were able to partially suppress myeloid differentiation, implying that they activate erythroid differentiation in uncommitted bone marrow progenitors. Both LMO2 and LMO4 interacted strongly to LDB1, which was required for their localization to the nucleus (Hansson, 2007).

LMO4 controls the balance between excitatory and inhibitory spinal V2 interneurons

Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. This study provides a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with bHLH factor SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and LIM domain-binding protein NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord (Joshi, 2009).

LIM-HD codes are crucial in implementing cell-type-specific transcription by directing different types of LIM-complexes in a cell context-dependent manner. These studies expand the LIM codes to include bHLH and Gata proteins as these two factors form an atypical LIM-complex via a non-DNA binding LIM factor LMO4. Unlike typical LIM-complexes such as the V2-tetramer complex, which utilize LIM-HD proteins for recognition of specific DNA response elements (Lee, 2008), SCL and Gata2 serve as the major DNA-binding components in the V2b complex. A couple of unique advantages of assembling the V2b complex can be proposed in cell fate specification (Joshi, 2009).

First, these results suggest that the V2b complex allows integration of SCL and Gata2 functions by selecting a group of target genes that bear both SCL- and GATA-recognition sites. This should ensure the expression of V2b-target genes specifically in cells coexpressing SCL and Gata2. It was found that the enhancers of Gata2 and Gata3 genes display striking similarity in that they contain reiterated bipartite elements composed of E-box (CAnnTG) and/or atypical E-box (CAnnnTG) for SCL-binding and GATA sites for recruiting Gata proteins. E-boxes and GATA sites occur relatively often in the genome due to their short sequences and serve as binding motifs for multiple bHLH and Gata factors. Thus, simultaneous recognition of paired E-box-GATA composite elements by the V2b complex is expected to provide the required stringency in choosing the target genes coregulated by SCL and Gata2 (Joshi, 2009).

Second, this study found that formation of the V2b complex facilitates the transcriptional synergy among its components by enabling the recruitment of coactivators including SSDP1. Coexpression of SSDP1 allowed a potent transcriptional activation by the V2b complex on its physiological targets, Gata2/3-enhancers. Given that SCL and Gata2 are relatively weak transcriptional activators in Gal4-DBD fusion transcription assays, the transcriptional synergy between SCL and Gata2 resulting from forming a complex may be due, at least in part, to the recruitment of SSDP1. The facilitated recruitment of SSDP1 and possibly other coactivators may account for the necessity of the V2b complex formation for inducing the V2b-IN genes (Joshi, 2009).

Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes

Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. This study shows that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrβ clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes (Tremblay, 2010).


Search PubMed for articles about Drosophila Beadex

Aoyama, M., et al. (2005). LMO3 interacts with neuronal transcription factor, HEN2, and acts as an oncogene in neuroblastoma. Cancer Res. 65: 4587-4597. PubMed Citation: 15930276

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Bejarano, F., Smibert, P. and Lai, E. C. (2010). miR-9a prevents apoptosis during wing development by repressing Drosophila LIM-only. Dev Biol 338: 63-73. PubMed ID: 19944676

Biryukova, I., Asmar, J., Abdesselem, H. and Heitzler, P. (2009). Drosophila mir-9a regulates wing development via fine-tuning expression of the LIM only factor, dLMO. Dev. Biol. 327: 487-496. PubMed ID: 19162004

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Chang, D. F., et al. (2003). Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors. Dev. Cell 4: 107-118. 12530967

Chen, H. H., et al. (2002). Differential expression of a transcription regulatory factor, the LIM domain only 4 protein Lmo4, in muscle sensory neurons. Development 129: 4879-4889. 12397097

Chen, L., et al. (2002). Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development. Proc. Natl. Acad. Sci. 99: 14320-14325. PubMed Citation: 12381786

Deane, J. E., et al. (2003). Structural basis for the recognition of ldb1 by the N-terminal LIM domains of LMO2 and LMO4. EMBO J. 22: 2224-2233. PubMed Citation: 12727888

Deane, J. E., et al. (2004). Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex. EMBO J. 23: 3589-3598. PubMed Citation: 15343268

Deleuze, V., et al. (2007). TAL-1/SCL and its partners E47 and LMO2 up-regulate VE-cadherin expression in endothelial cells. Mol. Cell. Biol. 27: 2687-2697. PubMed Citation: 17242194

Gering, M., et al. (2003). Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1. Development 130: 6187-6199. 14602685

Grutz, G. G., et al. (1998). The oncogenic T cell LIM-protein lmo2 forms part of a DNA-binding complex specifically in immature T cells. EMBO J. 17(16): 4594-4605. PubMed Citation: 9707419

Hahm, K., et al. (2004). Defective neural tube closure and anteroposterior patterning in mice lacking the LIM protein LMO4 or its interacting partner Deaf-1. Mol. Cell. Biol. 24: 2074-2082. PubMed Citation: 14966286

Halder, R., Hennion, M., Vidal, R. O., Shomroni, O., Rahman, R. U., Rajput, A., Centeno, T. P., van Bebber, F., Capece, V., Garcia Vizcaino, J. C., Schuetz, A. L., Burkhardt, S., Benito, E., Navarro Sala, M., Javan, S. B., Haass, C., Schmid, B., Fischer, A. and Bonn, S. (2016). DNA methylation changes in plasticity genes accompany the formation and maintenance of memory. Nat Neurosci 19(1): 102-110. PubMed ID: 26656643

Hansson, A., et al. (2007). The Lim-only protein LMO2 acts as a positive regulator of erythroid differentiation. Biochem. Biophys. Res. Commun. 364: 675-681. PubMed Citation: 17964543

Hinks, G. L., et al. (1997). Expression of LIM protein genes Lmo1, Lmo2, and Lmo3 in adult mouse hippocampus and other forebrain regions: differential regulation by seizure activity. J. Neurosci. 17(14): 5549-5559. PubMed Citation: 9204936

Hirano, Y., Ihara, K., Masuda, T., Yamamoto, T., Iwata, I., Takahashi, A., Awata, H., Nakamura, N., Takakura, M., Suzuki, Y., Horiuchi, J., Okuno, H. and Saitoe, M. (2016). Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies. Nat Commun 7: 13471. PubMed ID: 27841260

Joshi, K., Lee, S., Lee, B., Lee, J. W. and Lee, S. K. (2009). LMO4 controls the balance between excitatory and inhibitory spinal V2 interneurons. Neuron 61(6): 839-51. PubMed Citation: 19323994

Kairamkonda, S. and Nongthomba, U. (2014). Beadex function in the motor neurons is essential for female reproduction in Drosophila melanogaster. PLoS One 9: e113003. PubMed ID: 25396431

Kenny, D. A., et al. (1998). Identification and characterization of LMO4, an LMO gene with a novel pattern of expression during embryogenesis. Proc. Natl. Acad. Sci. 95(19): 11257-11262. PubMed Citation: 9736723

Krivega, I., Dale, R. K. and Dean, A. (2014). Role of LDB1 in the transition from chromatin looping to transcription activation. Genes Dev 28: 1278-1290. PubMed ID: 24874989

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Lee, S. K., et al. (2005). The LIM domain-only protein LMO4 is required for neural tube closure. Mol. Cell. Neurosci. 28: 205-214. PubMed Citation: 15691703

Lee, S., et al. (2008). A regulatory network to segregate the identity of neuronal subtypes. Dev. Cell 14: 877-889. PubMed Citation: 18539116

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Lahlil, R., et al. (2004). SCL assembles a multifactorial complex that determines glycophorin A expression. Mol. Cell. Biol. 24: 1439-1452. PubMed Citation: 14749362

Manaia, A., et al. (2000). Lmo2 and GATA-3 associated expression in intraembryonic hemogenic sites. Development 127(3): 643-653. PubMed Citation: 10631184

Mattox, W. W. and Davidson, N. (1984). Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene. Mol. Cell. Biol. 4(7): 1343-1353. 85061239

Mead, P. E., et al. (2001). Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins. Development 128: 2301-2308. PubMed Citation: 11493549

Milan, M., Diaz-Benjumea, F. J. and Cohen, S. M. (1998). Beadex encodes an LMO protein that regulates Apterous LIM-homeodomain activity in Drosophila wing development: a model for LMO oncogene function. Genes Dev. 12(18): 2912-2920. PubMed Citation: 9744867

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Biological Overview

date revised: 30 December 2014

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