u-shaped : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - u-shaped
Cytological map position - 21C6--21C6
Function - transcription factor
Symbol - ush
FlyBase ID: FBgn0003963
Genetic map position - 2-0.1
Classification - zinc finger protein
Cellular location - nuclear
|Recent literature||Gao, H., Baldeosingh, R., Wu, X. and Fossett, N. (2016). The Friend of GATA transcriptional co-regulator, U-Shaped, is a downstream antagonist of Dorsal-driven prohemocyte differentiation in Drosophila. PLoS One 11: e0155372. PubMed ID: 27163255
Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, this study identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. It was shown that Dorsal is required to limit the size of the progenitor pool. Additionally, activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. The study also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. It was found that Dorsal represses Ush expression levels to promote differentiation, whereas Cactus maintains Ush levels to block differentiation. Additionally, another Toll antagonist, Lesswright, also maintains the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.
|Baldeosingh, R., Gao, H., Wu, X. and Fossett, N. (2018). Hedgehog signaling from the Posterior Signaling Center maintains U-shaped expression and a prohemocyte population in Drosophila. Dev Biol. PubMed ID: 29966604
The hematopoetic lymph gland is zonally arranged, with progenitors located in medullary zone, differentiating cells in the cortical zone, and the stem cell niche or Posterior Signaling Center (PSC) residing at the base of the medullary zone (MZ). This arrangement facilitates investigations into how signaling from the microenvironment controls progenitor choice. The Drosophila Friend of GATA transcriptional regulator, U-shaped, is a conserved hematopoietic regulator. To identify additional novel intrinsic and extrinsic regulators that interface with U-shaped to control hematopoiesis, this study conducted an in vivo screen for factors that genetically interact with u-shaped. Smoothened, a downstream effector of Hedgehog signaling, was one of the factors identified in the screen. This study reports studies that characterized the relationship between Smoothened and U-shaped. The PSC and Hedgehog signaling are required for U-shaped expression, and U-shaped is an important intrinsic progenitor regulator. These observations identify a potential link between the progenitor regulatory machinery and extrinsic signals from the PSC. Furthermore, both Hedgehog signaling and the PSC were shown to be required to maintain a subpopulation of progenitors. This led to a delineation of PSC-dependent versus PSC-independent progenitors and provided further evidence that the MZ progenitor population is heterogeneous. Overall, a connection has been identified between a conserved hematopoietic master regulator and a putative stem cell niche, which adds to understanding of how signals from the microenvironment regulate progenitor multipotency.
|Harrison, B. R., Wang, L., Gajda, E., Hoffman, E. V., Chung, B. Y., Pletcher, S. D., Raftery, D. and Promislow, D. E. L. (2020). The metabolome as a link in the genotype-phenotype map for peroxide resistance in the fruit fly, Drosophila melanogaster. BMC Genomics 21(1): 341. PubMed ID: 32366330
This study used metabolomics to explore the nature of genetic variation for hydrogen peroxide (H(2)O(2)) resistance in the sequenced inbred Drosophila Genetic Reference Panel (DGRP). First genetic variation was studied for H(2)O(2) resistance in 179 DGRP lines and along with identifying the insulin signaling modulator u-shaped and several regulators of feeding behavior, it is estimated that a substantial amount of phenotypic variation can be explained by a polygenic model of genetic variation. Then a portion of the aqueous metabolome was profiled in subsets of eight 'high resistance' lines and eight 'low resistance' lines. These lines were used to represent collections of genotypes that were either resistant or sensitive to the stressor, effectively modeling a discrete trait. Across the range of genotypes in both populations, flies exhibited surprising consistency in their metabolomic signature of resistance. Importantly, the resistance phenotype of these flies was more easily distinguished by their metabolome profiles than by their genotypes. Furthermore, a metabolic response was found to H(2)O(2) in sensitive, but not in resistant genotypes. Metabolomic data further implicated at least two pathways, glycogen and folate metabolism, as determinants of sensitivity to H(2)O(2). A confounding effect was found of feeding behavior on assays involving supplemented food. This work suggests that the metabolome can be a point of convergence for genetic variation influencing complex traits, and can efficiently elucidate mechanisms underlying trait variation.
|Buchberger, E., Bilen, A., Ayaz, S., Salamanca, D., Matas de Las Heras, C., Niksic, A., Almudi, I., Torres-Oliva, M., Casares, F. and Posnien, N. (2021). Variation in pleiotropic hub gene expression is associated with interspecific differences in head shape and eye size in Drosophila. Mol Biol Evol. PubMed ID: 33386848
Revealing the mechanisms underlying the breath-taking morphological diversity observed in nature is a major challenge in Biology. It has been established that recurrent mutations in hotspot genes cause the repeated evolution of morphological traits, such as body pigmentation or the gain and loss of structures. To date, however, it remains elusive whether hotspot genes contribute to natural variation in the size and shape of organs. Since natural variation in head morphology is pervasive in Drosophila, the molecular and developmental basis of differences in compound eye size and head shape was studied in two closely related Drosophila species. Differences were shown in the progression of retinal differentiation between species, and comparative transcriptomics and chromatin accessibility data were applied to identify the GATA transcription factor Pannier (Pnr) as central factor associated with these differences. Although the genetic manipulation of Pnr affected multiple aspects of dorsal head development, the effect of natural variation is restricted to a subset of the phenotypic space. Data is presented suggesting that this developmental constraint is caused by the co-evolution of expression of pnr and its co-factor u-shaped (ush). It is proposed that natural variation in expression or function of highly connected developmental regulators with pleiotropic functions is a major driver for morphological evolution, and implications on gene regulatory network evolution are discussed. In comparison to previous findings, the data strongly suggests that evolutionary hotspots are not the only contributors to the repeated evolution of eye size and head shape in Drosophila.
|Lenz, J., Liefke, R., Funk, J., Shoup, S., Nist, A., Stiewe, T., Schulz, R., Tokusumi, Y., Albert, L., Raifer, H., Forstemann, K., Vazquez, O., Tokusumi, T., Fossett, N. and Brehm, A. (2021). Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis. PLoS Genet 17(2): e1009318. PubMed ID: 33600407
U-shaped (Ush), a multi-zinc finger protein, maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches this study has revealed that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. Complementary biochemical approaches were employed to characterise the molecular mechanisms of Ush-mediated gene regulation. Distinct Ush isoforms were uncovered, one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex (see HDAC1) using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, genetic assays were used to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. These findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.
|Moussalem, D., Auge, B., Di Stefano, L., Osman, D., Gobert, V. and Haenlin, M. (2021). Two Isoforms of serpent Containing Either One or Two GATA Zinc Fingers Provide Functional Diversity During Drosophila Development. Front Cell Dev Biol 9: 795680. PubMed ID: 35178397
GATA transcription factors play crucial roles in various developmental processes in organisms ranging from flies to humans. In mammals, GATA factors are characterized by the presence of two highly conserved domains, the N-terminal (N-ZnF) and the C-terminal (C-ZnF) zinc fingers. The Drosophila GATA factor Serpent (Srp) is produced in different isoforms that contains either both N-ZnF and C-ZnF (SrpNC) or only the C-ZnF (SrpC). This study investigated the functional roles ensured by each of these isoforms during Drosophila development. Using the CRISPR/Cas9 technique, new mutant fly lines were generated deleted for one (ΔsrpNC) or the other (ΔsrpC) encoded isoform, and a third one with a single point mutation in the N-ZnF that alters its interaction with its cofactor, the Drosophila FOG homolog U-shaped (Ush). Analysis of these mutants revealed that the Srp zinc fingers are differentially required for Srp to fulfill its functions. While SrpC is essential for embryo to adult viability, SrpNC, which is the closest conserved isoform to that of vertebrates, is not. However, to ensure its specific functions in larval hematopoiesis and fertility, Srp requires the presence of both N- and C-ZnF (SrpNC) and interaction with its cofactor Ush. These results also reveal that in vivo the presence of N-ZnF restricts rather than extends the ability of GATA factors to regulate the repertoire of C-ZnF bound target genes.
Mutations in the gene u-shaped and the associated embryonic recessive lethal phenotype were first described by Nüsslein-Volhard in a characterization of mutations that affect the pattern of larval cuticle in Drosophila. In ush null embryos, the germ band fails to retract and lateral fusion occurs between anterior and posterior ectoderm. A large number of ushmutant alleles have been isolated; these include hypomorphic mutants that affect the pattern of bristles on the head and thorax (Cubadda, 1997 and references).
A number of dominant alleles for another gene, pannier, result in the development of extra thoracic bristles as well as an overexpression of achaete and scute. Mutations of ush have been isolated in a screen for second-site modifiers of the pannierD phenotype. Alleles of ush, causing a loss of function, act as dominant enhancers of pannierD heterozygotes, resulting in an increase in the number of ectopic thoracic bristles. This suggests that pannier and u-shaped might act in the same developmental pathway to regulate the achaete and scute genes. Such evidence would be of great interest, particularly because pannier's mammalian GATA homologs have been associated with neural development in only a very few studies. The discovery of a mammalian homolog of u-shaped, FOG, that acts as a co-factor for transcription factor GATA-1 in erythroid and magakaryotic differentiation, suggests a conservation of GATA protein interactions in both flies and humans (Cubadda, 1997 and references).
Evidence has been provided for a direct physical interaction between Pannier and U-shaped, explaining the observed genetic interaction. Usp antagonizes Pnr by physically interacting with the Pnr DNA-binding domain. The number of ectopic bristles in pannierD/+ flies increases in flies bearing only a single copy of u-shaped+ but decreases when three copies are present. Activation of a chicken alpha-globin promoter sequence by Pannier (via Pannier's GATA type DNA binding domain) in cultured cells is inhibited by Ush. When both Ush and wild-type Pnr are expressed simultaneously, promoter activation is abolished. Because Pnr and vertebrate globin regulating protein GATA-1 have no homology outside their GATA DNA-binding domain, and because Ush alone has no effect on globin promoter activity, these observations suggest that the function of Ush in inhibiting Panier's activation of globin is mediated through the GATA DNA-binding domain of Pnr (Haenlin, 1997).
Pnr and Ush heterodimerize through the amino-terminal zinc finger of Pnr; when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. In summary, these results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development (Haenlin, 1997).
In the developing Drosophila notum, wingless expression is regulated by positive and negative Decapentaplegic signaling so that only notal cells receiving optimal levels of Decapentaplegic signal express wingless. This Decapentaplegic-dependent regulation of notal wingless expression includes multiple mechanisms involving pannier and u-shaped. In the medial notum, Pannier and U-shaped form a complex. The expression of pannier and u-shaped is positively regulated by Decapentaplegic signals emanating from the dorsal-most region. The Pannier/U-shaped complex serves as a repressor and a transcriptional activator, respectively, for wingless and u-shaped expression. In the more lateral region, wingless expression is up-regulated by U-shaped-unbound Pannier. wingless expression is also weakly regulated by its own signaling (Sato, 2000).
achaete-scute (ac-sc) expression in the notum is affected in several allelic combinations of pnr, whose function is prevented by Ush as a result of direct binding to Pnr. Since pnr appears involved in notal wg expression, and pnr and ush are expressed in the future medial notum in a graded fashion with peaks within the dpp expressing dorsal-most region, the late third instar notum was examined by staining for wg protein and pnr or USH RNA. ush and wg expression areas were found to abut on each other except for the future scutellum, while almost all wg expressing cells were situated in a ventral-most region of the pnr expression domain. Although somewhat ambiguous, a similar relationship among wg, pnr and ush expression areas was detected in small discs at an early third instar stage, the earliest stage of notal wg expression. It may thus follow that wg expression occurs in lateral-notal cells expressing pnr but not ush throughout third instar larval notal development. wg-LacZ expression occurs in a region much broader than the authentic wg expression domain; wg-LacZ expression was always observed to be expanded medially or dorsally, suggesting that the authentic wg expression domain shifts ventrally as a disc grows (Sato, 2000).
To determine the role of pnr in wg expression, examination was made of wg expression on various pnr mutant backgrounds. Strong wg misexpression occurs in medial pnr-null-mutant (pnrVX6) clones. wg-LacZ or Wg signals are detected in the entire medial notum transheterozygous for pnrVX6 and pnrVI, from which most, if not all, pnr activity is absent. In contrast, a significant reduction of wg expression occurs in pnr-null-mutant (pnrVX6) clones generated within the authentic wg expression domain. These findings indicate that pnr is involved in both negative and positive regulation of notal wg expression; Pnr serves as a positive regulator of wg expression in the future lateral notum including the authentic wg expression domain, while it is a negative factor of wg expression in the medial notum. Consistent with this, ubiquitous or clonal expression of wild-type pnr induces wg misexpression in the notum ventral to the authentic wg expression domain, while no or little wg misexpression occurs in the future medial notum (Sato, 2000).
As in the case of ac-sc expression, ush appears to serve as a negative factor for notal wg expression, since (1) wg is misexpressed in ush-null-mutant (ush1) clones in the future medial notum and (2) virtually all endogenous wg expression is abolished when wild-type ush is overexpressed throughout the notal region. In contrast to medial ush1clones, no appreciable change in wg expression is detected in ush1clones generated within the authentic wg expression domain. That the authentic wg expression domain is demarcated by medial ush expression may indicate that medial ush expression is involved in the establishment of the dorsal boundary of the authentic wg expression domain. Based on the fact that Pnr mutants such as PnrD1 and PnrD4, lacking ability to bind to Ush, are still capable of activating ac-sc in the presence of ush activity, wild-type Pnr has been proposed to be inactivated by ush through direct interactions of Ush with Pnr. However, the results presented here show that this may not be the case in notal wg expression and ac-sc expression for the DC macrochaetae formation. If Ush serves only as the inhibitor of Pnr as predicted, a wild-type copy of pnr added in trans to PnrD1 would not decrease the area of wg expression, since wild-type Pnr is considered to either activate wg expression or neutralize the negative function of Ush or both. The results presented here are apparently at variance with this consideration. Both wg and ac-sc misexpression found in the future medial notum of Pnr14/PnrVI discs are abolished in PnrD1/+ discs with no loss of wg and ac-sc expression in the authentic wg expression domain. This negative effect of wild-type pnr is reversed by halving the copy number of endogenous ush. It is concluded that, in the medial notum, Pnr forms a complex with Ush and the resultant Pnr/Ush complex represses wg and ac-sc expression directly or indirectly to establish the dorsal boundaries of the authentic wg expression domain and the ac-sc expression area for the DC macrochaetae formation (Sato, 2000).
Notal wg expression is regulated not only by dpp signaling but also by Pnr and Ush. Thus, pnr and ush expression may be under the control of Dpp signaling or conversely, Dpp signaling is regulated by pnr and ush. The second possibility, however, seems to be unlikely, since neither pnr nor ush mutant clones exhibit any appreciable change in brinker (brk)-LacZ expression. brk is a general Dpp target gene whose expression is negatively regulated by Dpp signaling. Loss of Dpp signaling causes cell-autonomous brk misexpression in the wing pouch and notum of wing imaginal discs. To determine the feasibility of the first possibility, pnr and ush expression was examined in tkva12, Mad1-2 or tkvQ253D(tkvQD) clones; tkvQD is a constitutively active form of tkv. pnr and ush are misexpressed in lateral UAS-tkvQD clones generated in late second instar, an observation indicating that pnr and ush expression is under the control of Dpp signaling. Unlike wg expression, pnr and ush expression are abolished not only in early tkva12 clones but also in late tkva12 and early Mad1-2 clones, both expressing wg, suggesting that pnr and ush expression requires higher levels of Dpp-signaling activity than those required for wg expression. Loss of ush expression in tkva12 and Mad1-2 clones might be a secondary effect due to the loss of pnr expression, since the maintenance of ush expression requires both pnr and ush activities. pnr and ush expression may be independently initiated by Dpp signaling, since pnr expression normally occurs in ush mutant clones and no ush misexpression is induced by ubiquitous pnr expression. It is concluded that the graded expression of pnr and ush is determined by Dpp signaling and hence, Pnr and Ush act downstream of Dpp (Sato, 2000).
In the larval notal region, dpp expression is not continuous but is broken by the authentic wg expression domain, thus suggesting that notal development could be regulated by Dpp signals emanating separately from dorsal and ventral sources up to the wg expression domain. As anticipated, the expression of dad (dad-LacZ), a downstream component of Dpp signaling whose expression is positively regulated by Dpp signaling, is detected not only in medial but also in lateral notum. However, double-staining of dad-LacZ and either PNR or USH RNA expression shows that unlike dad-LacZ, pnr and ush are not induced in the postero-lateral notum in spite of the presence of active Dpp signals. In addition, ectopic wg expression induced by tkvQD is restricted to the antero-lateral notum. It may thus follow that an unidentified factor represses the expression of a fraction of Dpp target genes, which include pnr, ush and wg but not dad, in the postero-lateral notum (Sato, 2000).
Wg signaling represses wg transcription for refinement of its own expression domain in the wing margin. Thus, an examination has been made of notal wg expression on Wg-signaling mutant backgrounds. In contrast to wing-margin, wg expression in the notum is activated by its own signaling though much less effectively. armadillo (arm) and disheveled (dsh) encode Wg signal transducers and wgts is a temperature-sensitive Wg secretion mutant. Weak partial wg misexpression is noted in about 50% of lateral clones (19 of 40 clones) expressing Deltaarm, which constitutively activates Wg signaling. Ectopic wg expression was also detected in a cell non-autonomous fashion when wg misexpressing clones were induced in the lateral notum. In contrast, wg transcription is considerably reduced in dsh null mutant clones. When wgts mutant discs are incubated at a non-permissive temperature, for 48 h, an appreciable reduction of wg expression is detected in the authentic wg domain without any significant change in pnr and ush expression. Taken together, these results indicate that Wg signaling weakly activates wg transcription in the future lateral notum. The failure of induction of wg misexpression in Deltaarm and wg clones in future medial notum may indicate that wg expression due to auto-activation is repressed by Pnr/Ush complexes in the medial notum. One unexpected finding is that, in the hinge region, strong wg misexpression occurred only in cells surrounding wg expressing cells, suggesting possibly a new type of Wg-dependent wg expression (Sato, 2000).
The entire notal ush expression area is included in the notal pnr expression domain and hence, notal ush expression might be positively regulated by pnr. This possibility using a pnr hypomorphic mutant and a significant reduction of notal ush expression was in pnr hypomorphic mutant flies. Thus, it is concluded that Pnr is involved in the up-regulation of notal ush expression. In the case of wg expression, Ush free of Pnr serves as an activator, while a Pnr/Ush complex serves as a repressor. To determine which forms of Pnr are involved in ush expression, examination was made of USH RNA expression in the notum expressing pnr ubiquitously and the notum transheterozygous for pnrD1 and pnrVl. Neither wild-type Pnr free of Ush nor PnrD1, incapable of binding to Ush but capable of activating wg expression, could induce ush expression. It may thus follow that a Pnr/Ush complex (but not Pnr free of Ush) is required for ush expression as a positive transcriptional regulator (Sato, 2000).
A summary is presented of wg regulation in the notum. In both future medial and lateral notal regions, dpp is expressed and Dpp signaling is active. However, ventral Dpp signals are neutralized by an unknown mechanism as far as pnr, ush and wg expression is concerned. Notal wg expression, except for that in the scutellum, is regulated through four different pathways, three under the control of Dpp signals emanating from the dorsal-most region. pnr and ush expression is up-regulated by Dpp signaling, but ush expression is much narrower than that of pnr, possibly because of the requirement of higher Dpp-signaling activity for ush expression than that for pnr expression. In the future medial notum, Pnr and Ush form a complex repressing wg expression, while Ush-unbound Pnr activates lateral wg expression. The authentic wg domain and the medial notum abut one another. Unlike wg expression, ush expression in the future medial notum is positively regulated by the Pnr/Ush complex. This regulation appears required for the maintenance of medial ush expression. Dpp signaling is also capable of activating notal wg expression through an unidentified factor X. This route includes neither Pnr nor Ush. In addition, wg expression is weakly up-regulated by its own signaling in the lateral notum (Sato, 2000).
Drosophila has emerged as an important model system to discover and analyze genes controlling hematopoiesis. One regulatory network known to control hemocyte differentiation is the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signal-transduction pathway. A constitutive activation mutation of the Janus kinase Hopscotch (hopscotchTumorous-lethal; hopTum-l) results in a leukemia-like over-proliferation of hemocytes and copious differentiation of lamellocytes during larval stages. Friend of GATA (FOG) protein U-shaped (Ush) is expressed in circulating and lymph gland hemocytes, where it plays a critical role in controlling blood cell proliferation and differentiation. These findings demonstrate that a reduction in ush function results in hematopoietic phenotypes strikingly similar to those observed in hopTum-l animals. These include lymph gland hypertrophy, increased circulating hemocyte concentration, and abundant production of lamellocytes. Forced expression of N-terminal truncated versions of Ush likewise leads to larvae with severe hematopoietic anomalies. In contrast, expression of wild-type Ush results in a strong suppression of hopTum-l phenotypes. Taken together, these findings demonstrate that U-shaped acts to control larval hemocyte proliferation and suppress lamellocyte differentiation, likely regulating hematopoietic events downstream of Hop kinase activity. Such functions appear to be facilitated through Ush interaction with the hematopoietic GATA factor Serpent (Srp) (Sorrentino, 2007).
In wild-type lymph glands, Ush is not detectable in the second instar larva (L2) but is expressed in L3, beginning in the cortical zone and eventually spreading to the entire lymph gland. It stands to reason that during the normal dispersal of the hematopoietic organs in late L3, those lymph gland hemocytes in the cortical zone will be the first to enter circulation. In such a model, Ush-expressing lymph gland hemocytes enter the circulating hemocyte population, which (with the exception of crystal cells) already express Ush. Thus Ush can be viewed as a hemocyte maturation marker. This begs the question of why Ush is expressed in what are apparently the most mature hemocytes. The current observations strongly implicate Ush as being present in order to suppress proliferation and lamellocyte differentiation among mature plasmatocytes (Sorrentino, 2007).
The strongest evidence for a mechanism in which Ush suppresses hemocyte proliferation and lamellocyte differentiation comes from analyses of ush mutants. Reducing Ush function causes lymph gland hypertrophy, which is a direct result of an increase in the number of lymph gland hemocytes. Furthermore, in a manner similar in quality (but somewhat less in intensity) to those of hopTum-l larvae, ush mutant lymph glands disperse precociously, and cortical zone hemocytes appear to be morphologically consistent with lamellocytes. Additionally, total circulating hemocyte concentration (CHC) of ush mutants is over four-fold greater than that of wild-type larvae, and two different alleles of ush when heterozygous, induce a less severe but nonetheless significant hematopoietic phenotype. High CHC is consistent with the mechanism of a large number of hemocytes, including lamellocytes, leaving the lymph gland and entering circulation. The possibility cannot be ruled out that circulating hemocytes, which are of a different embryonic origin than lymph gland hemocytes, can also over-proliferate and/or differentiate into lamellocytes in ush mutants. Since cortical zone hemocytes (predominantly plasmatocytes) express Ush and can differentiate into lamellocytes, the fact that circulating plasmatocytes also express Ush would support the notion of circulating plasmatocytes also being able to differentiate into lamellocytes (Sorrentino, 2007).
Importantly, it was observed that wild-type L2 lymph glands do not express Ush, while L3 organs do (in the cortical zone first, then throughout the lymph gland). Clearly, a mechanism that represses a developmental decision is not necessary unless a cell has the potential to actually make the choice. Thus the existence of a Ush-regulated mechanism for suppression of hemocyte proliferation and lamellocyte differentiation in L3 cortical zone hemocytes is interpreted as supportive of the hypothesis that Ush+ cells are in a different genetic state in which they can, given the proper cues, hyperproliferate and differentiate into lamellocytes. It follows that wild-type L2 lymph gland hemocytes cannot hyperproliferate and become lamellocytes. Such a putative mechanism is consistent with previous findings; Jung (2005) observed that L3 lymph gland hemocyte proliferation takes place primarily within the cortical zone, while Sorrentino (2002) observed that, in larvae parasitized by the wasp Leptopilina boulardi, L2 lymph glands are immune-unresponsive (as indicated by mitotic index, crystal cell population size, and a lamellocyte marker) whereas L3 lymph glands do respond (Sorrentino, 2007).
Additional strong evidence for the role of Ush is provided by transgene expression data. Expression of wild-type Ush in hopTum-l/Y larvae produced an effect opposite in quality to that of ushVX22/ushr24, that being a significant 90% reduction in the hopTum-l-induced circulating lamellocyte population. The CgGAL4 driver is active in hopTum-l/Y L2 hemocytes, thus transgenic Ush has an opportunity to act on hemocytes prior to lymph gland dispersal. The significant reduction could be explained by the suppression of lamellocyte differentiation and/or the suppression of the proliferation of pro-lamellocytes. An apoptotic mechanism may also be partially involved. The reason for the observation that the hopTum-l non-lamellocyte population was not significantly affected by transgenic Ush is unknown, but one explanation would be a dosage-dependent mechanism in which experimental expression of just one copy of a UASush transgene is insufficient to suppress hopTum-l over-proliferation. It is also possible that hemocyte over-proliferation is a secondary effect of lamellocyte differentiation, and thus not under the direct control of Ush (Sorrentino, 2007).
If Ush normally suppresses crystal cell differentiation why do ush mutants not exhibit a severe overabundance of crystal cells? Using the strong amorphic ush1 background, an approximately 30% increase in mean crystal cell counts has been observed in stage-16 embryos. However, since the wild-type mean number of crystal cells in stage-16 embryos is about 24-25 per embryo, a 30% increase amounts to about 8 additional crystal cells. Since there are hundreds of plasmatocytes in an embryo, the overwhelming majority of plasmatocytes do not become crystal cells in the absence of Ush (Sorrentino, 2007).
Such findings can be explained by a model in which Ush suppresses crystal cell differentiation in a small subset of hemocytes, with the primary role of Ush in hematopoiesis being to control lamellocyte differentiation and hemocyte proliferation. In this model, the down-regulation of ush expression in embryonic crystal cells occurs because hemocytes committed to the crystal cell lineage cannot become lamellocytes, and thus require no mechanism to suppress lamellocyte differentiation (Sorrentino, 2007).
Srp is expressed in all hopTum-l/Y hemocytes, both lamellocytes and non-lamellocytes, in circulation and in the lymph gland. Thus, all Drosophila hemocyte classes studied thus far express this hematopoietic GATA factor, and the role of Srp in the differentiation of all hemocyte types is worthy of investigation. The fact that the lamellocyte population observed in ushVX22/+ larvae is strongly reduced by srpneo45/+ and completely reduced to wild-type levels by srp3/+ suggests Srp plays an active role in lamellocyte differentiation. In humans, GATA-3 determines the differentiation of Th2 cells, which like lamellocytes are the primary effectors in a cellular immune response against metazoan endoparasites. FOG-1 inhibits Th2 differentiation by inhibiting GATA-3 activity via physical interaction. However, if Srp is indeed necessary for lamellocyte differentiation, the finding that Srp is expressed in all hemocytes likely means it is not the sole determinant of lamellocyte differentiation. Thus it is considered likely that an additional transcriptional regulator, either another GATA factor (e.g., Grain, dGATAd, dGATAe) or a non-GATA factor, works in conjunction with Srp to specify the lamellocyte differentiation program (Sorrentino, 2007).
Ush232-1191, Ush302-1191, and Ush365-1191 driven by CgGAL4 are dominant inducers of hematopoietic tumor phenotypes, measurably stronger than that of the ushVX22/ushr24 loss-of-function condition. This finding is not interpreted as coincidental. In an important parallel all three of these constructs, when activated by the mesodermal twiGAL4 driver, exhibit a failure to suppress the expression of a cardiac-active Dmef2-lacZ reporter gene. Additionally, it was observed that Ush232-1191, though missing zinc finger 1, is still able to bind to Srp in vitro just as it is able to bind to Pnr. Such observations are consistent with the possibility that endogenous and transgenic Ush may compete in their binding to Srp. In such a situation, transgenic Ush232-1191, even if bound to Srp, might fail to suppress Srp-induced hemocyte proliferation and lamellocyte differentiation. Alternative explanations include: (1) Ush232-1191 bound to Srp may actually enhance normal Srp activity; (2) the Srp:Ush232-1191 complex may behave neomorphically; (3) Ush may normally dimerize while not bound to Srp, if so a Ush:Ush232-1191 complex may not be able to separate into active Ush monomers. The transgenic Ush proteins are assumed to be sufficiently stable and functional as to validate these observations, since the UASush constructs used have been shown to generate stable proteins in other cell types and also to induce measurable phenotypes (Sorrentino, 2007).
There is significantly more Ush present in the nuclei of hopTum-l/Y hemocytes than in nuclei of wild-type blood cells. Interestingly, Ush appears to be exclusively nuclear in hopTum-l/Y hemocytes, whereas there appears to be some cytoplasmic anti-Ush staining in wild-type hemocytes. It is possible that Ush function is in part determined by its cytoplasmic/nuclear ratio. Perhaps the qualitatively higher Ush concentration in hopTum-l hemocytes is the result of nuclear translocation of all cellular Ush. Based on work with human 293T and mouse erythroleukemia cell cultures, Garriga-Canut (2004) proposed a model in which TACC-3 and GATA-1 compete in binding to FOG-1, with FOG-1 bound to TACC-3 retained in the cytoplasm. Such a mechanism may also be at work in Drosophila hemocytes and the possibility of a Ush cytoplasmic sequestration phenomenon remains to be investigated (Sorrentino, 2007).
This study also found that there exist high concentrations of exclusively nuclear Ush in other tumorous backgrounds, those being in Tl10b/+ and CgGAL4>UAScol animals. Larvae carrying the dominant Tl10b allele exhibit a hematopoietic tumor phenotype similar to that of hopTum-l/Y larvae. In addition to srpDGAL4>UAScol, CgGAL4>UAScol is sufficient to induce lamellocyte differentiation (although it also induces L2 developmental arrest). All hemocytes, including lamellocytes, in both of these backgrounds also exhibit high concentrations of nuclear Ush. These observations are consistent with a model in which three different signaling pathways (Hop-Stat, Toll-Dorsal, and the early B-cell related factor Collier) all make use of a single common downstream lamellocyte induction program that involves Ush. Examination of hemocytes from additional tumorous backgrounds will reveal whether such a model is truly universal. An important question remains as to how Ush suppresses hemocyte proliferation and lamellocyte differentiation, yet is expressed so strongly in tumorous hemocytes. Taken together, the findings are supportive of Ush having an early function in repressing lamellocyte differentiation. Up-regulation of the protein in lamellocytes would be suggestive of a second, separate function for Ush within this differentiated hemocyte. Comparable multi-functional properties have been reported for FOG-1 in vertebrate hematopoiesis (Sorrentino, 2007).
Therefore, reduction of Ush function results in a classic hematopoietic tumor phenotype: lymph gland hypertrophy and early dispersal, a significant increase in total circulating hemocyte concentration, large-scale lamellocyte differentiation, and melanotic tumors. These anomalies can be partially induced by the loss-of-function of a single copy of ush. The identification of this FOG class protein as a tumor suppressor raises questions about the roles of other FOG proteins in mammalian leukemias. While mutations in murine fog1 have been associated with hematopoietic dysfunction such as the failure of megakaryopoiesis and the arrest of erythropoiesis, FOG proteins have not been implicated as a causal factor in any human leukemia. While there is no guarantee that the observations in Drosophila will directly translate to specific human hematopoietic pathologies, it may now be worthwhile to examine the state of fog gene expression and function in human leukemias (Sorrentino, 2007).
The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet (tup) and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).
The dpp stripe results in an expansion in both the hnt and ush expression patterns. The broadening of these patterns is particularly evident in anterior regions in the vicinity of the eve stripe. Increases in dpp+ gene dose do not expand the pnr expression pattern. For example, four copies of dpp+ result in augmented levels of pnr expression, but the dorsoventral limits of expression are essentially normal. The stripe2-dpp transgene has no obvious effect on the early sog and brk expression patterns (Ashe, 2000).
Previous studies have identified mutations in the Drosophila homolog of the mammalian CBP histone acetyltransferase gene, nejire. nej is maternally expressed so that the detection of early patterning defects depends on the analysis of embryos derived from females containing nej germline clones. The complete loss of nej+ activity results in a failure to make mature eggs. However, it is possible to obtain embryos from a strong hypomorphic allele, nej1. These embryos exhibit dorsoventral patterning defects. Recent studies have shown that CBP interacts with Smad proteins including the Drosophila protein Mad, a transcription factor downstream of Dpp signaling. In nej mutant embryos, there is a loss of the amnioserosa and other derivatives of the dorsal ectoderm. The expression of target genes requiring peak levels of Dpp signaling is essentially abolished. For example, hnt expression is lost in the presumptive amnioserosa, but persists at the posterior pole where it might be separately regulated by the torso signaling pathway (Ashe, 2000).
There is a similar loss of the dorsal rho pattern in mutant embryos. In contrast, the lateral, neurogenic stripes are unaffected, indicating that the nej mutant does not cause defects in the patterning of the neurogenic ectoderm. Moreover, the fact that the rho stripes are excluded from ventral regions, as seen in wild-type embryos, suggests that the patterning of the mesoderm is also normal. Thus, the nej mutation does not appear to cause a general loss of transcriptional activation, but instead results in specific patterning defects in the dorsal ectoderm. Target genes that are activated by lower levels of Dpp signaling such as ush and pnr are also affected by the nej mutation. In the case of ush, there is a loss of staining in central regions of the dorsal ectoderm. Moreover, the residual staining pattern is narrower than the wild-type pattern. This is reminiscent of the ush pattern seen in dpp/+ heterozygotes. However, the nej mutation also causes a narrowing of the pnr pattern, whereas expression is normal in dpp/+ embryos (Ashe, 2000).
A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity gradient presumably leads to a broad nuclear gradient of Mad and Medea across the dorsal ectoderm of early embryos. It is conceivable that the early lateral stripes of brk expression lead to the formation of an opposing Brk repressor gradient through the limited diffusion of the protein in the precellular embryo. Peak levels of Dpp and Scw activity lead to the activation of Race and hnt at the dorsal midline. The tup and ush patterns represent another threshold of gene activity. The similar patterns might involve different mechanisms of Dpp signaling since tup is repressed by Brk, whereas ush is not. Finally, the broad pnr pattern represents another threshold of gene activity. It is not inhibited by Sog but is repressed by Brk. It is possible that tup and pnr are differentially repressed by a Brk gradient. Low levels of Brk might be sufficient to direct the lateral limits of the tup pattern, whereas high levels may be required to repress pnr (Ashe, 2000).
The morphogen gradient of Wingless provides positional information to cells in Drosophila imaginal discs. Elucidating the mechanism that precisely restricts the expression domain of wingless is important in understanding the two-dimensional patterning by secreted proteins in imaginal discs. In the pouch region of the wing disc, wingless is induced at the dorsal/ventral compartment boundary by Notch signaling in a compartment-dependent manner. In the notum region of the wing disc, wingless is also expressed across the dorsal/ventral axis, however, regulation of notal wingless expression is not fully understood. Notal wingless expression is established through the function of Pannier, U-shaped and Wingless signaling itself. Initial wingless induction is regulated by two transcription factors, Pannier and U-shaped. At a later stage, wingless expression expands ventrally from the pannier expression domain by a Wingless signaling-dependent mechanism. Interestingly, expression of pannier and u-shaped is regulated by Decapentaplegic signaling that provides the positional information along the anterior/posterior axis, in a concentration-dependent manner. This suggests that the Decapentaplegic morphogen gradient is utilized not only for anterior/posterior patterning but also contributes to dorsal/ventral patterning through the induction of pannier, u-shaped and wingless during Drosophila notum development (Tomoyasu, 2000).
A hierarchy of the activity of these genes during notum development is presented. dpp is initially induced at the dorsal region of the A/P compartment boundary by Hh signaling. Dpp signaling induces two target genes, pnr and ush. Analyses of pnr expression in put-ts and tkva12 cells suggest that different thresholds are set for the induction of these genes: low levels for pnr and high levels for ush. wg is induced by Pnr where ush is not expressed. Simultaneously, the Pnr-Ush complex represses wg expression at the dorsal-most region of the presumptive notum. In the later stage, the wg expression domain expands ventrally from the pnr expressing region and wg starts to be expressed in the non-pnr-expressing cells. During this process, Wg signaling plays a crucial role and this separation does not occur in the Wg signaling mutants. The Pnr-Ush complex acts as a repressor for the induction of wg and of DC enhancer-lacZ expression (DC enhancer is an enhancer of the achaete-scute proneural gene complex that activates gene expression in the dorsocentral area). It is interesting that Ush does not simply inhibit Pnr function but switches the activator function of Pnr to a repressor function. Based on the result that the extra doses of Pnr cannot revert the repressor activity of Pnr-Ush, it has been proposed that the activator function of Pnr and the repressor function of the Pnr-Ush complex do not simply compete with each other on the notal wg enhancer element. However, it also seems to be possible that Pnr and the Pnr-Ush complex compete for the binding site at the notal wg enhancer, but the ability of Pnr-Ush complex to bind this site may be greater than that of Pnr. It is also worth noting that FOG-1, a mammalian homolog of Ush, represses the transactivation of alpha-globin and EKLF promoter by GATA-1, but enhances the transactivation of NF-E2 p45 promoter by GATA-1 in a culture cell system. Dorsocentral (DC) bristles are ectopically formed but postvertical bristles on the head are missing in a loss-of-function allelic combination for ush or in pnrD1 heterozygous flies. These observations suggest that the Pnr-Ush complex acts as a repressor for the DC enhancer, but acts as an activator for the enhancer of postvertical bristles. Only a cis-regulatory element of the DC enhancer has been analyzed at the nucleotide level. Additional studies of the molecular analyses of the cis-regulatory elements of both wg and DC or other enhancers of the achaete-scute complex seem to be necessary in order to reveal the functions of Pnr and Ush (Tomoyasu, 2000).
Generally, at least two different coordinate axes are necessary for positional specification in a two-dimensional field. Morphogen gradients of Dpp and Wg provide this axial information during Drosophila imaginal disc development. In both wing and leg discs, Dpp is induced at the A/P compartment boundary by Hh signaling. In the leg disc, wg is also induced by Hh signaling. Mutual repression between Dpp and Wg signaling separates each expression territory, localizing dpp in the dorsal and wg in the ventral regions abutting the A/P border (a compartment-independent manner). In contrast, wg is induced by Notch signaling only at the D/V compartment boundary in the wing pouch (a compartment-dependent manner). Then, secreted Dpp and Wg proteins provide positional information along the A/P and D/V axes, respectively, to establish Cartesian-like coordinates in the pouch field. Relative positions of dpp and wg expression domains in the notum are more similar to those in the wing pouch (in both cases, the expression domains are orthogonal). However, a D/V compartment boundary does not exist in the notum. The results described here reveal that another compartment-independent mechanism acts to pattern the presumptive notum. Namely, the D/V axis, provided by Pnr, Ush, and Wg, is initially established by the Dpp gradient, which mainly contributes the positional information along the A/P axis. One of the key issues of this patterning model is that Dpp signaling seems to act preferentially along the A/P axis of the notum. This is because two target genes, pnr and ush, are induced farther from the Dpp source along the A/P axis than along the D/V axis. One possible explanation for this phenomenon is that the diffusion of Dpp protein may be positively regulated along the A/P axis. However, such asymmetric induction is not observed on the dad induction; dad is one of the Dpp signaling targets in the wing disc. This suggests that diffusion of Dpp protein is not directionally regulated in the notum region. An alternative explanation would be that an effective range of Dpp morphogen gradient is established in a relatively short range. Cells that respond to Dpp would proliferate or migrate preferentially along the A/P axis. pnr mRNA is detected mainly in the posterior-dorsal region of the presumptive notum. GFP expression of UAS-gfp pnrmd237 is seen along the entire dorsal side of the presumptive notum. This difference between the staining pattern of pnr mRNA and GFP expression of UAS-gfp pnrmd237 in the late third larval stage seems to be caused by a long half-life of gal4 and/or gfp products, suggesting that cells that once have expressed pnr mRNA proliferate preferentially along the A/P axis. However, it seems to be difficult to explain the determination of pnr and ush expression domains only by the Dpp morphogen gradient. The existence of Tkv*-insensitive cells for inducing pnr and ush indicates that some regional subdivision may occur independently of Dpp signaling. Discontinuous expression of dpp in the A/P border of the notum also suggests the existence of a Dpp-independent subdivision. D/V subdivision of the presumptive notum seems to be achieved by several parallel mechanisms, including Dpp signaling (Tomoyasu, 2000).
Because Drosophila is a holometabolous insect, it should destroy larval tissues and replace them with a different population of cells to form the adult structure during the pupal stage. Thus, formation of epidermal structure should occur reiteratively during embryogenesis and metamorphosis. Patterning of larval epidermal structure takes place during embryogenesis; however, patterning of adult structure is mainly performed in larval stage imaginal discs. The Dpp morphogen gradient has been shown here to regulate pnr and ush expression to pattern the presumptive notum, which forms the dorsal structure of the adult, in the wing imaginal disc. pnr and ush are necessary for the formation of amnioserosa, the dorsal structure of the embryo, and both pnr and ush expressions are also positively regulated by Dpp in a concentration-dependent manner during embryogenesis. Furthermore, dorsal closure during embryogenesis and thorax closure in metamorphosis is also analogous, because both processes are regulated by the same signaling cascade, JNK signaling. These similarities between embryogenesis and metamorphosis presumably reflect the evolutionary history of the development in holometabolous insects (Tomoyasu, 2000).
How body size is determined is a long-standing question in biology, yet its regulatory mechanisms remain largely unknown. This study finds that a conserved microRNA miR-8 and its target, U-shaped (USH), regulate body size in Drosophila. miR-8 null flies are smaller in size and defective in insulin signaling in fat body that is the fly counterpart of liver and adipose tissue. Fat body-specific expression and clonal analyses reveal that miR-8 activates PI3K, thereby promoting fat cell growth cell-autonomously and enhancing organismal growth non-cell-autonomously. Comparative analyses identify USH and its human homolog, FOG2, as the targets of fly miR-8 and human miR-200, respectively. USH/FOG2 inhibits PI3K activity, suppressing cell growth in both flies and humans. FOG2 directly binds to p85α, the regulatory subunit of PI3K, and interferes with the formation of a PI3K complex. This study identifies two novel regulators of insulin signaling, miR-8/miR-200 and USH/FOG2, and suggests their roles in adolescent growth, aging, and cancer (Hyun, 2009).
Animal body size is a biological parameter subject to considerable stabilizing selection; animals of abnormal size are strongly selected against as less fit for survival. Thus, the way in which body size is determined and regulated is a fundamental biological question. Recent studies using insect model systems have begun to provide some clues by showing that insulin signaling plays an important part in modulating body growth. The binding of insulin (insulin-like peptides in Drosophila) to its receptor (InR) triggers a phosphorylation cascade involving the insulin receptor substrate (IRS; chico in Drosophila), phosphoinositide-3 kinase (PI3K), and Akt/PKB. An active PI3K complex consists of a catalytic subunit (p110; dp110 in Drosophila) and a regulatory subunit (p85α; dp60 in Drosophila). Phosphorylated Akt (p-Akt) phosphorylates many proteins -- including forkhead box O transcription factor (FOXO) -- which are involved in cell death, cell proliferation, metabolism, and life span control. Once activated, the kinase cascade enhances cell growth and proliferation (Hyun, 2009).
Organismal growth is achieved not only by cell-autonomous regulation but also by non-cell-autonomous control through circulating growth hormones. Recent studies in insects indicate that several endocrine organs, such as the prothoracic gland and fat body, govern organismal growth by coordinating developmental and nutritional conditions. However, detailed mechanisms of how body size is determined and modulated remain largely unknown (Hyun, 2009).
microRNAs (miRNAs) are noncoding RNAs of ~22 nt that act as posttranscriptional repressors by base-pairing to the 3' untranslated region (UTR) of their cognate mRNAs. The physiological functions of individual miRNAs remain largely unknown. Studies of miRNA function rely heavily on computational algorithms that predict target genes. In spite of their utility, however, these target prediction programs generate many false-positive results, because regulation in vivo depends on target message availability and complementary sequence accessibility. To overcome the difficulties in identifying real targets, various experimental approaches have been developed, including microarrays, proteomic analyses, and biochemical purification of the miRNA-mRNA complex. Genetic approaches using model organisms can also be useful tools for studying the biological roles of miRNAs at both the organismal and molecular levels. Despite these advances, however, it is still a daunting task to understand the biological function of a given miRNA and to identify its physiologically relevant targets (Hyun, 2009).
This study found using Drosophila as a model system that conserved miRNA miR-8 positively regulates body size by targeting a fly gene called u-shaped (ush) in fat body cells. It was further discovered that this function of miR-8 and USH is conserved in mammals and that the human homolog of USH, FOG2, acts by directly binding to the regulatory subunit of PI3K (Hyun, 2009).
The phenotype of the miR-8 null fly was first analyzed using mir-8δ2. It has been shown that mir-8 mutation results in increased apoptosis in the brain and frequent occurrence of malformed legs and wings (in about one-third of the mutants). Interestingly, in addition to these phenotypes, it as found that miR-8 null flies are significantly smaller in size and mass than their wild-type counterparts (Hyun, 2009).
The determination of the final body size in insects during the larval stage is analogous to that which occurs during the human juvenile period. It is generally known that reduced body size in insects is caused by either slow larval growth, precocious early pupariation that shortens the larval growth period, or both. It was observed that, at 100 hr after egg laying (AEL), miR-8 null larvae exhibit a significantly smaller body volume than do wild-type larva. The onset of pupariation in miR-8 null flies was not significantly different from that in wild-type flies, and adult emergence was slightly delayed (~12 hr). Thus, the smaller body size of miR-8 null flies is likely to be caused by slower growth during the larval period rather than by precocious pupariation. Insufficient food intake has been reported to accompany either precocious or delayed pupariation, depending on the onset of reduced feeding. However, the levels of Drosophila insulin-like peptides (Dilps), which are known to be reduced in starvation conditions, were not downregulated in miR-8 null larvae. Given the unaffected onset time of pupariation and the levels of Dilps in this animal, the small body size of miR-8 null flies is unlikely due to reduced feeding (Hyun, 2009).
Next, it was asked whether the small body phenotype was caused by a reduction in cell size, cell number, or both. Cell size and number were measured and it was found that cell number was reduced in the wing in miR-8 null flies, whereas cell size was not significantly different from that of wild-type. Thus, assuming that similar regulation takes place in other body parts, the reduced growth in the peripheral tissues of the miR-8 null flies may be ascribed to decreased cell number rather than reduced cell size (Hyun, 2009).
To understand why miR-8 null animals grow slowly, the activities of the proteins involved in insulin signaling were examined in the miR-8 null flies. The level of activated Akt was measured by Western blotting using a p-Akt-specific antibody. The p-Akt level was reduced in the mutant flies, suggesting that Akt signaling is impaired in the absence of miR-8. Activated p-Akt is known to inactivate FOXO via phosphorylation. Phosphorylation prevents nuclear localization of FOXO, which, in turn, results in the reduction of transcription of FOXO target genes. Consistent with the reduced level of p-Akt, the FOXO target gene, 4EBP, was increased in mir-8 mutant larvae, indicating that insulin signaling is indeed significantly reduced in the miR-8 null animal (Hyun, 2009).
Recent studies suggested that Drosophila fat body may be an important organ in the control of energy metabolism and growth. Therefore, it was reasoned that if miR-8 in the larval fat body is critical for body size control, exclusive expression of miR-8 in the fat body alone should alleviate the whole body size defect observed in the mir-8 mutants. To test this idea, transgenic flies were generated to specifically reintroduce miR-8 into the fat bodies of mir-8 mutant larvae using a fat body-specific GAL4 driver, Cg gal4 (CgG4). Remarkably, miR-8 expression in the fat body alone rescued the phenotype to near wild-type levels in both body weight and body size, suggesting that miR-8 in the fat body is important for systemic body growth. Another interesting observation was that the miRNAs from the human miR-200c cluster, which includes miR-200c and miR-141, could also yield a comparable rescue effect. Human miR-200 family miRNAs, which are located in two chromosomal clusters, have extensive homology to miR-8. The fact that miRNAs of the human miR-200c cluster effectively compensate for the loss of miR-8 suggests that these human miRNAs can be processed by the Drosophila miRNA processing machinery and that they share a conserved biological function. Because CgG4 is expressed in the anterior lymph gland as well as in the fat body, an additional GAL4 driver, ppl gal4 (pplG4), was used that is active mainly in the fat body and slightly in the salivary gland (Zinke, 1999). Similar rescue effects were observed with pplG4, in support of the fat body-specific function of miR-8 (Hyun, 2009).
To examine which targets among the candidates are physiologically relevant to the phenotype observed, the candidate genes were knocked down in the fat body of miR-8 null flies and it was asked whether the knockdown could rescue the small body phenotype. Using the UAS-RNA interference (RNAi) lines obtained from the Vienna RNAi Library Centre, dsRNAs of five candidate genes were expressed in the fat body of mir-8 mutants using CgG4. Lap1 knockdown was unsuccessful and, thus, did not rescue the mir-8 mutant phenotype. Among the RNAi lines tested, the one against ush rescued the dwarf phenotype most dramatically. RNAi of ush in wild-type background did not significantly increase body weight, ruling out the possibility that the effects of ush knockdown and mir-8 mutation are additive (Hyun, 2009).
Because a previous study showed that miR-8 targets atrophin (atro) to prevent neurodegeneration, whether atro is also involved in body size regulation was tested. Knockdown of atro in the fat body, however, failed to rescue the small body phenotype of miR-8 null flies. Thus, the reported function of miR-8 in the prevention of neurodegeneration may be separate from its function in body growth, not only spatially but also at the molecular level. To exclude possible off-target effects of ush RNAi, the ush1513 hypomorph, which expresses a reduced level of ush as the result of a mutation in the promoter region, was used. Consistent with the results of the ush RNAi, ush1513 heterozygotes have larger adult bodies than do the control flies. This result indicates that USH may indeed suppress body growth (Hyun, 2009).
Next, whether the level of USH was elevated in miR-8 null animals was examined. The endogenous ush mRNA level was determined by qRT-PCR analysis of the RNAs from whole larva or larval fat body. The ush mRNA is, indeed, significantly upregulated in the fat body of miR-8 null larvae (δ2.0 fold), suggesting that miR-8 suppresses ush in the fat body. Upregulation of ush mRNA in whole larval RNA was less prominent (~1.3 fold). Thus, ush may be more strongly suppressed in the fat body than in other body parts. Notably, USH protein levels are more dramatically affected than the mRNA levels, indicating that miR-8 represses USH production by both mRNA destabilization and translational inhibition. Furthermore, a point mutation of the miR-8 target site in the 3' UTR of ush abolished the suppression of the 3' UTR reporter, indicating that the suppression is mediated through the direct binding of miR-8 to the predicted target site. Putative target sites for miR-8 are found in all Drosophila species examined, including distant species such as D. virilis and D. grimshawi. Together, these results demonstrate that ush is an authentic target of miR-8 (Hyun, 2009).
To more precisely analyze miR-8's function in fat cells, flip-out GAL4 overexpressing clones of miR-8 were generated in the fat body of mir-8 heterozygote. In the mosaic fat cells overexpressing miR-8, the tGPH signals was augmented in the membrane, indicating that miR-8 promotes PI3K activity in a cell-autonomous manner. Cell size also increased with miR-8 overexpression (Hyun, 2009).
Next mitotic null clones were generated to observe the loss of function phenotype. Cells of the miR-8 null clone were smaller than the adjacent cells in the twin spot -- the cells harboring wild-type copies of miR-8. This suggests that miR-8 promotes fat cell growth in a cell-autonomous manner, as expected if miR-8 enhances insulin signaling in the fat body. Fewer (or no) null clone cells were often observed next to the twin spot cells when the mitotic clones were induced at embryonic stage or newly hatched larval stage. This suggests the frequent failure of proliferation and survival of miR-8 null cells during larval development. It is noted that null clones of miR-8 were generated in the wing or eye disc but little growth defect was found in these organs. Therefore, the effect of miR-8 on cell growth is dependent on tissue type, which may be explained by the fact that USH is present in the fat body but not in wing precursor cells or the eye disc (Hyun, 2009).
To determine whether USH negatively regulates insulin signaling, mosaic clones of fat cells overexpressing USH were generated. USH-overexpressing cells were smaller in size and showed significantly lower tGPH signals in the membrane and higher FOXO signals in the nucleus than did the neighboring wild-type cells. Also mosaic fat cells expressing dsRNA against ush were created to observe the knockdown phenotype. The tGPH signal was significantly enhanced in the mosaic cells depleted of USH. In mosaic ush mutant cells, the nuclear FOXO signals decreased. Together, these observations indicate that USH inhibits insulin signaling upstream of or in parallel with PI3K in a cell-autonomous manner (Hyun, 2009).
Whether reduced insulin signaling caused by the absence of miR-8 could be rescued by knockdown of USH was further examined. Excessive insulin signaling is known to reduce the levels of insulin receptor (Inr) and cytohesin Steppke (step) through negative feedback by FOXO. These two targets of FOXO were upregulated in the fat body of miR-8 null larvae, whereas reintroduction of miR-8 dramatically reduced their expression. Notably, ush RNAi also restores the mRNA levels of the FOXO target genes Inr and step in mir-8 mutant fat bodies. Thus, the defect of insulin signaling in the fat body of miR-8 null larvae is at least partially attributable to elevated ush levels (Hyun, 2009).
Given that FOG2 suppresses PI3K and colocalizes with p85α, it is suspected that FOG2 may interact with PI3K. Notably, a significant amount of p85α, the regulatory subunit of PI3K, was coprecipitated with anti-FOG2 antibody. Interaction between FOG2 and p85α was also observed when the FOG2 was ectopically expressed in a FLAG-tagged form (Hyun, 2009).
To map the interaction domain of FOG2, several truncated mutants of FOG2 were generated. The mutants containing a FLAG-tag in the N termini were coexpressed with V5-tagged p85α and were analyzed by immunoprecipitation using anti-FLAG antibody. The results indicate that the middle region of FOG2 (507-789 aa) mediates the interaction with p85α. It was then asked whether the middle region is sufficient to inhibit PI3K activity when it is ectopically expressed in HepG2 cells. The middle region suppressed PI3K, whereas neither the N-terminal part nor the C-terminal part had a significant effect on PI3K activity (Hyun, 2009).
To test whether FOG2 binds to p85α directly, the FOG2 protein was expressed and purified from bacteria and was used in an in vitro binding assay, along with purified recombinant p85α protein fused to GST. The recombinant FOG2 protein containing the middle region of FOG2 (413-789 aa) specifically bound to recombinant p85α (Hyun, 2009).
Finally, it was asked whether FOG2 can directly inhibit p85α by performing an in vitro PI3K assay using recombinant FOG2. Addition of the recombinant FOG2 protein containing the middle region (FOG2[413-789]) to the immunoprecipitated PI3K complex significantly inhibited the PI3K activity. This finding suggests that direct binding of FOG2 to p85α leads to the inhibition of PI3K activity. Notably, it was also found that Drosophila USH physically interacts with Drosophila p60 (dp60, the fly ortholog of p85α) when dp60 is coexpressed with USH in human HEK293T cells. Therefore, the action mechanism of USH/FOG2 may be conserved across the phyla (Hyun, 2009).
This study has revealed two novel regulatory components of insulin signaling: miR-8/miR-200 and USH/FOG2. miR-8/200 negatively regulates USH/FOG2 through direct base-pairing to the 3' UTR of the ush/FOG2 mRNA. USH/FOG2, in turn, inhibits the formation of an active PI3K complex via direct interaction with dp60/p85α, the regulatory subunit of PI3K. In fly fat bodies, miR-8 suppresses ush, which causes cell-autonomous increase of fat cell growth. The roles of miR-8 and USH are conserved in mammals; miR-200 miRNAs target FOG2 to upregulate insulin signaling and cell proliferation in human cells. Given that the PI3K-Akt-FOXO pathway plays central roles in many developmental processes and that defects of this pathway have been associated with cancer, diabetes, neuropathology, and aging, further investigation of the miR-8/200 family and USH/FOG2 may contribute to the understanding and amelioration of such human diseases (Hyun, 2009).
In Drosophila, miR-8 posttranscriptionally represses USH, thereby activating insulin signaling, which results in cell-autonomous growth of fat body cells. This process also causes nonautonomous organismal growth, likely through the induction of humoral factors. In human liver cells, miR-200 posttranscriptionally represses FOG2, which directly binds to p85α and blocks the formation of an active PI3K complex. As such, the repression of FOG2 by miR-200 stimulates insulin signaling and cell proliferation (Hyun, 2009).
The results support and extend the emerging theory that the fat body is a central organ coordinating metabolic condition and global growth of the organism. It is proposed that miR-8 regulates the growth of peripheral tissues in a non-cell-autonomous manner by modulating the secretion of the humoral factors that are under the control of insulin signaling (see A model for the functions of miR-8/miR-200 and USH/FOG2). Future investigation is needed to identify the humoral factors that mediate the communication between the fat body and other tissues. Because the larval fat body is considered the Drosophila counterpart of mammalian liver and adipose tissues, it will be interesting to study whether miR-200 and FOG2 play a similar role in liver and adipose tissues to control body growth during the human juvenile period (Hyun, 2009).
Previous studies suggest that USH/FOG2 may function as either transcriptional coactivators or corepressors by partnering with various GATA transcription factors. However, FOG2 is localized to the cytoplasm in some tissues. FOG1, the other human homolog of Drosophila USH, was also reported to remain in the cytoplasm of skin stem cells that lack GATA-3 and was shown to be sequestered in the cytoplasm by a cytoplasmic protein TACC3. USH/FOG2 have been studied mainly in hematopoiesis and heart development in both flies and mammals. However, it was recently shown that USH suppresses cell proliferation in Drosophila hemocytes. It is also noteworthy that FOG2 is frequently downregulated in human cancers of the thyroid, lung, and prostate, which suggests a role of FOG2 as a tumor suppressor. This study is the first report that FOG2 acts as a negative modulator of the PI3K-Akt pathway via direct binding to p85α. It remains to be determined whether the newly discovered molecular function of USH/FOG2 is related to the previously described phenotypes of ush/FOG2 (Hyun, 2009).
This study also offers a comprehensive way of discovering the physiological function of conserved miRNAs. By systematically mapping the protein homologs of miRNA targets and by validating them experimentally, seven gene pairs were identified as conserved targets of the miR-8/200 family. Also fly genetics and human cell biology were used to identify ush/FOG2 as the target gene that is responsible for one particular phenotype. Of note, six other genes (Lap1/ERBB2IP, CG8445/BAP1, dbo/KLHL20, Lar/PTPRD, Ced-12/ELMO2, and CG12333/WDR37) may also be authentic targets of miR-8/200, although they need to be further verified by additional methods. These six genes may function in different organs and/or at different developmental stages. It has been reported that miR-8 prevents neurodegeneration by targeting atro. This study observed that atro knockdown does not rescue the small body phenotype of mir-8 mutants and that ush knockdown cannot reverse the wing and leg defects attributed to atro. Thus, a single miRNA may have several distinct functions in different cell types, likely depending on the availability of specific targets or downstream effectors. In a recent study, miR-8 gain of function was shown to affect the WNT pathway, although this finding was not sufficiently supported by the phenotype resulting from miR-8 loss of function. The miR-200 family has also been shown to interfere with epithelial to mesenchymal transitions in humans to enhance cancer cell colonization in distant tissues and to regulate olfactory neurogenesis and osmotic stress in zebrafish. It remains to be determined whether these previously described functions of the miR-8/200 microRNAs are systemically interconnected in a single organism and how widely each of these functions is conserved among animals expressing miR-8/200 microRNAs (Hyun, 2009).
Body size determination is a process that is tightly linked with developmental maturation. Ecdysone, an insect maturation hormone, contributes to this process by antagonizing insulin signaling and thereby suppressing juvenile growth. This study reports that the microRNA miR-8 and its target, u-shaped (USH), a conserved microRNA/target axis that regulates insulin signaling, are critical for ecdysone-induced body size determination in Drosophila. The miR-8 level is reduced in response to ecdysone, while the USH level is up-regulated reciprocally, and miR-8 is transcriptionally repressed by ecdysone's early response genes. Furthermore, modulating the miR-8 level correlatively changes the fly body size; either overexpression or deletion of miR-8 abrogates ecdysone-induced growth control. Consistently, perturbation of USH impedes ecdysone's effect on body growth. Thus, miR-8 acts as a molecular rheostat that tunes organismal growth in response to a developmental maturation signal (Jin, 2012).
This study reveals the mechanism by which ecdysone suppresses insulin signaling and thereby decelerates larval growth. During larval development, ecdysone regulates the levels of miR-8 and its target, USH, a PI3 kinase inhibitor, through the EcR downstream pathway, and quantitative regulation of miR-8 by ecdysone leads to determining the final fly size. Since the ecdysone level is low during the larval stage not engaged in the molting process, a mild response of miR-8 increase by EcR inhibition in this stage would be expected. Notably, however, it was found that this increase in miR-8 level by EcRDN is constantly sustained throughout the third instar larval stage, which is the period of exponential growth. Interestingly, among early response genes of EcR signaling, E74 and BR-C are also persistently expressed during this period; these gene products repress miR-8 expression at the transcriptional level. Thus, throughout the third instar larval period, EcR downstream signaling keeps miR-8 (and, concomitantly, insulin signaling) under control. Because the duration of this regulation lasts several days, the effect of miR-8 modulation accumulates and manifests a significant impact on final body size. This cumulative effect accounts for the effect of ecdysone signaling on body size despite only modest changes in the miR-8 level. When EcR signaling was hampered throughout the larval stage, leading to a sustained increase of miR-8, the final body size became noticeably bigger (Jin, 2012).
This function of miR-8 in shaping body size provides a novel example in which an animal uses the inherent ability of miRNAs in fine-tuning target gene expression. Previously, several studies have shown that such a strategy has been used with miRNAs in diverse biological contexts. Specific examples include maintaining the optimal level of miRNA target proteins, which is critical for organismal survival, and setting the thresholds of target gene activity to prevent inappropriate development. The current data show the application of this type of strategy in a continuous process of organismal growth. By tuning the activity of insulin signaling, miRNAs could regulate organismal growth and ensure the attainment of appropriate body size. It is currently unclear whether similar regulatory mechanisms exist in other organisms. However, in humans and rodents, the miR-200 family of miRNAs are predominantly expressed in organs such as the pituitary, thyroid, testes, ovary, and breast, most of which are major target organs of steroid hormones. Moreover, the miR-200 family of miRNAs are significantly down-regulated by the estrogen hormone in breast cancer cells and uterus tissues, suggesting that the miR-200 family may also be controlled by steroids in mammals. It would be interesting to investigate whether a comparable regulatory axis of steroid hormone/miR-200/insulin signaling is conserved through metazoan evolution (Jin, 2012).
The pattern of the large sensory bristles on the notum of Drosophila arises as a consequence of the expression of the achaete and scute genes. U-shaped acts as a transregulator of achaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression of achaete and scute. A synergism between ac-sc and ush has been observed: animals heterozygous for both a deletion of ush and a deletion of the Achaete-Scute complex lack the posterior vertical bristles on the head. ac-sc mutants are epistatic over ush for the bristle phenotype: in the absence of ac-sc function, no sensory organs develop, even in ush flies. This indicates that Ush functions upstream of Ac and Sc. The additional macrochaetes seen in ush mutants may therefore be attributable to overexpression of ac and sc. Overexpression of u-shaped causes a loss of achaete-scute expression and consequently a loss of dorsocentral bristles (Cubadda, 1997).
The effects of u-shaped on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The dorsocentral proneural cluster of ac-sc expression is known to depend on enhancer sequences, as for example, the dorso-central (DC) enhancer, located 4.0-9.8 kb upstream of the ac start site (Cubadda, 1997). The effects on u-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute. A study of the sites at which the dorsocentral bristles arise in mosaic u-shaped nota, suggests that the levels of the u-shaped protein are crucial for the precise positioning of the precursors of these bristles (Cubadda, 1997).
On each half of the dorsal mesothorax (heminotum), 11 large bristles (macrochaetae) occupy precisely constant positions. The location of each macrochaeta is specified during the third instar larval and early pupal stages by the emergence of its precursor cell (sensory mother cell: SMC) at a precise position in the imaginal wing discs, the precursors of the epidermis of most of the mesothorax and wings. The accurate positioning of SMCs is thought to be the culmination of a multistep process in which positional information is gradually refined. The GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles. Pannier has been shown to directly activate the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by U-shaped, a Zn-finger protein, sets its limit dorsally. Therefore, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. Evidence is provided indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum (Garcia-Garcia, 1999).
An enhancer that directs expression specifically at the DC proneural cluster is present within a 5.7 kb fragment of AS-C DNA. Different subfragments were assayed for enhancer activity in vivo. A 1.4 kb subfragment (AS1.4DC) directs lacZ transcription from a minimal hsp70 promoter in the DC proneural cluster: beta-galactosidase and Scute endogenous accumulations precisely colocalized at this cluster. This fragment and the corresponding region of the AS-C from D. virilis were sequenced. Stretches of conserved DNA were present throughout the fragment, although they appeared to cluster within three regions. Subfragments containing each one of these regions were assayed for DC enhancer activity. Only the most 3' subfragment (PB0.5DC) shows such an activity, but to a much lesser extent than AS1.4DC. Interestingly, the activity is usually limited to only one cell, which is the posterior DC SMC. However, when assayed with the sc promoter, the PB0.5DC fragment directs lacZ activity in most cells of the DC cluster. Consequently, the sequences essential for specifying transcription in the DC cluster are contained within the PB0.5DC subfragment, although additional sequences that reinforce this expression are present in the larger AS1.4DC fragment. The AS1.4DC fragment was used to study DC enhancer activity (Garcia-Garcia, 1999).
The Pnr protein is known to regulate ac-sc expression at the DC cluster by acting directly or indirectly through the DC enhancer. The sequence of AS1.4DC was examined: within it, seven putative GATA-1 factor binding sites were found. Three of them fit the vertebrate consensus sequence (WGATAR: sites 1, 2 and 4); three comply with the consensus obtained in a random oligonucleotide selection experiment performed with Pnr protein (GATAAG: sites 3, 5 and 6), and one fits both consensus sequences (site 7). In the prospective notum, the stripe of diffusible Wg protein straddles the lateral border of the domain of expression of pnr. This is compatible with the location of the Wg source being on the border of, but still within, the pnr domain. In accordance with this location, pnr appears to activate wg, since it has been found that a wg-lacZ construct, which reproduces the notal band of Wg accumulation, is not expressed in pnr mutant discs and is ectopically expressed in the dorsalmost area of the disc in a pnr dominant gain-of-function combination. In contrast, other data suggest that Pnr represses wg. Thus, the notal wg stripe is expanded dorsally in strong hypomorphic pnr combinations. Moreover, in flies in which pnr is overexpressed there was no expansion of the domain of WG mRNA, which in fact accumulates in a stripe that is even narrower than that seen in the wild type. The repressing effects appeared to be restricted to the domain of accumulation of Ush, which suggests the participation of Pnr/Ush heterodimers in the repression. Consistent with this assumption, the PnrD1 mutant protein, which is incapable of interacting with Ush, promotes wg expression within the entire dorsalmost area of the disc in pnr mutants animals. Interestingly, Pnr D1 can not induce the expansion of the wg expression domain in the presence of wild-type Pnr, suggesting that Pnr+/Ush heterodimers interfere with the Ush-resistant function of PnrD1. Such interference may also account for the repression of the PnrD1-mediated dorsal expansion of DC-lacZ expression by Pnr+. Taken together, these results suggest that during development of the wing disc, Pnr is necessary both for activation of wg and (together with Ush) for its repression in the dorsalmost region of the presumptive notum. This dorsal repression probably takes place from the start of wg expression, since the earliest detectable accumulation of WG mRNA is already restricted to the presumptive mid notal region. A wg-lacZ enhancer trap line, which shows expression throughout the dorsalmost part of the early third instar wing discs and posterior refinement to the notal stripe, might have a reduced sensitivity to the repression by Pnr/Ush (Garcia-Garcia, 1999).
A model is provided for the dorsal-lateral patterning of the DC area by Pnr and Ush. In the third instar wing disc and in the dorsalmost part of the prospective notum, Ush is present at high concentrations and the Pnr/Ush heterodimers are relatively abundant. These heterodimers would act as repressors and prevent activation of downstream genes. In the DC area, defined along the dorso-lateral axis by lower concentrations of Ush and the presence of Pnr, there is sufficient free Pnr to activate genes like ac-sc, DC-lacZ and wg. ac-sc is transcribed in the more dorsal part of the area because its activation requires relatively high concentrations of Pnr. wg is only transcribed at the edge of the Pnr domain because its expression is very sensitive to both Pnr and Pnr/Ush, and consequently low concentrations of the former are sufficient for activation and low concentrations of the latter, even in the presence of high concentrations of free Pnr, impose repression. The inability of extra doses of the activator Pnr to revert the repression by Pnr/Ush in the dorsalmost region of the notum suggests that activator and repressor do not compete for overlapping sites at the DC as-sc and notal wg enhancers. The presence of Pnr/Ush at their site(s) would block the activating effect of bound Pnr. Additional inputs, notably decapentaplegic, are known to act on the DC enhancer (Garcia-Garcia, 1999).
In Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).
Pnr, a GATA-binding protein normally functions as a transcriptional activator and is antagonized by Ush in its function. Loss of function pnr mutants show no sr expression in the domain covered by pnr. This, along with sr expansion in mutants of ush, would suggest that pnr activates sr in the notum, and is inhibited by ush. However, there is also loss of sr expression in pnr `gain of function' mutants. The reason for this is not completely clear. One possibility is that since the mutation causes an increase in wg activity in the region this may cause a down-regulation of sr. This is supported by a similar effect seen on misexpression of activated armadillo in the pnr domain. Results with both pnr and ush have been taken into account to suggest that pnr positively regulates sr and is antagonized by ush (Ghazi, 2003).
These results indicate that each sr domain is regulated by a combination of prepattern genes and signaling molecules. But, a precise description of the 'combinatorial code' for regulation of each sr domain is beyond the scope of this work and can be achieved by generation of domain specific markers for sr. Based on expression pattern data, and existing literature, it is suggested that high levels of Pnr, low (or absence of) Ush and moderate levels of Wg determine the initial induction of domain a. The distinction between medial (a) and lateral (b-d) domains is established by the presence of very high levels of Wg (the cells where the Wg gradient originates). Lateral expression domains are probably induced in domains controlled by the lateral prepattern gene iro. The differences between different lateral domains arise as a result of expression of different genes in the region. For instance, the lateral-most domain d appears to be regulated by ush and does not encounter Wg at all. Whereas, all cells of b receive uniformly moderate levels of Wg, only cells at the borders of c receive high Wg levels, and these differences result in the distinct identities of the two domains. Dpp, either through its effects on these regulatory genes and/or through direct effects on sr influences the process (Ghazi, 2003).
The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, a PSC-active transcriptional enhancer was characterized that drives hh expression in supportive niche cells. The findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance (Tokusumi, 2010).
The lymph gland hematopoietic organ is formed near the end of embryogenesis from two clusters of cells derived from anterior cardiogenic mesoderm (Crozatier, 2004; Mandal, 2004). About 20 pairs of hemangioblast-like cells give rise to three distinct lineages that will form the lymph glands and anterior part of the dorsal vessel. Notch (N) pathway signaling serves as the genetic switch that differentially programs these progenitors towards cell fates that generate the lymph glands (blood lineage), heart tube (vascular lineage), or heart tube-associated pericardial cells (nephrocytic lineage). An essential requirement has also been proven for Tailup (Islet1) in lymph gland formation, in which it functions as an early-acting regulator of serpent, odd-skipped and Hand hematopoietic transcription factor gene expression (Tokusumi, 2010).
By the end of the third larval instar, each anterior lymph gland is composed of three morphologically and molecularly distinct regions (Jung, 2005). The posterior signaling center (PSC) is a cellular domain formed during late embryogenesis due to the specification function of the homeotic gene Antennapedia (Antp) (Mandal, 2007) and the maintenance function of Collier, the Drosophila ortholog of the vertebrate transcription factor early B-cell factor. PSC cells selectively express the Hedgehog (Hh) and Serrate (Ser) signaling molecules and extend numerous thin filopodia into the neighboring medullary zone. This latter lymph gland domain is populated by undifferentiated and slowly proliferating blood cell progenitors (Mandal, 2007). Prohemocytes within the medullary zone express the Hh receptor Patched (Ptc) and the Hh pathway transcriptional effector Cubitus interruptus (Ci). Medullary zone cells also express components of the Jak/Stat signaling pathway. By contrast, the third lymph gland domain -- the cortical zone -- solely contains differentiating and mature hemocytes, such as plasmatocytes and crystal cells. Upon wasp parasitization, or in certain altered genetic backgrounds, lamellocytes will also appear in the cortical zone as a third type of differentiated hemocyte (Tokusumi, 2010).
Two independent studies have provided compelling data to support the contention that the PSC functions as a hematopoietic progenitor niche within the lymph gland, with this cellular domain being essential for maintaining normal hemocyte homeostasis (Krzemien, 2007; Mandal, 2007). These investigations showed that communication between the PSC and prohemocytes present in the medullary zone is crucial for the preservation of the progenitor population and to prevent these cells from becoming abnormally programmed to differentiate into mature hemocytes. Seminal findings from these studies can be summarized as follows: Col expression must be restricted to the PSC by the localized expression of Ser; Hh must be expressed selectively in the PSC, coupled with the non-autonomous activation of the Hh signaling pathway in prohemocytes of the medullary zone; and the PSC triggers activation of the Jak/Stat pathway within cells of the medullary zone. With the perturbation of any of these molecular events, the precursor population of the medullary zone is lost owing to the premature differentiation of hemocytes, which swell the cortical zone. Although the exact interrelationship of Ser, Hh and Jak/Stat signaling within the lymph gland is currently unknown, the cytoplasmic extensions emanating from PSC cells might facilitate instructive signaling between these niche cells and hematopoietic progenitors present in the medullary zone (Krzemien, 2007; Mandal, 2007). A more recent study showed that components of the Wingless (Wg) signaling pathway are expressed in the stem-like prohemocytes to reciprocally regulate the proliferation and maintenance of cells within the supportive PSC niche (Sinenko, 2009). The cellular organization and molecular signaling of the Drosophila lymph gland are remarkably similar to those of the hematopoietic stem cell niches of vertebrate animals, including several mammals (Tokusumi, 2010 and references therein).
Through detailed molecular and gene expression analyses this study has identified the PSC-active transcriptional enhancer within hh intron 1 and delimited its location to a minimal 190 bp region. The hh enhancer-GFP transgene faithfully recapitulates the niche cell expression of Hh derived from the endogenous gene, as double-labeling experiments with the GFP marker and Antp or Hh show a clear co-expression in PSC domain cells. Appropriately, GFP expression is not detected in Antp loss-of-function or TCFDN genetic backgrounds, which culminate in an absence of niche cells from the lymph gland. The hematopoietic GATA factor Srp serves as a positive activator of hh PSC expression, as mutation of two evolutionarily conserved GATA elements in the enhancer abrogates its function and Srp functional knockdown via srp RNAi results in hh enhancer-GFP transgene inactivity and the absence of Hh protein expression. An additional intriguing phenotype was observed in lymph glands expressing the srp RNAi transgene, that being a strong reduction in the number of filopodial extensions emerging from cells of the PSC. This phenotype suggests a functional role for Srp in the correct differentiation of niche cells, via a requirement for normal Hh presentation from these cells and/or the transcriptional regulation by Srp of additional genes needed for the formation of filopodia (Tokusumi, 2010).
As Srp accumulates in all cells of the lymph gland, a question arose as to how hh expression is restricted to cells of the PSC. This paradox could be explained by a mechanism in which hh expression is also under some means of negative transcriptional control in non-PSC cells of the lymph gland. This possibility proved to be correct, with the analyses identifying two negative regulators of hh lymph gland expression. The first is Su(H). Mutation of the evolutionarily conserved GTGGGAA element, a predicted recognition sequence for this transcriptional repressor, resulted in an expanded activity of the hh PSC enhancer-GFP transgene; that is, the de novo appearance of GFP was observed in prohemocytes of the medullary zone. Likewise, ectopic medullary zone expression of the wild-type PSC enhancer-GFP transgene and of Hh protein was seen in lymph glands mutant for Su(H). These findings, coupled with the detection of Su(H) in blood cell progenitors, strongly implicate this factor as a transcriptional repressor of the hh PSC enhancer, restricting its expression to niche cells (Tokusumi, 2010).
Additional studies identified Ush as a second negative regulator of hh expression. Ush is expressed in most cells of the lymph gland, with the exception of those cells resident within the PSC domain. Previous research demonstrated that ush expression in the lymph gland is under the positive control of both Srp. Why Ush protein fails to be expressed in the PSC remains to be determined. Forced expression of ush in niche cells resulted in inactivation of the hh PSC enhancer and reduced the formation of filopodia. It was hypothesized that Ush might be forming an inhibitory complex with the SrpNC protein, changing Srp from a positive transcriptional activator to a negative regulator of hh lymph gland expression. Such a mechanism has been demonstrated previously in the negative regulation by Ush of crystal cell lineage commitment. The expansion of wild-type hh enhancer-GFP transgene and Hh protein expression to prohemocytes within the medullary zone and to differentiated hemocytes within the cortical zone in lymph glands mutant for ush is also supportive of Ush functioning as a negative regulator of hh expression (Tokusumi, 2010).
Bringing these results together, a model can be proposed for the regulatory events that culminate in the precise expression of the vital Hh signaling molecule in niche cells. Srp is a direct transcriptional activator of hh in the lymph gland and Hh protein is detected in niche cells due to this activity. hh expression is inhibited in prohemocytes of the medullary zone by Su(H) action, while a repressive SrpNC-Ush transcriptional complex prevents Srp from activating hh expression in prohemocytes and in differentiated hemocytes of the medullary zone and cortical zone. Together, these positive and negative modes of regulation would allow for the niche cell-specific expression of Hh and facilitate the localized presentation of this crucial signaling molecule to neighboring hematopoietic progenitors (Tokusumi, 2010).
The identification of Srp and Su(H) as key regulators of Hh expression in the larval hematopoietic organ prompted an investigation into the functional requirement of these proteins in the control of blood cell homeostasis. Since Srp knockdown by RNAi leads to an absence of the crucial Hh signal, it was not surprising to find that normal Srp function is required for prohemocyte maintenance and the control of hemocyte differentiation within the lymph gland; that is, a severe reduction of Ptc-positive hematopoietic progenitors and a strong increase in differentiated plasmatocytes and crystal cells was observed in srp mutant tissue (Tokusumi, 2010).
Likewise, Ptc-positive prohemocytes were lost and large numbers of plasmatocytes were prematurely formed in Su(H) mutant lymph glands. This disruption of prohemocyte maintenance occurred even though Hh protein expression was expanded throughout the medullary zone. This raised the question as to why expanded Hh protein and possible Hh pathway activation did not increase the progenitor population in Su(H) mutant lymph glands, instead of the observed loss of prohemocytes and appearance of differentiated plasmatocytes. One explanation might be that the PSC niche is not expanded in Su(H) mutant lymph glands and Hh might only function in promoting blood cell precursor maintenance within the context of the highly ordered progenitor-niche microenvironment. It has been hypothesized that the filopodial extensions that emanate from differentiated niche cells are crucial for Hh signal transduction from the PSC to progenitor cells of the medullary zone. The possibility exists that ectopic Hh protein, which is not produced or presented by niche cells, is unable to positively regulate prohemocyte homeostasis. An experimental result consistent with this hypothesis is that expression of UAS-hh under the control of the medullary zone-specific tepIV-Gal4 driver failed to expand the blood cell progenitor population. A second possibility is that the Hh pathway transcriptional effector Ci might require the co-function of Su(H) in its control of prohemocyte maintenance. This model would predict that, in the absence of Su(H) function, Hh signaling would be less (or non) effective in controlling the genetic and cellular events needed for the maintenance of the prohemocyte state. Third, Su(H) might regulate additional target genes, the expression (or repression) of which is crucial for normal blood cell precursor maintenance and the prevention of premature hemocyte differentiation. Finally, it cannot be ruled out that the expression of ectopic Hh in medullary zone cells, in the context of the adverse effects of Su(H) loss of function in these cells, culminates in the disruption of normal Hh pathway signaling due to an unforeseen dominant-negative effect (Tokusumi, 2010).
In summary, these findings add significantly to knowledge of hematopoietic transcription factors that function to control stem-like progenitor maintenance and blood cell differentiation in the lymph gland. An additional conclusion from these studies is that the hh enhancer-GFP transgene can serve as a beneficial reagent to identify and characterize genes and physiological conditions that control the cellular organization of the hematopoietic progenitor-niche cell microenvironment. RNAi-based genetic screens could be undertaken using this high-precision marker to determine signaling pathways and/or environmental stress conditions that might alter niche cell number and function, leading to an alteration in hematopoietic progenitor maintenance coupled with the robust production of differentiated blood cells. Much remains to be determined about the regulated control of these critical hematopoietic changes and their likely relevance to hematopoietic stem cell-niche interactions in mammals (Tokusumi, 2010).
Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR-8, functions in the fat body to non-autonomously regulate organismal growth, suggesting that fat body-derived humoral factors are regulated by imiR-8. This study found that several putative peptide hormones known to have mitogenic effects are regulated by imiR-8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase-related growth factors are up-regulated in the absence of imiR-8. Drosophila insulin-like peptide 6 (Dilp6) and Imaginal morphogenesis protein-late 2 (Imp-L2), a binding partner of Dilp, are also up-regulated in the fat body of miR-8 null mutant larvae. The fat body-specific reintroduction of miR-8 into the miR-8 null mutants revealed six peptides that showed fat-body organ-autonomous regulation by miR-8. Amongst them, only Imp-L2 was found to be regulated by U-shaped, the miR-8 target for body growth. However, a rescue experiment by knockdown of Imp-L2 indicated that Imp-L2 alone does not account for miR-8's control over the insect's growth. These findings suggest that multiple peptide hormones regulated by miR-8 in the fat body may collectively contribute to Drosophila growth (Lee, 2014).
The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. The number of ectopic bristles in pannierD/+ flies increases in flies bearing only a single copy of u-shaped+ but decreases when three copies are present. Activation of a chicken alpha-globin promoter sequence by Pannier in cultured cells is inhibited by Ush. When both Ush and wild-type Pnr are expressed simultaneously, activation is abolished. Stimulation by Pnr is lost progressively in a concentration-dependent manner. Similarly, activation by chicken GATA-1 is also lost after cotransfection with the Ush expression vector. Because Pnr and GATA-1 have no homology outside their GATA DNA-binding domain, and since Ush alone has no effect on globin promoter activity, these observations suggest that the function of Ush is mediated through the GATA DNA-binding domain (Haenlin, 1997).
Additional genetic evidence is provided for an antagonistic interaction of Pannier and U-shaped. Pnr proteins deleted in the C-terminal region do not activate transcription from a heterologous alpha-globin promoter. Overexpression of wild-type and mutant Pnr proteins in transgenic flies regulates achaete-scute expression through the dorsocentral enhancer. Heterozygous flies mutant for pannierD alleles differentiate extra dorsocentral bristles resulting from overexpression of achaete-scute, whereas heterozygous flies mutant for C-terminally deleted Pnr proteins differentiate fewer dorsocentral bristles attributable to decreased ac-sc expression. Overexpression of the U-shaped protein in transgenic flies reduces achaete-scute expression in wild-type but not in pannierD mutants. It is concluded that Ush antagonizes the effects of Pnr, leading in consequence to reduced achaete-scute expression and reduced bristle development (Haenlin, 1997).
Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr; when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development (Haenlin, 1997).
serpent encodes a GATA transcription factor essential for hematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C-terminal type. srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). The respective functions of SrpC and SrpNC during embryonic hematopoiesis were examined. Both isoforms individually rescue blood cell formation, which is lacking in a srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. These results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development (Waltzer, 2002).
In a systematic search for GATA zinc finger-coding sequences in the Drosophila genome, five genes were found: dGATA-E (CG10278), dGATA-D (CG5034), pnr, grain and srp. dGATA-E and dGATA-D appear to include only a C-finger, while Pnr and Grain contain both an N- and a C-finger. Interestingly, while Srp has been reported to contain a single C-finger, the presence of a putative exon (E4A) coding for an N-finger motif in srp is also evident. Using RTPCR assays with various combinations of oligonucleotides, it has been shown that E4A is expressed and that E4A and E4B are alternatively spliced to exon 5. In the course of these experiments, an additional splice acceptor site was also identified within E7. This downstream acceptor site in E7 is out-of-frame and leads to the deletion of the Srp glutamine-rich C-terminal region. The data indicate that four alternatively spliced mRNAs are transcribed from srp, two encoding products with a single C-finger (SrpC and SrpCd) and two encoding products with both N- and C-fingers (SrpNC and SrpNCd). Interestingly, in SrpNC and SrpNCd, the two fingers present the same conserved organization as in other GATA factors. Notably, they are separated by 29 amino acids, as in all vertebrate GATA. The two isoforms that contain the full-length exon 7, i.e. srpC and srpNC, have been used to address the functional consequences of the alternative splicing of E4A and E4B (Waltzer, 2002).
Whether SrpC and SrpNC display different properties in vitro was investigated. While the C-finger is necessary and sufficient for specific DNA binding, it has also been shown in vertebrates that the N-finger can stabilize the binding to particular double GATA sites. By electophoretic mobility shift assays (EMSAs), it was determined if SrpNC and SrpC have similar DNA-binding properties. Both in vitro translated SrpC and SrpNC proteins bind to an oligonucleotide containing a consensus GATA site. The binding is specific, since it can be competed out efficiently by an excess of cold GATA oligonucleotide, but not by an excess of the GATC oligonucleotide. The stability of the SrpN and SrpNC complex on a single or on a palindromic GATA site was assessed by dissociation experiments. While the rate of dissociation is similar for SrpC and SrpNC on a single GATA probe, SrpNC bound more stably than SrpC to the palindromic GATA sites (Waltzer, 2002).
The GATA N-finger allows interaction with cofactors of the FOG family. Key residues that are required for the interaction between GATA and FOG are conserved in the Srp N-finger. In order to test the binding between Ush and srp products, pull-down assays were performed in vitro. in vitro translated [35S]methionine-labelled Ush binds to GSTSrpNC, but not GSTSrpC. Thus, Ush specifically interacts with Srp isoforms that contain the N-finger. In addition, like its vertebrate homologs, Ush interacted with the transcriptional corepressor dCtBP in this assay (Waltzer, 2002).
Taken together, the results indicate that SrpNC displays features characteristic of two-fingered GATA factors. The two types of naturally occurring isoforms encoded by srp (with or without the N-finger) have different DNA-binding properties, and only the isoforms including an N-finger can interact with Ush (Waltzer, 2002).
In order to determine whether a spatial regulation of the alternative splicing leading to SrpC and SrpNC occurs during embryonic development, the distribution of the corresponding srp transcripts was assessed by in situ hybridization using specific probes for exon 4A or 4B. At the blastoderm stage and during gastrulation, srpC and srpNC show the same expression pattern. They are expressed in the procephalic mesoderm, the hemocyte primordium, at the anterior and posterior pole, in the primordium of the anterior and posterior midgut as well as in the amnioserosa and in the yolk cells. Later, during germ band extension, and after germ band retraction, srpC and srpNC are expressed identically in the developing fat body. Thus, srpC and srpNC transcripts are not differentially regulated spatially during embryonic development. However, the level of the transcripts is not identical. Indeed, by means of semi-quantitative RTPCR, it was determined that exon 4B-containing mRNA is five times more abundant than exon 4A-containing mRNA, suggesting that two-fingered isoforms of Srp are less abundant than single-fingered isoforms (Waltzer, 2002).
In order to analyse SrpC and SrpNC activities, their capacities to activate gene expression in vivo were tested during Drosophila embryonic hematopoiesis. Using the UAS-GAL4 system, they were ectopically expressed in the mesoderm and the expression pattern of various hematopoietic markers was assessed. The two genes ush and gcm play critical roles in embryonic hematopoiesis. Their expression in the hematopoietic primordium occurs early and appears to depend on srp activity. Therefore, it was determined whether they are transcriptional targets of SrpC and/or SrpNC. Whereas in a wild-type early embryo, ush expression is restricted to the anterior mesoderm, twist-driven expression of SrpC (twist-SrpC) or SrpNC (twist-SrpNC) induces strong expression of ush throughout the mesoderm. In contrast, twist-SrpC induces gcm expression poorly and in a limited number of mesodermal cells of stage 5 embryos, whereas twist-SrpNC strongly activates gcm expression segmentally from stage 5 to 9 (Waltzer, 2002).
The expression of hematopoietic lineage-specific markers was examined. As plasmatocyte markers, peroxidasin (pxn) and croquemort (crq) were used. Since, crystal cells are the only source of prophenoloxidase (pro-PO) in Drosophila, expression of this gene was used to monitor crystal cell formation. pro-PO transcripts were indeed detected in these cells from early stage 11 to the end of embryogenesis. Analysing these markers, two situations were observed. twist-SrpC and twist- SrpNC have similar abilities to induce expression of the plasmatocyte marker pxn and of the crystal cell marker pro-PO, however expression of crq was induced by twist-SrpC and not by twist-SrpNC. Note that pxn and crq were induced through most of the mesoderm, while pro-PO activation was restricted to the head region (Waltzer, 2002). Taken together, these data show that SrpC and SrpNC have both common and different activities during hematopoiesis. Indeed, both isoforms activate the expression of ush, pxn and pro-PO in a similar manner. However, SrpC and SrpNC differentially stimulate the expression of crq and gcm, respectively, in the mesoderm (Waltzer, 2002).
It is remarkable that srp encodes both single and dual zinc finger-containing products. The results provide strong evidence that this alternative splicing allows production of transcription factors with specific activities. The two isoforms activate the expression of ush and pxn with similar efficiency, suggesting that SrpC and SrpNC have similar transactivating properties in vivo, yet, SrpC (but not SrpNC) activates crq expression, while SrpNC is a much stronger activator of gcm expression than SrpC. The domain coded by exon 4B that is present only in SrpC has no known motif and it is not known if and how it participates in SrpC-specific function. However, the presence of the N-terminal zinc finger encoded by exon 4A may explain some of the distinct features of SrpNC as discussed below (Waltzer, 2002).
As in the case of vertebrate GATA-1, the presence of the N-finger in Srp stabilizes binding to double palindromic GATA sites. Although the N-finger of GATA-1 modulates the binding and the transactivating properties of GATA-1 on synthetic promoters, the functional importance of these effects has remained elusive, particularly since no GATA-1 isoform contains only the C-finger. In the case of srp, these distinct binding properties may have direct functional consequences. For instance, the fact that SrpC and SrpNC activate a common target, ush, whereas only SrpNC strongly activates a specific target, gcm, could be related to the DNA-binding specificity of the two isoforms. A scan of the ush upstream regulatory region shows that it contains several GATA consensus sequences, nine of which are clustered in <1 kb and are organized as three repetitions of three sites. In contrast, GATA sites are far less frequent in gcm regulatory regions and are often organized in palindromes. Considering that ush and gcm are likely to be direct target genes for srp, the different organization of their regulatory regions may explain the differential effect observed (Waltzer, 2002).
The lack of plasmatocyte and crystal cell formation due to an srp null mutation can be rescued by expressing SrpC or SrpNC in the mesoderm. No difference between the two isoforms was seen in this assay, suggesting that the N-finger is not absolutely required for srp function in embryonic blood cell formation. However, in the absence of a functional test, to what extent the formation of embryonic blood cells is fully rescued cannot be determined. Interestingly, rescue experiments with the mouse GATA-1 mutant indicate that the GATA-1 N-finger is dispensable for primitive erythropoiesis but is required for definitive erythopoiesis. In Drosophila, a second wave of hematopoiesis, occurring at the larval stage, gives rise to four different lineages: plasmatocytes, crystal cells, secretory cells and lamellocytes. srp is expressed in the dorsal lymph gland (i.e. the main larval hematopoietic organ) and it probably controls larval hematopoiesis. By analogy to vertebrate GATA-1, the Srp N-finger may provide an additional function for larval hematopoiesis, perhaps during formation of the new cell types (Waltzer, 2002).
In the assay used, the expression of the transgene was limited to the mesoderm but it still rescued blood cell formation. This finding suggests that the early expression of srp in the hematopoietic primordium is sufficient to initiate the genetic program that controls hemocyte formation and differentiation. Interestingly, in the wild-type embryo, srp transcripts are not expressed detectably in hemocytes after stage 11, but Srp protein is detected in plasmatocytes and crystal cells throughout most of embryogenesis. Persistence of srp products in hemocytes might be critical for srp function, and control of srp products at the post-translational level may play a crucial role in the correct regulation of blood cell differentiation. Rescue of crystal cell formation by mesodermal expression of SrpC and SrpNC contrasts with the observation that later expression driven by lz-Gal4 in crystal cells represses their development. Srp levels are reduced in crystal cells compared with surrounding plasmatocytes. Therefore, the results are consistent with a two-step model in which Srp expression is first necessary to induce lz expression and subsequently is downregulated to allow crystal cell differentiation (Waltzer, 2002).
One of the best characterized features of GATA N-fingers is their dimerization with cofactors of the FOG family. Consistent with this feature, it was found that SrpNC interacts with the Drosophila FOG Ush, but SrpC does not. Previous analysis showed that ush regulates the number of crystal cells. It was proposed that this function of ush could be mediated by a putative isoform of Srp containing an N-finger. The current findings strongly support this hypothesis. However, it was not possible to address this issue directly, since both SrpC and SrpNC display a strong Ush-independent repressive effect on crystal cell formation and differentiation (Waltzer, 2002).
A new function of ush revealed here is the regulation of the level of expression of the macrophage receptor crq, suggesting that ush displays a broader function in hematopoiesis than previously assumed. Notably, evidence is provided that Ush modulates SrpNC transactivation of crq. Since Ush interacts with the corepressor dCtBP in vitro, the UshSrpNC complex could repress crq expression. However, it is not known whether crq is a direct target of srp, so the possibility that the UshSrpNC complex activates a transcriptional repressor that regulates crq cannot be ruled out. Vertebrate FOGs can act as either a coactivator or a corepressor of GATA factors. In Drosophila, Ush is a repressor of Pannier-induced activation in cell culture, and it probably also represses the expression of achaete in the dorso-central proneural cluster in vivo. Furthermore, in a heterologous assay in Drosophila, the CtBP-binding region of mFOG2 is required for repressing the formation of crystal cells but not cardiac cells. Thus several mechanisms seem to regulate the function of the GATAFOG complex (Waltzer, 2002).
Remarkably, some functions of SrpNC appear to be independent of Ush. Thus, gcm-specific activation by SrpNC is not affected in an ush mutant embryo. Moreover, SrpNC still represses crystal cell formation in the absence of ush. This is reminiscent of mouse erythropoiesis, where both FOG-dependent and FOG-independent regulation of gene expression by GATA-1 have been observed. The molecular mechanisms underlying the regulation by Ush/FOG-1 of SrpNC/GATA-1 activity on some specific targets remain to be elucidated. It is tempting to speculate that the N-finger of SrpNC is involved in the recognition of promoter sequences, on gcm for example, and thus is not available to recruit Ush. Alternatively, other cofactors already localized to the promoter or bound to SrpNC might prevent Ush binding to the N-finger (Waltzer, 2002).
This study has focussed on hematopoiesis, but srp also participates in other developmental processes, such as germ band retraction, midgut differentiation, fat body formation, induction of the immune response and the ecdysone response. It will be interesting to determine the respective roles of SrpC and SrpNC in these different phenomena. Phylogenetic analysis shows that SrpNC is closely related to vertebrate GATA factors. It has been suggested that srp is a functional homolog of the entire vertebrate GATA family, since srp is required in Drosophila for hematopoiesis, like GATA-1/2/3 in mice, and for endodermal development, like GATA-4/5/6. Nevertheless, this hypothesis was at odds with the fact that Srp seemingly had a single zinc finger while all the vertebrate GATAs have two. The present identification of Srp isoforms with two fingers gives new force to this hypothesis. Further, the expression of isoforms of Srp with distinct activities helps to account for the broad range of functions ensured by this gene (Waltzer, 2002).
It is worth noting that alternative splicing eliminating the N-finger has also been described in Bombyx mori GATAß and in chicken GATA-5 genes. Moreover, a BLAST search analysis revealed alternatively spliced human expressed sequence tags coding for two isoforms of a potential GATA factor with either one or two zinc fingers. This suggests that alternative splicing of GATA genes could be more general than previously thought, and as yet unnoticed splice variants of GATA vertebrate genes may generate proteins with only a C-finger (Waltzer, 2002).
In conclusion, these results shed further light on the molecular control of hematopoiesis by the GATA factor Srp. The alternative splicing of srp gives rise to different Ush-interacting and non-interacting Srp proteins with different target gene specificities, thereby contributing to the exquisite control of Drosophila blood cell formation. It is speculated that alternative splicing of the GATA N-finger might be an important mechanism regulating the activity of other GATA genes from insects to man (Waltzer, 2002).
C-terminal binding protein (CtBP) is an evolutionarily and functionally conserved transcriptional corepressor known to integrate diverse signals to regulate transcription. Drosophila CtBP (dCtBP) regulates tissue specification and segmentation during early embryogenesis. This study investigated the roles of dCtBP during development of the peripheral nervous system (PNS). This study includes a detailed quantitative analysis of how altered dCtBP activity affects the formation of adult mechanosensory bristles. dCtBP loss-of-function was shown to result in a series of phenotypes with the most prevalent being supernumerary bristles. These dCtBP phenotypes are more complex than those caused by Hairless, a known dCtBP-interacting factor that regulates bristle formation. The emergence of additional bristles correlated with the appearance of extra sensory organ precursor (SOP) cells in earlier stages, suggesting that dCtBP may directly or indirectly inhibit SOP cell fates. It was also found that development of a subset of bristles was regulated by dCtBP associated with U-shaped through the PxDLS dCtBP-interacting motif. Furthermore, the double bristle with sockets phenotype induced by dCtBP mutations suggests the involvement of this corepressor in additional molecular pathways independent of both Hairless and U-shaped. It is therefore proposed that dCtBP is part of a gene circuitry that controls the patterning and differentiation of the fly PNS via multiple mechanisms (Stern, 2009).
This study provides evidence that dCtBP is required for different aspects of PNS development. In addition, extensive genetic characterization demonstrates how altered dCtBP activity can influence the formation of the adult dorsal thoracic mechanosensory organs. The data show that overexpression of dCtBP impairs mechanosensory formation. In contrast, reduction of dCtBP activity leads to variable bristle phenotypes, suggesting that dCtBP is likely operating in different molecular complexes. Namely, the mechanisms by which dCtBP regulates cell fate specification within the PNS may involve protein–protein interactions between dCtBP and at least two factors: Ush and possibly H (Stern, 2009).
The data strongly suggest that dCtBP associates with the Ush-Pnr repressor complex through the Ush PxDLS motif to inhibit the expression of achaete and scute in particular PNCs. This model is supported by the following evidence. First, the ush loss-of-function and gain-of-function phenotypes were phenocopied by the corresponding genetic alterations to dCtBP activity. Second, Ush interacts with Pnr and the Ush-Pnr complex inhibits expression of the achaete and scute genes through GATA sites located within the DC enhancer. Third, the additional SOP cells were formed in both the dCtBP and ush mutant imaginal discs. Fourth, both ush and pnr alleles exhibited dominant genetic interactions with dCtBP. Finally, disruption of the PxDLS motif of Ush partially mitigated the effects of ush overexpression on particular bristles (Stern, 2009).
The evolutionarily conserved physical interaction of dCtBP with Ush is essential for the propagation of certain cell lineages, such as blood cells (crystal cells) of the fruit fly, but not for heart development, processes known to be regulated by Ush and the GATA factors, Pnr and Serpent. Surprisingly, the interaction between CtBP and FOG-1 is not required for erythroid development in mice, despite the fact that this interaction was found to be important in tissue culture experiments and in frog embryos. The current results from the ush overexpression assay suggest that Ush may utilize both the PxDLS motif and another repression domain(s) to fully function, since particular bristles are affected by disruption of the PxDLS motif of Ush. A putative corepressor that interacts with the additional repression domain may act additively or cooperatively with dCtBP or function in different tissue/cell-type contexts. In fact, recently other repression domains in Ush, required for repression of the D-mef2 cardiac gene, were identified and these seemed to cooperatively work with the dCtBP-dependent motif. Consistent with this hypothesis, some dCtBP-interacting factors contain multiple repression domains. Knirps (nuclear receptor), Snail (zinc-finger protein), and H all have two repression domains, dCtBP-dependent and -independent, which can function additively in transgenic flies and/or in tissue culture. It has been also demonstrated that H has an additional repression activity independent of Groucho and dCtBP-binding. Krüppel (zinc-finger protein) has two evolutionarily conserved repression domains. The dCtBP-dependent domain is functional in tissue culture and in transgenic embryos, while the other repression domain is only active in tissue culture but not in transgenic embryos, suggesting a cell-type specific effect. Finally, Brinker (a helix-turn-helix protein) contains at least three repression domains (dCtBP-dependent, Groucho-dependent, and the third repression domain) that are important for repression of different target genes (Stern, 2009).
The physical interaction of dCtBP with H is implicated in sensory organ formation, wing formation, and embryonic patterning. H acts as an adaptor protein to bridge the Groucho and dCtBP corepressors to the DNA-binding factor Su(H), to ultimately inhibit Notch target genes. Vertebrate Notch target genes are similarly repressed by a complex consisting of CtBP with RBP-Jkappa (the mammalian counterpart to Su(H)) and the SHARP/CtIP corepressors. This study demonstrates that the bristles that are affected in dCtBP mutants also show defects in H loss-of-function mutants, although the effect of H is stronger than that of dCtBP. H mutations induce two distinct phenotypes associated with loss of bristles; one is the bald phenotype (a complete loss of both sockets and bristles) due to lack of SOP cells, and the other is the double-socket phenotype (also lack of bristles). A similar bald phenotype was observed in dCtBP mutant backgrounds, such as dCtBP RNAi, the dCtBP87De-10/dCtBP03463 transheterozygote, the dCtBP87De-10 clonal backgrounds. Although compared to what is seen in dCtBP mutants, reduction of H activity interferes more uniformly with the formation of all 11 bristles that were analyzed, the bald phenotype further supports previous observations that dCtBP is involved in H-mediated repression. The double-socket phenotype seen in H loss-of-function mutants was never observed in dCtBP mutants. This distinct phenotype suggests that H may play a role independent of dCtBP, possibly by interacting with another corepressor Groucho. Interestingly, the bald phenotype was also induced by overexpression of dCtBP. The mechanism by which overexpression causes the bald phenotype in all regions except the DC region remains unclear, although one simple explanation could be that overproduction of dCtBP may disrupt the stoichiometric balance of the H/dCtBP/Groucho repression complex (Stern, 2009).
The double bristle phenotype observed in dCtBP mutants suggests that dCtBP may be required to execute cell fate decisions within the SOP lineage. A similar phenotype seen in the H gain-of-function background was the result of a socket-to-bristle cell fate transformation. Of note, this phenotype is clearly distinct from the double bristle phenotype observed in dCtBP mutants, which is always associated with a socket(s). This dCtBP phenotype implies that cousin-to-cousin cell fate conversions may be occurring within the sensory organ lineage. This type of cell fate switch could be similar to the conversion of sheath to bristle observed in hamlet mutants. Hamlet is a zinc-finger transcription factor and interestingly contains a PLDLS peptide sequence located between amino acid 747 and 751, identical to the CtBP-interacting motif. Future experiments will address whether dCtBP and Hamlet can physically interact and function together within the same biological process (Stern, 2009).
Based on the results, it is concluded that dCtBP regulates the development of the mechanosensory organs likely via multiple mechanisms. This highlights the centrality of this transcriptional corepressor in integrating multiple inputs to define boundaries and thereby control pattern formation during development (Stern, 2009).
The 4.7 kb transcript is detected with a peak of expression during early embryonic stages at 4-8 hours (Cubadda, 1997).
Expression of ush in the late third-instar wing imaginal disc appears to be restricted to specific domains. Staining is detected in territories corresponding to the future dorsal-most region of the thorax, as well as in part of the hinge region and the posterior region of the pleura. In the hinge region, ush expression is found in a domain comprising the sites of appearance of the anterior notal wing process and the proximal tegula, expanding up to the border where anterior and posterior notopleural bristles develop. In the dorsal part of the notum, staining covers the site of appearance of the scutellar bristles and extends to the border of the site at which the dorsocentral bristles form. Therefore, in the notum, the area of ush expression corresponds well with the region where ush is required for normal development (Cubadda, 1997).
Maintenance of hematopoietic progenitors ensures a continuous supply of blood cells during the lifespan of an organism. Thus, understanding the molecular basis for progenitor maintenance is a continued focus of investigation. A large pool of undifferentiated blood progenitors are maintained in the Drosophila hematopoietic organ, the larval lymph gland, by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. This study examined the function of the Drosophila fibroblast growth factor receptor (FGFR), Heartless, a critical regulator of early lymph gland progenitor specification in the late embryo, during larval lymph gland hematopoiesis. Activation of Heartless signaling in hemocyte progenitors by its two ligands, Pyramus and Thisbe, is both required and sufficient to induce progenitor differentiation and formation of the plasmatocyte-rich lymph gland cortical zone. Two transcriptional regulators were identified that function downstream of Heartless signaling in lymph gland progenitors, the ETS protein, Pointed, and the Friend-of-GATA (FOG) protein, U-shaped, which are required for this Heartless-induced differentiation response. Furthermore, cross-talk of Heartless and target of rapamycin signaling in hemocyte progenitors is required for lamellocyte differentiation downstream of Thisbe-mediated Heartless activation. Finally, the Drosophila heparan sulfate proteoglycan, Trol, was identified as a critical negative regulator of Heartless ligand signaling in the lymph gland, demonstrating that sequestration of differentiation signals by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance that is of potential relevance to many other stem cell niches (Dragojlovic-Munther, 2013).
Germband retraction in Drosophila, like most embryonic morphogenetic events in this organism and in higher eukaryotes, is not well understood. Several approaches have been taken to study the relationships between previously identified mutations (u-shaped, serpent, hindsight and tailup) that selectively cause germband retraction defects in homozygous embryos, and a more pleiotropically acting locus, DER/faint little ball, the Drosophila Epidermal growth factor receptor. The former four loci are elements of at least two parallel and partially redundant cellular pathways that affect germband retraction by acting in amnioserosal development or maintenance. An additional discrete and unique pathway, represented by DER/faint little ball, is likely to function in the germband itself. While the role of the amnioserosa during germband retraction appears to be permissive, the action of DER in the germband may be mediated by the cytoskeleton (Goldman-Levi, 1996).
The amnioserosa is an extraembryonic, epithelial tissue that covers the dorsal side of the Drosophila embryo. The initial development of the amnioserosa is controlled by the dorsoventral patterning genes. A group of genes, referred to as the U-shaped-group (ush-group), is required for maintenance of the amnioserosa tissue once it has differentiated. Using several molecular markers, amnioserosa development was developed in the ush-group mutants: u-shaped (ush), hindsight (hnt), serpent (srp) and tail-up (tup). The amnioserosa in these mutants is specified correctly and begins to differentiate as in wild type. However, following germ-band extension, there is a premature loss of the amnioserosa. This cell loss is a consequence of programmed cell death (apoptosis) in ush, hnt and srp, but not in tup. Thus, the ush-group genes are implicated in the maintainance of the amnioserosa's viability. In light of these mutants' unretracted phenotype, the amnioserosa could be involved in signal reception or the initiation of signal transduction with respect to the adjacent ectoderm (Frank, 1996).
A large number of mutant alleles of ush have been isolated; these include hypomorphic mutants that affect the pattern of bristles on the head and thorax. Viable transallelic combinations of ush display additional dorsocentral and scutellar bristles on the notum and a loss of postverticle bristles on the head. Loss of function mutants affect the development of the dorsal half of the notum only, clones in the lateral part of the notum differentiate normally. Clones extending into the scutellum fail to differentiate, generating large gaps in this region. Clones touching the dorsal midline are associated with a cleft in the thorax, whereas clones extending into the dorsocentral area are associated with absence or abnormal positioning of the dorsocentral bristles. Remarkably, in almost all cases of mosaicism in the dorsocentral area, the dorsocentral bristles formed by wild-type cells are also found to be displaced from their normal postions. Therefore, there seems to be a nonautonomous effect of mutant cells at positions where wild-type bristles would be expected to form (Cubadda, 1997).
Multitype zinc-finger proteins of the class Friend of GATA/U-shaped (Ush) are known to function as transcriptional regulators of gene expression through their modulation of GATA factor activity. To better understand intrinsic properties of these proteins, the expression and function of the ush gene during Drosophila embryogenesis has been investigated. ush is dynamically expressed in the embryo, including several cell types present within the mesoderm. The gene is active in the cardiogenic mesoderm, and a loss of function results in an overproduction of both cardial and pericardial cells, indicating a requirement for the gene in the formation of these distinct cardiac cell types. Conversely, ectopic expression of ush results in a decrease in the number of cardioblasts in the heart and the inhibition of a cardial cell enhancer normally regulated by the synergistic activity of the Pannier and Tinman cardiogenic factors. These findings suggest that, similar to its known function in thoracic bristle patterning, Ush functions in the control of heart cell specification through its modulation of Pannier transcriptional activity. ush is also required for mesodermal cell migration early in embryogenesis, where it shows a genetic interaction with the Heartless fibroblast growth factor receptor gene. Taken together, these results demonstrate a critical role for the Ush transcriptional regulator in several diverse processes of mesoderm differentiation and heart formation (Fossett, 2000).
The ush gene exhibits a dynamic pattern of expression during embryogenesis. Gene transcripts are first detected at high levels in the primordium of the amnioserosa at stage 5. Additional expression is observed in germ band extending embryos, in cells of the developing anterior and posterior midgut, and in hemocyte precursors present in the cephalic mesoderm. By stage 11, ush RNA is detected in the dorsal ectoderm and in precursor cells of the hemocytes and fat body. By late embryogenesis, ush expression is greatly diminished, but transcripts are still observed in the dorsal ectoderm during dorsal closure and cells within, or associated with, the central nervous system. To investigate the possible expression of ush in mesodermal cells underlying the dorsal ectoderm, cross sections of embryos at stage 11 were examined. ush RNA is detected in a changing pattern in this germ layer, initially throughout most of the mesoderm and then in subpopulations of cells, including precursors of the fat body, visceral mesoderm, and cardiogenic mesoderm. Therefore, ush is expressed in the dorsal mesoderm, where it could function in the early stages of heart formation (Fossett, 2000).
pnr mutant embryos show a loss of contractile cardial cells and an overproduction of certain nonmuscle pericardial cells in the heart-forming region. To identify a possible role for ush in these cardiogenic processes, alterations in cardiac cell production were sought in mutant embryos. The ush alleles used in this analysis were ush1 and ush2, believed to be hypomorphic mutations of the gene, and Df(2L)al, a chromosome deletion that represents a ush null mutation. The D-mef2 heart enhancer-lacZ fusion gene serves as a cardial cell marker, since it is detected in progenitors of these cells around stage 11 and thereafter in two, then four cardioblasts per hemisegment of the forming dorsal vessel. Embryos homozygous for either a point mutation or deletion of the gene show an increase in the number of cells expressing the reporter gene, as compared with the wild-type embryo. In ush1 embryos, a few hemisegments contain up to nine positive cells with an average of six cardial cells present in many clusters. In ush-deficiency embryos, a comparable increased density of cardial cells is found (Fossett, 2000).
Mef2 protein also marks cardioblasts; it is detected in the nuclei of all cardial, but not pericardial cells of the forming dorsal vessel. In wild-type embryos at stage 13, the germ band has retracted with cardioblasts migrating dorsally, separating from the dorsal somatic muscles. A lateral view at this stage shows a single row of cells that contains six stained nuclei per hemisegment. In contrast, ush mutant embryos possess supernumerary cardioblast nuclei. ush1 and ush2 embryos contain up to 12 nuclei per hemisegment with eight cells per cluster observed on average. Similar results have also been obtained with ush-deficiency embryos. Therefore, reducing or completely eliminating ush function leads to an increased production of cardial cells. Intriguingly, the ush heart phenotype uncovered by the analysis of these two markers directly contrasts the absence of cardial cells observed in pnr loss-of-function embryos (Fossett, 2000).
Production of pericardial cells was quantitated in ush mutant embryos, using Eve protein as a marker for a subset of these cells. In wild-type embryos at stage 12, there exist 11 Eve-positive clusters within the dorsal mesoderm, each containing about three cells. In contrast, the number of Eve-expressing pericardial cells increases in homozygous ush1 and ush2 embryos to an average of 5-6 per cluster. A similar increase in pericardial cell number is also observed in homozygous Df(2L)al embryos. Thus, ush gene activity is required to prevent the overproduction of this pericardial cell type, a function that has also been ascribed to the pnr gene (Fossett, 2000).
Because the loss of ush function resulted in a supernumerary cardial cell phenotype, the effect of expressing the gene throughout the mesoderm was monitored using the Gal4/UAS binary system. Mef2 was used to assess the status of cardial cells, with two contiguous rows of 52 cells present in the forming or mature dorsal vessel of wild-type embryos. In comparably staged embryos expressing ush throughout the mesoderm, a significant reduction in cardial cells is observed. The D-mef2 heart enhancer-lacZ fusion gene was used as a second marker for the cardial cells and also to assay the effect of ush expression on enhancer activity. In wild-type embryos at stage 16, the enhancer is active in eight cardial cells in most segments of the dorsal vessel. In contrast, beta-galactosidase activity is greatly diminished in the hearts of ush-expressing embryos, most likely a combination of the decrease in cardial cell number and the reduced activity of the D-mef2 cardiac enhancer. Thus, forced expression of ush has a potent negative effect on cardial cell formation and enhancer function (Fossett, 2000).
It has been shown that Pnr can function combinatorially with Tin in the regulation of the D-mef2 heart enhancer in Drosophila embryos. This synergistic activation was examined using a transient transfection assay in cultured Drosophila cells. The activation of a CAT reporter gene linked to the D-mef2 enhancer was monitored in cells transfected independently with Pnr, Tin, and Ush or with various combinations of the factors. The expression of Tin alone activated the enhancer about 2-fold above the basal level, whereas neither Pnr nor Ush affected enhancer activity. Coexpression of Pnr and Tin resulted in a synergistic activation of the enhancer to a level 5-6 times that of the basal activity, and this strong induction requires the binding of Tin to the Mef2 enhancer; a Tin DNA binding mutant, Tin (N-Q), failed to synergize with Pnr in the assay. In contrast, adding Ush as a third transfected factor significantly inhibits the synergistic activation of the enhancer by Pnr and Tin. This result demonstrates that Ush can antagonize the positive functional interaction of Pnr and Tin in the regulation of the cardial cell enhancer, which is consistent with the in vivo data (Fossett, 2000).
ush mutants contain an increased number of cardial cells. However, in about half of the embryos a disparity was noticed in the cardial cell populations, ranging from a high of 8-12 per hemisegment down to regions completely devoid of cells. This complex phenotype is observed with both ush hypomorphic and null alleles. This sporadic loss of cells from the dorsal-most part of the mesoderm is reminiscent of a htl mutant phenotype, where the absence of the encoded fibroblast growth factor receptor homolog results in an incomplete dorsal migration of mesodermal cells. In this event, cells fail to receive the ectodermal signal needed for their further commitment, resulting in a loss of dorsal mesodermal derivatives, including cardioblasts (Fossett, 2000).
To determine whether the variable absence of cardial cells in ush embryos is because of a cell migration defect, wild-type and mutant embryos were stained for Mef2 protein present in the invaginated population of mesodermal cells. In cross sections of normal embryos at stage 10, a uniform layer is observed where the dorsal-most mesodermal cells have migrated to a position adjacent to the dorsal-most ectodermal cells. In contrast, both htl and ush homozygous embryos display an irregular layer where mesodermal cells remain clustered and fail to undergo their complete dorsal migration. This result suggests ush function is required for the directional migration of the mesoderm. To investigate a potential genetic interaction of htl and ush in this process, embryos were examined that were heterozygous for mutations in each of the genes. These embryos also present a strong mesodermal migration phenotype, suggesting the two genes function in a common genetic pathway that controls this aspect of mesoderm differentiation. As was observed with homozygous ush embryos, slightly less than half of the transheterozygous embryos show a loss of cells from the dorsal mesoderm (Fossett, 2000).
It is thus thought that pnr has a dual requirement in the cardiogenic mesoderm because it is needed for the formation of cardial cells although simultaneously limiting the production of Eve-expressing pericardial cells. Based on these dissimilar phenotypes, it is postulated that Pnr might work with different combinations of factors to promote or repress the formation of cells within the distinct lineages. Recent studies have shown that Pnr and Tin act synergistically to induce cardial cells and activate gene expression, and the loss of function of either of these genes results in an absence of cardial cells. Therefore, the two work together in cardial cell specification (Fossett, 2000).
In contrast, Ush is a factor that normally suppresses cardial cell production. ush homozygous and hemizygous embryos show an increase in cardial cell number: the latter finding suggests Ush control of this cell population is dose-dependent, as is the case for Ush regulation of Pnr during sensory bristle development. Furthermore, forced expression of Ush decreases cardial cell production and D-mef2 heart enhancer activity, whereas ectopic expression of Pnr produces extra cardial cells and expands the domain of enhancer activity. Thus, Ush displays phenotypes that are in direct opposition to those of Pnr, suggesting that it can function as an antagonist of Pnr's cardiogenic activity. This conclusion is supported by the ability of Ush to inhibit the synergy of Pnr and Tin in the activation of the D-mef2 heart enhancer in cell transfection studies. As for the second cardiac phenotype, both pnr and ush are required to limit the number of Eve-expressing pericardial cells, consistent with a model in which Ush and Pnr function as corepressors in the control of these cells. To summarize, these genetic studies predict that, in the wild-type embryo, pnr is expressed and functions independent of ush in precursors of the cardioblast lineage. However, in neighboring cells that include the Eve lineage precursors, the expression and function of the two most likely overlaps. Future expression analyses of the two regulatory proteins at the resolution of single mesodermal cells will be required to elaborate on this genetic model in molecular terms (Fossett, 2000).
Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homolog U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homolog Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages (Fossett, 2001).
Using an antibody directed against Ush synthetic peptides, Ush protein was detected in an expression pattern similar to that of the gene transcript. Around embryonic stage 8, both ush RNA and protein can be detected in blood cell precursors. By stage 10, Ush-positive hemocyte precursors have spread throughout the lateral and ventral head mesoderm. As embryogenesis progresses, Ush is detected in stage 13 plasmatocytes migrating throughout the head mesoderm and down the ventral midline. During the late stages of embryogenesis, Ush continues to be expressed in plasmatocytes circulating throughout the embryonic hemolymph (Fossett, 2001).
lz expression in crystal cells is detected first during stage 10 and is maintained in this lineage until the late stages of embryogenesis. Fluorescent antibody staining and confocal microscopy were used to determine whether Ush and lz are coexpressed in the crystal cell lineage. To detect lz expression in hemocyte precursors and crystal cells, the expression of a UASlacZ reporter gene driven by lzGal4 (lzlacZ) was monitored. This reporter is active in hemocyte precursors as early as stage 10 and is expressed in the crystal cell lineage throughout embryogenesis. During embryonic stage 10, a number of hemocyte precursors express both Ush and lz. Later, during stage 13, the number of cells that expressed both lz and Ush decreases. Finally, during the late stages of embryogenesis, Ush is not detected in crystal cell lineage, evidenced by its failure to colocalize with the lzlacZ crystal cell marker. These results are consistent with a role for ush as a repressor of crystal cell production and suggest that ush expression is down-regulated in hemocyte precursors during crystal cell lineage commitment (Fossett, 2001).
During larval development, hematopoiesis takes place in the larval lymph glands, which flank the dorsal vessel. Plasmatocytes are specified and develop in the primary and secondary lobes of the gland, whereas crystal cells develop exclusively in the primary lobe. Ush is detected in most cells of primary and secondary lobes, consistent with expression in the plasmatocyte lineage. The protein is expressed in a differential pattern in the cells of the lymph glands, perhaps indicative of down-regulation during hemocyte precursor commitment. This may be analogous to the down-regulation of murine FOG-1 that is required for eosinophil and myeloid lineage differentiation (Fossett, 2001).
Srp function is required for hemocyte development and for differentiation of plasmatocytes and crystal cells. Furthermore, studies using amorphic alleles of srp indicate that it is required for hemocyte precursor specification. Srp is expressed first in the hemocyte precursors during embryonic stage 5, and, similar to Ush, its expression is maintained in plasmatocytes throughout embryogenesis. To determine whether an epistatic relationship exists between srp and ush, Ush expression was assayed in srp mutant embryos and Srp expression in ush mutant embryos. The hypomorphic allele srp3, which results in the production of hemocyte precursors, even with the reduction of Srp function, was used. In srp embryos, Ush is not detected in hemocyte precursors, plasmatocytes, or midgut, unlike the wild-type expression pattern. In contrast, Srp is observed in hemocyte precursors and plasmatocytes in both wild-type and ush mutant embryos. This result suggests ush resides downstream of srp in the hematopoiesis hierarchy and ush expression requires Srp function. Furthermore, ush is not required for the specification of hemocyte precursors or plasmatocytes, because these Srp-positive cells are detected in ush mutant embryos. Finally, wild-type levels of ush are present in the dorsal ectoderm of srp mutant embryos, indicating that dynamic ush expression is under the control of multiple regulators during embryogenesis (Fossett, 2001).
Previous studies have shown that ush functions to prevent the overproduction of sensory bristles, cardial cells, and pericardial cells. These observations, together with the findings that ush appears to be down-regulated during crystal cell lineage commitment, have suggested that Ush may act to limit crystal cell production. To test this hypothesis, assays were carried out for increased numbers of crystal cells in ush mutant embryos. Crystal cells are localized in a bilateral cluster of cells within the head mesoderm and require lz expression from embryonic stage 10 through 14 for their development. The lzlacZ genotype served as a crystal cell marker. Expression of lzlacZ was assayed in stage 13 embryos because during this stage the germ-band retraction phenotype can be used to distinguish ush mutants from wild-type embryos. Homozygous ush embryos show an increase in the number of lzlacZ-expressing cells compared with the wild-type control. These results were confirmed by using embryos harboring the Bc mutation, which renders crystal cells visible in late-stage homozygous embryos. Crystal cell production in ush Bc embryos was compared with the Bc parental strain, which has the wild-type ush allele. Again, homozygous ush Bc embryos have an increase in the number of crystal cells compared with the Bc embryos from the parental strain. Because the number of crystal cells in wild-type embryo populations can vary more than 2-fold, 20 wild-type and 20 ush embryos were sampled and a 30% overall increase in the number of crystal cells was seen by using either the lzlacZ or Bc marker. These results indicate that Ush functions to repress crystal cell production during hematopoiesis (Fossett, 2001).
To demonstrate further that Ush represses crystal cell production, Ush was expressed in crystal cells by using the Gal4/UAS binary system. The lzGal4 driver was used to express UASUsh in crystal cells, and their production was monitored by using the lzlacZ marker. Embryos with forced expression of Ush in crystal cells have a significant reduction in the number of these cells. Compared with similarly staged wild-type controls, UASUsh stage 13 and 16 embryos had a 30% and 85% reduction in number of crystal cells, respectively. A sample of 40 stage 13-16 UASUsh embryos averaged a 30% reduction in the number of crystal cells compared with wild-type controls. The phenotype of individual embryos within this population ranged from being completely devoid of crystal cells to wild-type cell numbers. These results indicate that down-regulation of ush during crystal cell lineage commitment is required for development of these cells. Together with the observed increase in crystal cell number in ush loss-of-function assays, these findings suggest that Ush functions during hematopoiesis to limit the number of hemocyte precursors that enter the crystal cell lineage (Fossett, 2001).
Recent studies have indicated that FOG proteins may function to regulate the commitment of several hematopoietic lineages. Ectopic expression of FOG proteins mFOG-1, mFOG-2, and xFOG (Xenopus FOG) early in Xenopus development represses red blood cell formation, possibly by down-regulating Gata-1 expression. These data suggest that FOG proteins may act to limit the differentiation of erythrocytes to prevent depletion of pluripotent stem cells. Furthermore, by using an in vitro avian hematopoietic differentiation system, it has been shown that FOG-1 represses eosinophil-specific gene expression and that forced expression of FOG-1 in eosinophils produces a multipotent precursor phenotype. Thus, down-regulation of FOG-1 in multipotent hematopoietic precursors is an essential step in eosinophil differentiation. In addition to findings that misexpressed Ush repressed crystal cell production, FOG-1 and FOG-2 also repress crystal cell number, indicating that the mechanism by which these proteins limit crystal cell number may be conserved. Taken together, studies using the Drosophila and vertebrate systems suggest that FOG proteins function to preserve the multipotent hemocyte precursor pool by controlling the lineage commitment of specific cell types (Fossett, 2001).
An additional factor that may be required to control lineage commitment is CtBP. This transcriptional corepressor may interact with FOG-1 and FOG-2 to repress erythrocyte differentiation, because a mutant version of FOG-2 lacking the consensus PXDL sequence fails to repress erythrocyte differentiation when ectopically expressed during Xenopus development. CtBP may be required for FOG protein repression of crystal cell production. It is noteworthy that CtBP likely functions during Drosophila hematopoiesis because a lacZ reporter gene inserted in the enhancer region of the CtBP gene is expressed in the larval plasmatocyte lineage. Thus, the FOG and CtBP class of transcriptional regulators may act together to control hemocyte lineage commitment in a pathway that is conserved evolutionarily (Fossett, 2001).
FOG function involves binding to its GATA partner's N-terminal zinc finger. Srp is the only known hematopoietic GATA factor in Drosophila and reportedly contains a single C-terminal zinc finger. However, a survey of the srp genomic sequence shows an ORF within the third intron of the gene that putatively encodes an N-terminal zinc finger with 96% homology to that of Pnr. This raises the possibility that Ush interacts with an alternatively spliced isoform of Srp during hematopoiesis (Fossett, 2001).
Ush appears to negatively regulate the cardiogenic function of the GATA-4 homolog Pnr, converting Pnr from a transcriptional activator to a repressor as observed during sensory bristle development. As with Ush, forced mesodermal expression of FOG-1, FOG-2, and DeltaFOG-2 (lacking a conserved motif that binds the corepressor C-terminal binding protein) also produces a diminution of cardial cells. These results demonstrate a functional conservation of the FOG proteins during Drosophila cardiogenesis, which most likely involves negative regulation of the cardiogenic activity of Pnr. In addition, forced expression of FOG proteins disrupts eye development-producing phenotypes that mimic pnr loss of function mutants, presumably by repressing Pnr activation of its downstream effector genes (Fossett, 2001).
The disruption of eye development and the repression of cardial cell production by FOG proteins occurred in the absence of the CtBP-binding motif. This is consistent with the work that shows that FOG-1 and FOG-2 repression of GATA-4 activation of cardiac promoters does not require the corepressor CtBP. Rather, conserved N-terminal regions of the murine FOG proteins were required for the repression of GATA-4 transcriptional activation, indicating that an alternative repressor mechanism may be used to negatively regulate GATA-4. An emerging hypothesis suggests that CtBP may be a hematopoietic corepressor, and an alternative corepressor may be required during heart development. Results showing that DeltaFOG-2 does not repress crystal cell production but does repress cardial cell production is evidence for this dual mechanism of FOG gene regulation during heart development and hematopoiesis in a single experimental organism (Fossett, 2001).
In conclusion, Ush and Lz function antagonistically during crystal cell lineage commitment and Ush is required to limit the overproliferation of crystal cells. This demonstrates a possible intersection between the FOG and AML-1 gene pathways, which may prove important for understanding vertebrate hematopoiesis. Furthermore, this study expands the molecular characterization of the earliest events of hematopoiesis in Drosophila, identifying additional conserved genes that establish the fly as a model organism for hematopoiesis (Fossett, 2001).
Inductive signaling is of pivotal importance for developmental patterns to form. In Drosophila, the transfer of TGFß (Dpp) and Wnt (Wg) signaling information from the ectoderm to the underlying mesoderm induces cardiac-specific differentiation in the presence of Tinman, a mesoderm-specific homeobox transcription factor. Evidence that the Gata transcription factor, Pannier, and its binding partner U-shaped, also a zinc-finger protein, cooperate in the process of heart development. Loss-of-function and germ layer-specific rescue experiments suggest that pannier provides an essential function in the mesoderm for initiation of cardiac-specific expression of tinman and for specification of the heart primordium. u-shaped also promotes heart development, but unlike pannier, only by maintaining tinman expression in the cardiogenic region. By contrast, pan-mesodermal overexpression of pannier ectopically expands tinman expression, whereas overexpression of u-shaped inhibits cardiogenesis. Both factors are also required for maintaining dpp expression after germ band retraction in the dorsal ectoderm. Thus, it is proposed that Pannier mediates as well as maintains the cardiogenic Dpp signal. In support, it is found that manipulation of pannier activity in either germ layer affects cardiac specification, suggesting that its function is required in both the mesoderm and the ectoderm (Klinedinst, 2003).
pnr and ush are both expressed in the mesoderm at the time of cardiac mesoderm formation, in addition to their expression in the dorsal ectoderm. Mesodermal expression of pnr is restricted to the dorsal cardiogenic margin, whereas ush extends more laterally. In order to assess the requirement for pnr and ush in initiating cardiac mesoderm and cardiac cell type-specific differentiation, tin expression was examined at progressively later developmental stages in null mutants for both pnr and ush. During mid-stage 11, tin is expressed segmentally in two regions of the mesoderm. The dorsal clusters of cells correspond to the cardiac precursor cells, whereas the lateral clusters will become part of the visceral mesoderm. In same stage pnr mutant embryos, tin expression is dramatically reduced in the clusters that correspond to the cardiac precursors, indicating that cardiogenesis is not being initiated. tin expression in the visceral mesodermal clusters, as well as tin expression earlier in development, is unaffected, suggesting the heart is a focal point for pnr function, which is consistent with its cardiac-restricted expression in the mesoderm. By contrast, ush mutant embryos initially seem to exhibit normal tin expression. At later stages, when tin expression is solely restricted to the heart cells, ush mutants display a progressively more severe reduction in tin expression, approaching the phenotype of pnr mutants. Thus, both pnr and ush are required for heart-specific tin expression, although ush seems to be initially dispensable (Klinedinst, 2003).
Even though tin is initially expressed in all heart progenitors, its expression is later turned off in some specific lineages, but continues to be expressed in many myocardial and pericardial cells. To determine which heart cells are affected in pnr and ush mutants, mutant embryos were examined with various markers. eve, for example, is co-expressed with tin in 11 clusters of heart progenitors, and these lineages give rise to a subset of pericardial cells. eve expression is only moderately reduced in pnr and hardly at all in ush mutants at early as well as later stages; this is accompanied by patterning defects at progressively later stages. By contrast, the lbe-expressing heart progenitors, which produce both myocardial and pericardial cells, are dramatically reduced in pnr but less so in ush mutants. Moreover, the svp-expressing cells, which also give rise to a mixed lineage, but cease to co-express tin at later stages, are dramatically reduced in both mutants. Thus, all lineage markers assayed are reduced in both mutants, but each is affected with disproportional severity, which is consistent with the idea that the formation of each cell type has a direct requirement for pnr and ush (Klinedinst, 2003).
Both tin and pnr have been shown to be targets of Dpp signaling at stage 9/10. It is proposed that dpp is necessary again at stage 11 to activate and maintain pnr and tin expression in the cardiogenic region of the mesoderm. First, pnr is activated with the help of early stage 11 tin, which is expressed broadly throughout the dorsal mesoderm, and dpp, which is expressed in a narrow dorsal ectodermal stripe. Then, at mid-stage 11, tin is restricted to the cardiogenic region with the help of mesodermal pnr as well as continuous ectodermal Dpp signaling. Once both are activated in the cardiogenic mesoderm, they are likely to contribute to the maintenance of each other's expression, probably aided again, but only moderately, by ectodermal Dpp signaling. This interpretation is consistent with mesodermal versus ectodermal expression of dominant-negative pnrEnR and the dpp target repressor encoded by brk. They are both equally effective in reducing cardiac-specific tin when expressed in the mesoderm, but ectodermal repression is more effective when dorsal-stripe dpp at stage 11 is also affected (as in the case of ZKr-Gal4>UAS-brk, but not with ZKr-Gal4>UAS-pnrEnR) (Klinedinst, 2003).
Mesodermal overexpression of ush and co-overexpression with pnr results in a decrease in the amount of cardiac-specific tin expression, suggesting that ush may not only be required along with pnr for heart development, but also play an inhibitory role. To test this hypothesis further, pnrD4, an allele that abolishes Ush binding to Pnr was overexpressed; not only ectopic tin expression was found at early stages of cardiogenesis, but also undiminished and even increased levels of expression at later stages. A similar phenotype was observed when both pnrD4 and ush were expressed throughout the mesoderm, suggesting that ush plays an anti-cardiogenic role by antagonizing the activity of wild-type Pnr, but not that of PnrD4. It would be interesting to see if pan-mesodermal overexpression of wild-type pnr in a ush mutant background results in ectopic tin expression similar to pnrD4, or if a minimal amount of ush activity is required to maintain normal and ectopic tin expression even with forced pnr expression. Interestingly, overexpression of both pnr and tin together in the mesoderm also causes a pnrD4-like phenotype, as assayed with Hand expression, suggesting that pnr and tin collaborate during initiation and subsequent differentiation of the heart progenitors (Klinedinst, 2003).
Although in vitro the Ush-related FOG factors are primarily known for their role as transcriptional repressors, they apparently can also function as co-activators: Fog2 can synergistically activate or repress the transcriptional activity of Gata4, depending on the (cardiac) promoter and cell line used, and FOG-1 can cooperate with Gata1 to transactivate NF-E2, an erythroid cell-specific promoter. Moreover, the ventricular hypoplasia and other heart defects observed in Fog2-deficient mice suggest a deficit rather than an excess in heart development. In addition, mice with an equivalent mutation to PnrD4 knocked into the Gata4 locus, thus eliminating binding to Fog2, exhibit in many ways a similar phenotype to Fog2-deficient mice. These data are consistent with the idea that Fog2 is normally involved in promoting rather than antagonizing cardiogenesis, similar to what was found with genetic studies during Drosophila heart development (Klinedinst, 2003).
The dual role of Ush suggests that the amount of Ush may be crucial for whether it exerts its function as a an activator or repressor, perhaps by binding to different sets of co-factors in a concentration-dependent manner. Alternatively, the mode of transcriptional regulation by Ush could be stage-dependent: at stage 11, Pnr and Ush cooperate as transcriptional activators in initiating cardiac-specific tin expression and heart development, but later Ush becomes a repressor to limit the transcriptional activation of tin by Pnr (Klinedinst, 2003).
pnr and ush are initially broadly expressed in the dorsal ectoderm of the early embryo, but by germband retraction the ectodermal expression of pnr is confined to a narrow stripe of cells along the border of the amnioserosa, which overlaps with the thin dorsal dpp stripe. The early ectodermal expression of ush is restricted to the presumptive amnioserosa, and by germband extension, ush also overlaps with the dorsalmost region of the ectoderm. These patterns of expression suggest that pnr and ush may be acting in both germ layers. The genetic data, including germ layer-specific expression of wild-type and dominant-negative pnr constructs, as well as germ layer-specific rescue experiments suggest strongly that pnr and ush function is not only needed in the mesoderm, but also in the ectoderm for heart formation. The ectodermal requirement for pnr and ush in heart development is probably achieved via the maintenance of dpp expression, since dorsal stripe dpp expression diminishes in pnr and ush mutants and ectodermal interference with pnr, ush and/or dpp-signaling function compromises the normal progression of heart development (Klinedinst, 2003).
The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, the extent of amnioserosa and dorsal ectoderm was altered genetically, and LE cell fate was assayed. No expansion of LE fate is observed in dorsalized or ventralized mutants. Furthermore, the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together these data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. It is proposed that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells (Stronach, 2001).
Using mutations that influence DV patterning, it is possible to alter the size and distribution of BMP target gene expression patterns, which indicate the extent of amnioserosa and dorsal ectodermal cell fates. If LE fate was specified directly by a particular threshold level of BMP signal, then one would expect LE fate to be perturbed in concert with amnioserosa and dorsal ectoderm fates in DV mutants. Mutations in genes such as dorsal, Toll, brinker and short gastrulation alter the size of BMP target gene expression domains; however, these mutants failed to alter specification of LE fate. Among these genotypes, brk and sog specifically modulate the shape of the BMP signaling gradient in a region where LE fate might arise, yet LE formation in these mutants is fundamentally normal. Furthermore, in dorsalized embryos, LE cells were observed regularly at the boundary between amnioserosa and dorsal ectoderm even when the morphology of these tissues was severely disrupted. Islands of amnioserosa cells within a field of ectoderm were consistently surrounded with a single row of LE cells, independent of the number of amnioserosa cells constituting the island. The converse situation also occurred; again, a single row of LE cells formed at the boundary between the ectoderm and amnioserosa (Stronach, 2001).
DV mutants were also analyzed to determine whether a decrease in BMP signaling activity converts amnioserosa to LE as predicted by a gradient patterning model. A range of ventralizing mutations (cactus, sog, screw, dpp) displaying progressive loss of amnioserosa tissue did not give rise to embryos with an expanded domain of LE cells. In fact, LE cells were not detected in the absence of amnioserosa. No situation was found in which an altered BMP gradient was associated with expanded LE fate, thus the prediction of a direct gradient response model does not explain LE fate specification (Stronach, 2001).
Notably, DV mutant embryos that perturb the BMP gradient, also perturb the expression domains of target genes, including u-shaped and hindsight, without altering LE specification. However, loss of ush and hnt function results in specific and distinct perturbations in LE formation. Thus, the interpretation that LE fate specification is not a direct early response to the BMP gradient is favored, but rather is a secondary consequence of the specification of dorsal fates through the action of BMP target genes like ush and hnt (Stronach, 2001).
Taken together, these results raise the possibility that amnioserosa may be required for LE formation. To address the function of amnioserosa for LE specification, puc enhancer expression was examined in several mutants of the U-shaped class, including u-shaped (ush) and hindsight (hnt). Incidentally, the dorsal expression domains of these genes are directly regulated by DV patterning signals. In these mutant embryos, the amnioserosa tissue is fated normally and begins to differentiate up to stage 11, but then degenerates prematurely. In both ush and hnt mutants, programmed cell death takes place over the course of a few hours, with elimination of amnioserosa cells by stage 13 -- the time when dorsal closure would normally commence. Unexpectedly, different patterns of expression were observed with the puc enhancer in the two mutants. In ush embryos, ß-gal-positive cells were not detected. In contrast, hnt mutant embryos displayed Puc-positive LE cells at the edge of the dorsal ectoderm, albeit with less uniform expression than normally observed. To confirm these observations, the accumulation of dpp mRNA in the LE was examined. Similar to puc enhancer expression, differential expression of dpp was observed in ush versus hnt mutant embryos. ush mutant embryos show a consistent and significant reduction in LE dpp expression, although residual dpp transcripts are seen. dpp expression appears relatively normal in hnt mutant embryos (Stronach, 2001).
In addition to the differential expression of two LE markers in the U-shaped mutants, ectopic expression of LE markers is observed only in hnt mutant embryos. ß-gal-positive cells were observed in the region of the amnioserosa in hnt mutants as early as stage 11, raising the possibility that this could be an example of expanded LE cell fates. These cells adopt only partial LE cell fate, for the following reasons. These cells do not express the LE marker Fasciclin III, but do express two other LE molecules, albeit aberrantly. puc, for example, is expressed precociously in these cells, preceding Fasciclin III expression in the ectoderm, and dpp is rarely but reproducibly expressed. Additionally, cells in this region express amnioserosa fate markers such as race, through stage 11. Thus, based on the possibility that these cells may co-express LE and amnioserosa markers during stage 11, their identity cannot be unequivocally determined. These results may indicate that these cells are of mixed fate. The presence of ectopic LE-like cells in hnt mutant embryos, coupled with the severe reduction of LE fate markers in ush mutants, suggest that the distinction between amnioserosa and LE is a secondary consequence of Hnt and Ush functions, not a direct result of specific BMP signaling thresholds (Stronach, 2001).
If LE cells are specified as a secondary consequence of DV patterning gradients, then what additional mechanisms are at work to define LE as a single row of cells? The data are consistent with several mechanisms. One possibility is that specification of the LE involves the combinatorial action of nested sets of transcriptional regulators, including Hnt dorsally and Ush in a broader domain. Accordingly, loss of Hnt function is predicted to result in a failure to differentiate amnioserosa, coupled with dorsal expansion of more lateral fates, such as the LE. Consistent with this model, hnt mutant embryos display Puc-positive cells with partial LE character in the region of the dying amnioserosa during stage 11. These results suggest that Hnt may be necessary to distinguish amnioserosa from LE fate at the time of extended germ band stage. This timing is late, relative to the timing of the early BMP threshold response, further supporting the notion that LE specification is a secondary consequence of initial BMP signaling (Stronach, 2001).
Ush may play a role in differentiation of more lateral fates adjacent to the amnioserosa and the Hnt expression domain. Indeed, Ush function is essential for LE development because LE does not form in ush mutant embryos. Based on these results, it is imagined that Ush could define a competency zone from which LE cells arise, or Ush could participate in generating or modulating a signal(s) for communication between the differentiating amnioserosa and dorsal ectoderm. Ush is related to mammalian zinc-finger protein family, Friend of GATA (FOG), which has been shown to participate as a cofactor with GATA transcription factors. Together, these protein complexes regulate cell fate determination multiple times during both mammalian and Drosophila development. Interestingly, FOG2, a mammalian homolog of Ush, appears to be required during an inductive signaling event between two distinct tissues in the mouse heart, suggesting that inductive processes in development may commonly use the function of Ush family members. It has not been determined whether the function of Ush in LE cell specification is localized to the amnioserosa, the dorsal ectoderm, or both. Experiments to replace Ush function in a tissue-specific manner should address this issue (Stronach, 2001).
Although transcriptional targets of BMP signaling, such as ush and hnt, among others, define at least three specific threshold responses, the size difference between the nested expression domains of these markers still fails to account for a cell fate defined by a single row of cells. An additional mechanism to explain the spatially restricted stripe of LE cells is through an inductive signaling event. From the analysis of dorsalized mutants, it is observed that LE forms as a result of the juxtaposition of amnioserosa tissue with dorsal ectoderm, which may provide spatially limited activation of the JNK pathway. Thus, restricted expression of JNK target genes, such as puc and dpp may be a direct result of a signal that specifies LE (Stronach, 2001).
Communication between the amnioserosa and the dorsal ectoderm during embryogenesis has been suggested in two cases recently: (1) Hnt expression in the amnioserosa is required nonautonomously for proper cell rearrangements in the dorsal ectoderm, associated with retraction of the embryonic germband; (2) the raw gene product appears to be expressed in the amnioserosa, though it influences the activity of the JNK pathway in the ectoderm during dorsal closure. As amnioserosa and ectoderm develop, they may acquire different cell affinities, which cause them to sort into separate domains or islands (in the case of dorsalized embryos), displaying smooth borders at their interface. A difference in cell adhesion at the boundary may be sufficient to generate signaling for LE specification similar to inductive mechanisms at work at the compartmental boundaries of larval imaginal discs. The challenge now will be to identify molecules that may participate in an inductive signal (Stronach, 2001).
These results suggest that a multistep process determines the LE as a single row of cells. LE does not form directly in response to discrete intermediate levels of BMP signaling activity, but forms secondarily by the action of transcriptional regulators that are themselves BMP target genes. Among these targets, Hnt and Ush define a LE competency zone that is expanded in hnt mutants and eliminated in ush mutants. It is proposed that from within the competency zone, LE fate is further refined to a single row by an unknown inductive signal generated by the physical juxtaposition of amnioserosa with dorsal ectoderm. This signal activates the JNK pathway that regulates localized expression of dpp and puc (Stronach, 2001).
U-shaped is a zinc finger protein that functions predominantly as a negative transcriptional regulator of cell fate determination during Drosophila development. In the early stages of dorsal vessel formation, the protein acts to control cardioblast specification, working as a negative attenuator of the cardiogenic GATA factor Pannier. Pannier and the homeodomain protein Tinman normally work together to specify heart cells and activate cardioblast gene expression. One target of this positive regulation is a heart enhancer of the Drosophila mef2 gene and U-shaped has been shown to antagonize enhancer activation by Pannier and Tinman. Protein domains of U-shaped required for its repression of cardioblast gene expression were mapped. Such studies showed GATA factor interacting zinc fingers of U-shaped are required for enhancer repression, as well as three small motifs that are likely needed for co-factor binding and/or protein modification. These analyses have also allowed for the definition of a 253 amino acid interval of U-shaped that is essential for its nuclear localization. Together, these findings provide molecular insights into the function of U-shaped as a negative regulator of heart development in Drosophila (Tokusumi, 2007).
Through the use of an established assay to monitor Pannier-dependent cardioblast gene activity, and the generation and analysis of 20 different versions of the U-shaped protein, six U-shaped domains required for its repression of mef2 gene expression were identified. Three previously identified GATA-interacting zinc fingers of U-shaped are critical for this inhibitory property, which likely reflects the necessity of multiple zinc fingers forming a strong and stable interaction with the Pannier GATA factor. Whether Pannier-U-shaped complex formation interferes with the physical interaction of Pannier and Tinman in the synergistic activation of D-mef2 target sequences remains to be determined (Tokusumi, 2007).
U-shaped may also directly antagonize Pannier function as has been shown in the process of sensory bristle formation. Heterodimerization of U-shaped with Pannier converts the GATA transcriptional activator into a transcriptional repressor, an event that leads to the non-activation of target genes such as ac, sc, and wg in the dorsal notum of the wing disc. It is noteworthy that the results demonstrated the requirement of a binding site for the CtBP transcriptional co-repressor protein. In the context of the cardiogenic mesoderm, the combination of Pannier, U-shaped, and CtBP may prevent mesodermal cells from initiating gene expression programs needed for the specification of the cardioblast fate. In contrast, the combination of Pannier, Dorsocross, and Tinman is known to activate a regulatory network programming heart cell specification and cardioblast differentiation. Additional studies will be needed to elucidate the potential role of CtBP as an antagonist of cardiac gene expression and heart development. If U-shaped-CtBP interaction plays a crucial inhibitory role, then one would predict comparable dorsal vessel phenotypes for CtBP and U-shaped in loss- and gain-of-function genetic backgrounds (Tokusumi, 2007).
Finally, these studies have defined a 253 amino acid region required for nuclear localization of U-shaped. Within this interval, two highly basic amino acid sequences have been defined as being essential for U-shaped ability to inhibit Pannier-mediated cardiac gene expression. Perhaps, these motifs are required to facilitate the binding and stable interaction of co-repressor proteins with U-shaped. Another possibility is that these sequences serve as sites for post-translational modification, such as acetylation and/or methylation. Selective protein modification(s) may be a requisite for U-shaped to act as a negative modulator of Pannier transcription factor function during cardiogenesis in Drosophila (Tokusumi, 2007).
Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of immune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as 'non-self'. Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. This study shows that over-expression of the CoREST-interacting transcription factor Charlatan (Chn) in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. Double-positive intermediates between plasmatocytes and lamellocytes were observed, and it was shown that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, plasmatocytes were marked, and lineage tracing showed that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, these data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge (Stofanko, 2010).
Drosophila provide a genetically tractable model system to investigate cellular innate immune function. This report examined the origins of lamellocytes, which are Drosophila hemocytes that differentiate in response to parasite infestation. Over-expression of Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. The data indicate that Chn over-expression transforms plasmatocytes into lamellocytes. Consistent with this, double-positive intermediates between plasmatocytes and lamellocytes were detected, and it was shown that isolated plasmatocytes in vitro can be triggered to differentiate into lamellocytes following Chn over-expression. This property is not limited to Chn since it was observed that other stimuli, including activation of the JAK/STAT pathway and the natural response to parasitic wasp infestation, also induced lamellocyte formation from plasmatocytes (Stofanko, 2010).
The data suggest that Chn may control lamellocyte development. Previously defined regulators of lamellocyte development include the transcription factor STAT92E, the FOG-1 homologue Ush, and the NURF chromatin remodelling complex. STAT92E functions as an inducer of lamellocyte development, as gain-of-function hopTum-l mutants that activate the JAK/STAT pathway cause lamellocyte over-production. In contrast, both loss-of-function ush and Nurf mutants exhibit increased lamellocyte numbers. Like the homologous FOG-1-GATA-1 pairing, Ush modulates activity of the Drosophila GATA factor Srp to favour plasmatocyte differentiation. Recent data in mammalian systems indicates that FOG-1 mediates its effect on GATA-1 in part via recruitment of the transcriptional co-repressor NURD, suggesting that Ush functions similarly to repress expression of gene targets required for lamellocyte differentiation in plasmatocytes. Likewise, NURF also inhibits lamellocyte differentiation, in this case by preventing activation of targets of the JAK/STAT pathway (Stofanko, 2010).
The current biochemical data suggest that Chn is a transcription repressor since Chn recruits the co-repressor complex CoREST. Indeed it has been shown that Chn over-expression represses Delta expression in the eye imaginal disk, while this study has shown that Chn over-expression is accompanied by repression of some plasmatocyte markers. However, it was also shown that Chn over-expression leads to elevated expression of lamellocyte markers, and it has been demonstrated that Chn over-expression increases expression of the proneural genes Achaete and Scute. These data do not allow discrimination of whether Chn functions entirely as a transcriptional repressor or whether it may also activate transcription. However, the temporally-controlled Chn induction system (Pxn-Gal4 TARGET) that was utilized in this study will allow the primary gene targets of Chn to be determined. By analyzing transcriptional profiles of hemocytes at defined time points after Chn over-expression the primary responders to Chn over-expression will be able to be identified. It will be possible to discriminate whether these targets are preferentially activated or repressed, and also subsequently determine recruitment of transcription co-activator or co-repressor complexes such as CoREST at these targets using chromatin immunoprecipitation (Stofanko, 2010).
The data demonstrating that lamellocytes can originate from plasmatocytes sheds new light on hemocyte lineages. Current models of hemocyte lineages speculate that plasmatocytes, crystal cells and lamellocytes are distinct lineages that arise separately from a common stem cell or prohemocyte. This study proposes, however, that prohemocytes generate either crystal cells or plasmatocytes. It is suggested that plasmatocytes are a plastic population that can generate other frequently observed hemocyte types including lamellocytes. This model is strikingly reminiscent of the initial hemocyte lineages first proposed more than 50 years ago. According to that analysis prohemocytes were predicted to generate either crystal cells or plasmatocytes, with plasmatocytes differentiating further into activated cells (podocytes) and then lamellocytes. This model has support from a number of experimental studies including this study. Foremost among these are recent studies of hemocyte functions of Ush. Dominant-negative Ush variants are able to induce lamellocyte differentiation and it has been suggested that Ush regulates lamellocyte differentiation from a potential plasmatocyte. Secondly, lamellocyte differentiation in response to Salmonella infection is blocked in decapentaplegic mutants with a corresponding increase in plasmatocyte number, suggesting that lamellocytes arise from plasmatocytes or a common precursor (Stofanko, 2010).
Two recent studies also suggest that plasmatocytes are a plastic population that may be able to differentiate into lamellocytes. Marking of embryonic plasmatocytes using the gcm-GAL4 or sn-GAL4 drivers and an act5C>stop>GAL4 flip-out transgene shows that lamellocytes that arise in larvae after wasp infestation may originate from cells that had expressed gcm-GAL4 or sn-GAL4 in embryos. Similar results have also been observed using the act5C>stop>GAL4 flip-out transgene and Pxn-GAL4 and eater-GAL4. In both these cases the elicitor of the FLP/FRT activation event and the subsequent sustained marker are the same, namely GAL4 expression. However, in the current lineage tracing experiments, GAL4 expression initiates the FLP/FRT activation of a distinct marker, lacZ protein. These data, taken together with lineage tracing experiments and in vitro differentiation studies suggest that the plasmatocyte is an inherently plastic cell type that is capable of being reprogrammed to tailor immune responses to suit the infectious threats faced by the host. In humans, lymphocyte and leukocyte plasticity has a significant impact on immune responses. An important future challenge is to establish the full spectrum of Drosophila plasmatocyte heterogeneity and exploit the utility of the Drosophila genetic system to dissect the mechanisms that regulate such leukocyte plasticity (Stofanko, 2010).
Drosophila heart development is an invaluable system to study the orchestrated action of numerous factors that govern cardiogenesis. Cardiac progenitors arise within specific dorsal mesodermal regions that are under the influence of temporally coordinated actions of multiple signaling pathways. The Drosophila Iroquois complex (Iro-C) consists of the three homeobox transcription factors araucan (ara), caupolican (caup) and mirror (mirr). The Iro-C has been shown to be involved in tissue patterning leading to the differentiation of specific structures, such as the lateral notum and dorsal head structures and in establishing the dorsal-ventral border of the eye. A function for Iro-C in cardiogenesis has not been investigated yet. Loss of the whole Iroquois complex, as well as loss of either ara/caup or mirr only, affect heart development in Drosophila. The data indicate that the GATA factor Pannier requires the presence of Iro-C to function in cardiogenesis. A detailed expression pattern analysis of the members of the Iro-C revealed the presence of a possibly novel subpopulation of Even-skipped expressing pericardial cells and seven pairs of heart-associated cells that have not been described before. Taken together, this work introduces Iro-C as a new set of transcription factors that are required for normal development of the heart. As the members of the Iro-C may function, at least partly, as competence factors in the dorsal mesoderm, these results are fundamental for future studies aiming to decipher the regulatory interactions between factors that determine different cell fates in the dorsal mesoderm (Mirzoyan, 2013).
Tissue patterning requires the spatial and temporal coordinated action of signals providing instructive or permissive cues that result in the specification of different cell types and their subsequent differentiation into different lineages. This analyses of Iro-C deficient embryos demonstrate that ara/caup and mirr are required in the dorsal mesoderm for normal heart development. The heart phenotypes could be caused by alterations of the fine balance of the interactions between factors of the cardiac signaling network. In early stage Drosophila embryos the mesoderm is patterned along the anterior-posterior (AP) axis with cardiac and somatic mesodermal domains alternating with visceral mesodermal domains. The tin-positive mesoderm is specified as cardiac and somatic mesoderm under the influence of combined Dpp and Wg signaling. Subsequently, the cardiac and somatic mesodermal domains are further subdivided by the action of the Notch pathway and MAPK signaling activated by EGFR and FGFR. The Eve-expressing cell clusters that give rise to pericardial and DA1 somatic muscle cells, as well as the Doc expression pattern, distinguish the cardiac and somatic mesodermal domain from the visceral mesodermal domain. The early expression pattern of Ara/Caup and Mirr at stages 10/11 suggests a role for Iro-C in patterning the dorsal mesoderm along the AP axis. Consistent with their previously described functions in other developmental contexts, members of the Iro-C may integrate signaling inputs and interact with other transcription factors to specify different dorsal mesodermal derivatives. Activation of the Iro-C by the EGFR pathway is required for the specification of the notum. Mirr was shown to interpret EGFR signaling by eliciting a specific cellular response required for patterning the follicular epithelium. During Drosophila eye development, mirr expression can be regulated by Unpaired, a ligand that activates JAK/Stat signaling. In fact, the JAK/Stat signaling pathway has only recently been added to the signaling pathways that function in Drosophila cardiogenesis. In chromatin immunoprecipitation experiments caup was identified as a target of Stat92E, which is the sole transcriptional effector of the JAK/Stat signaling pathway in Drosophila. Interestingly, the increase of Odd-pericardial cells and the additional Tin-expressing cells that were the characteristic phenotypes in ara/caup (iroDFM1) and in mirr (mirre48) mutants are highly similar to the phenotypes in stat92E mutants described by Johnson (Johnson, 2011). Also, as described for stat92E mutants, cell adhesion defects were noticed in a number of embryos as determined by the distant location of some Tin-expressing cells from the forming heart tube. As for establishing a possible link between JAK/Stat and Iro-C in the dorsal mesoderm and specifically in cardiogenesis, it would be necessary to determine for example whether caup and mirr can rescue the heart phenotype of stat92E mutants. Also, it would be interesting to compare the expression of the other crucial heart marker genes, Tup, Doc and Pnr, in stat92E mutants at early stages to determine to what extent the phenotypes of embryos mutant for Iro-C and for JAK/Stat signaling are similar (Mirzoyan, 2013).
Members of the Iro-C were shown to be positively or negatively regulated by signaling pathways that play crucial roles in heart development. Conversely, the Iro-C factors can also regulate the activity of at least one of these pathways. Specifically, Ara/Caup, as well as Mirr were shown to regulate the expression of the glycosyltransferase fringe and as a consequence modulate Notch signaling activity in the eye. In the dorsal mesoderm, the lateral inhibitory function of Notch signaling establishes the proper number of heart and muscle progenitors. Given the fact that Iro-C can regulate Notch activity it may be that the loss of Iro-C leads to an imbalance of progenitor cell specification resulting in an abnormal number of heart cells. Further studies are required to decipher the molecular mechanism by which Iro-C could integrate diverse signaling inputs and thereby function in the specification and differentiation of the different dorsal mesodermal derivatives (Mirzoyan, 2013).
To determine whether Iro-C can be positioned into the early transcriptional network that determines a cardiac lineage, this study investigated the interdependency between crucial cardiac factors and Iro-C during cardiogenesis. Analyses of the expression of Ara/Caup and Mirr in tin346, Df(3L)DocA, pnrVX6 and tupisl-1 embryos demonstrated the dependency of Ara/Caup and Mirr on all four factors. The strongest loss of Ara/Caup and Mirr expression was observed in tin346 and Df(3L)DocA mutants, which clearly places tin and Doc upstream of Ara/Caup and Mirr. In tupisl-1 and in pnrVX6 mutant embryos, Ara/Caup and Mirr were strongly downregulated, however regarding Ara/Caup, some expression remained in segmental patches suggesting a different level of regulation. The currently available data indicates a positive and a negative regulatory effect of pnr on Iro-C. Whereas pnr restricts Iro-C expression in the dorsal ectoderm and in the wing disc, there is also evidence that pnr can positively regulate Iro-C in the wing disc. Whether Pnr activates or represses Iro-C appears to depend on the presence of U-shaped (Ush), a protein that modulates the transcriptional activity of Pnr. In the wing disc it was shown that an Iro-C-lacZ (IroRE2-lacZ) construct was activated in cells that contained Pnr but were devoid of Ush. The data demonstrate that in the dorsal mesoderm, the expression of Ara/Caup and Mirr depends on pnr. Additionally this analyses show that pnr expression is independent of Iro-C. This finding is intriguing with respect to the downregulation of Tup and Doc in Iro-C mutants. Pnr is required for the maintenance of Doc and for the initiation and/or maintenance of Tup. Since Iro-C mutants exhibit a reduction in Doc-positive cells despite the presence of pnr, members of the Iro-C appear to be required independently or in addition to pnr to maintain expression of Doc. This could be investigated by expressing ara, caup and/or mirr in the mesoderm of pnr mutants to determine whether these factors are able to restore Doc expression. Alternatively, it may be that Iro-C is required indirectly meaning that its main function is to provide a molecular context in which Pnr can be active. For example, it is known that Ush can bind to Pnr thereby inactivating Pnr function. It is conceivable that the absence of Iro-C affects the spatial expression of Ush. If, in the absence of Iro-C, the expression domain of Ush shifts into the Pnr expression domain, Ush could bind to Pnr and inactivate it in the region where Pnr is required to maintain the expression of Tup and Doc. Adding to the complexity of the interpretation of the observed phenotypes is the finding that the majority of embryos that are mutant for ara/caup or for mirr were characterized by supernumerary Tin-positive cells in the cardiac region by stage 11/12. This phenotype could still be observed at later stages when the heart tube forms. The additional Tin-positive cells are pericardial cells as determined by the expression of Prc around the Tin-expressing cells. Also, no increase was observed of Dmef2-positive myocardial cells. Hence, the data suggests a different level of regulation of Tin by the Iro-C. Similar to the findings of Johnson (2011), it may be that Iro-C is normally required to restrict Tin expression at an early stage. The regulation of Tin expression can be divided into four phases. The phenotype this study observed occurs when Tin expression becomes restricted to the myo- and pericardial cells in the cardiac region. In summary, the data adds Iro-C to tin, pnr, Doc and tup whose concerted actions establish the cardiac domains in the dorsal mesoderm. Further studies are required to re-evaluate the current understanding of the interactions between factors of the cardiac transcriptional network (Mirzoyan, 2013).
According to the expression pattern of Ara/Caup and Mirr it was possible to distinguish between an early and late role for these factors, the latter being a role in the differentiation of heart cells (Mirzoyan, 2013).
This analyses of the expression of Ara/Caup and Mirr during embryogenesis led to the identification of hitherto unknown heart-associated cells. Seven pairs of Ara/Caup and Mirr expressing cells and seven pairs of Mirr only expressing cells were detected that were located along the dorsal vessel. No co-expression was detected with any of the known pericardial cell markers. Because there are seven pairs of these cells segmentally arranged, it was tempting to speculate that these cells may function, for example, as additional attachment sites for the seven pairs of alary muscles. The alary muscles attach the heart to the dorsal epidermis and their extensions can be visualized by Prc. Due to the lack of markers little is known about the development of the alary muscles. Previous work demonstrated that the alary muscles attach to the dorsal vessel in the vicinity of the Svp pericardial cells and, in addition, more laterally to one of two distinct locations on the body wall. Maybe it is the Mirr-positive cells that identify the more lateral locations. Clearly, a detailed analysis is needed to identify the function of the Ara/Caup- and Mirr- as well as Mirr-expressing cells that are positioned along the heart tube and whose existence has now been revealed. Additionally, on each side at the anterior end of the dorsal vessel four pericardial cells were identified that co-express Ara/Caup and Eve. Their location at the anterior tip of the heart is intriguing. Further analysis is required to unambiguously determine whether these cells are, for example, the wing heart progenitor cells or the newly identified heart anchoring cells. It is also possible that they represent a yet undefined, novel subpopulation of pericardial cells. In any case, this finding suggests that Ara/Caup plays a role in the diversification of pericardial heart cell types. Future experiments aim to determine the developmental fate of these cells (Mirzoyan, 2013).
Taken together, this investigation of a role for Iro-C in heart development introduces ara/caup and mirr as additional components of the transcriptional network that acts in the dorsal mesoderm and as novel factors that function in the diversification of heart cell types (Mirzoyan, 2013).
The results show that the role of the Iro-C and its individual members, respectively, appears to be rather complex and awaits in-depth analyses. Nevertheless, this work raises important questions regarding the current understanding of interactions between the well-characterized transcription factors that will be addressed in future studies (Mirzoyan, 2013).
Germ band retraction involves a dramatic rearrangement of the tissues on the surface of the Drosophila embryo. As germ band retraction commences, one tissue, the germ band, wraps around another, the amnioserosa. Through retraction the two tissues move cohesively as the highly elongated cells of the amnioserosa contract and the germ band moves so it is only on one side of the embryo. To understand the mechanical drivers of this process, a series of laser ablations was designed that suggest a mechanical role for the amnioserosa. first, it was found that during mid retraction, segments in the curve of the germ band are under anisotropic tension. The largest tensions are in the direction in which the amnioserosa contracts. Second, ablating one lateral flank of the amnioserosa reduces the observed force anisotropy and leads to retraction failures. The other intact flank of amnioserosa is insufficient to drive retraction, but can support some germ band cell elongation and is thus not a full phenocopy of ush mutants. Another ablation-induced failure in retraction can phenocopy mys mutants, and does so by targeting amnioserosa cells in the same region where the mutant fails to adhere to the germ band. It is concluded that the amnioserosa must play a key, but assistive, mechanical role that aids uncurling of the germ band (Lynch, 2013).
The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, a novel, multitype zinc finger protein, Friend of GATA-1 (FOG) has been identified, which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors (Tsang, 1997).
GATA transcription factors are required for the differentiation of diverse cell types in several species. Recent evidence suggests that their biologic activities may be modulated through interaction with multitype zinc finger proteins, such as Friend of GATA-1 (FOG) and U-shaped (Ush). In cell culture, FOG cooperates with the hematopoietic transcription factor GATA-1 to promote erythroid and megakaryocytic differentiation. Mice lacking FOG die during mid-embryonic development with severe anemia. FOG-/- erythroid cells display a marked, but partial, blockage of maturation, reminiscent of GATA-1- erythroid precursors. In contrast to GATA-1 deficiency, however, megakaryocytes fail to develop in the absence of FOG. Although the FOG-/- erythroid phenotype supports the proposed role of FOG as a GATA-1 cofactor in vivo, the latter finding points to a pivotal, GATA-1-independent requirement for FOG in megakaryocyte development from the bipotential erythroid/megakaryocytic progenitor. It is speculated that FOG and other FOG-like proteins serve as complex cofactors that act through both GATA-dependent and GATA-independent mechanisms (Tsang, 1998).
Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. The interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA), has been investigated. This interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. The contact domains have been mapped to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy, key residues within the GATA-1 N-finger that are required for FOG binding have been identified. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1 (Fox, 1998).
Friend of GATA-1 (FOG-1) is a zinc finger protein that has been shown to interact physically with the erythroid DNA-binding protein GATA-1 and modulate its transcriptional activity. Recently, two new members of the FOG family have been identified: a mammalian protein, FOG-2 (which also associates with GATA-1 and other mammalian GATA factors), and U-shaped, a Drosophila protein that interacts with the Drosophila GATA protein Pannier. FOG proteins contain multiple zinc fingers and the sixth finger of FOG-1 is known to interact specifically with the N-finger but not the C-finger of GATA-1. Fingers 1, 5 and 9 of FOG-1, all atypical Cys-Cys:His-Cys fingers, also interact with the N-finger of GATA-1; FOG-2 and U-shaped also contain multiple GATA-interacting fingers and both FOG-2 and U-shaped contain several Cys-Cys:His-Cys zinc fingers. The key contact residues are defined and these residues are shown to be highly conserved in GATA-interacting fingers. The effects of selectively mutating the four interacting fingers of FOG-1 were examined and each is shown to contribute to FOG-1's ability to modulate GATA-1 activity. FOG-1 can repress GATA-1-mediated activation: evidence is presented that this ability involves the recently described CtBP co-repressor proteins that recognize all known FOG proteins (Fox, 1999).
The mechanism by which FOG-1 acts to repress GATA-mediated transcription was investigated. FOG-1 contains a motif that is bound by the CtBP family of co-repressors. This site PIDLSKR occurs immediately N-terminal to finger 7. Yeast two-hybrid and GST pull-down assays were used to test whether a small region of FOG-1 (residues 724-834, spanning the CtBP-binding motif) could interact with one family member, mCtBP2. GST-FOG-1(724-834) can retain in vitro-translated mCtBP2 efficiently, whereas a mutant FOG-1 containing a mutation in the core region (PIDLSKR to AIAASKR) is unable to retain mCtBP2. Similarly, in the yeast two-hybrid system, FOG-1(724-834) is able to interact with mCtBP2, whereas the mutant cannot. To test if this region of FOG-1 can act as a repression domain in vivo, fusions of FOG-1(724-834) (both wild-type and mutant) with the Gal4 DNA-binding domain were prepared and these were co-transfected with a construct harbouring a Gal4-dependent promoter upstream of the human growth hormone reporter gene. Gal4DBD-FOG-1(724-834) represses the basal reporter activity 20-fold. However, the mutant is unable to significantly repress transcription. This result indicates that FOG-1 contains a repression domain that can mediate repression by associating with CtBP family proteins (Fox, 1999).
The FOG-2 gene was disrupted in mice to define its requirement in vivo. FOG-2(-/-) embryos die at midgestation with a cardiac defect characterized by a thin ventricular myocardium, common atrioventricular canal, and the tetralogy of Fallot malformation. Remarkably, coronary vasculature is absent in FOG-2(-/-) hearts. Despite formation of an intact epicardial layer and expression of epicardium-specific genes, markers of cardiac vessel development (ICAM-2 and FLK-1) are not detected, indicative of failure to activate their expression and/or to initiate the epithelial to mesenchymal transformation of epicardial cells. Transgenic reexpression of FOG-2 in cardiomyocytes rescues the FOG-2(-/-) vascular phenotype, demonstrating that FOG-2 function in myocardium is required and sufficient for coronary vessel development. These findings provide the molecular inroad into the induction of coronary vasculature by myocardium in the developing heart (Tevosian, 2000).
Sox is a large family of genes related to the sex-determining region Y gene (designated as the SRY gene), In mammals, Sry expression in the bipotential, undifferentiated gonad directs the support cell precursors to differentiate as Sertoli cells, thus initiating the testis differentiation pathway. In the absence of Sry, or if Sry is expressed at insufficient levels, the support cell precursors differentiate as granulosa cells, thus initiating the ovarian pathway. The molecular mechanisms upstream and downstream of Sry are not well understood. The transcription factor GATA4 and its co-factor FOG2 are required for gonadal differentiation. Mouse fetuses homozygous for a null allele of Fog2 or homozygous for a targeted mutation in Gata4 (Gata4ki) that abrogates the interaction of GATA4 with FOG co-factors exhibit abnormalities in gonadogenesis. Sry transcript levels are significantly reduced in XY Fog2/ gonads at E11.5, which is the time when Sry expression normally reaches its peak. In addition, three genes crucial for normal Sertoli cell function (Sox9, Mis and Dhh) and three Leydig cell steroid biosynthetic enzymes (p450scc, 3ßHSD and p450c17) are not expressed in XY Fog2/ and Gataki/ki gonads, whereas Wnt4, a gene required for normal ovarian development, is expressed ectopically. By contrast, Wt1 and Sf1, which are expressed prior to Sry and necessary for gonad development in both sexes, are expressed normally in both types of mutant XY gonads. These results indicate that GATA4 and FOG2 and their physical interaction are required for normal gonadal development (Tevosian, 2002).
GATA transcription factors are important regulators of both hematopoiesis (GATA-1/2/3) and cardiogenesis (GATA-4) in mammals. The transcriptional activities of the GATA proteins are modulated by their interactions with other transcription factors and with transcriptional coactivators and repressors. Recently, two related zinc finger proteins, U-shaped (Ush) and Friend of GATA-1 (FOG) have been reported to interact with the GATA proteins Pannier and GATA-1, respectively, and to modulate their transcriptional activities in vitro and in vivo. In this report, the molecular cloning and characterization of a third FOG-related protein, FOG-2 is described. FOG-2 is an 1,151 amino acid nuclear protein that contains eight zinc finger motifs that are structurally related to those of both FOG and Ush. FOG-2 is first expressed in the mouse embryonic heart and septum transversum at embryonic day 8.5 and is subsequently expressed in the developing neuroepithelium and urogenital ridge. In the adult, FOG-2 is expressed predominately in the heart, brain, and testis. FOG-2 associates physically with the N-terminal zinc finger of GATA-4 both in vitro and in vivo. This interaction appears to modulate specifically the transcriptional activity of GATA-4 because overexpression of FOG-2 in both NIH 3T3 cells and primary rat cardiomyocytes represses GATA-4-dependent transcription from multiple cardiac-restricted promoters. Taken together, these results implicate FOG-2 as a novel modulator of GATA-4 function during cardiac development and suggest a paradigm in which tissue-specific interactions between different FOG and GATA proteins regulate the differentiation of distinct mesodermal cell lineages (Svensson, 1999).
Tricuspid atresia (TA) is a common form of congenital heart disease, accounting for 1%-3% of congenital cardiac disorders. TA is characterized by the congenital agenesis of the tricuspid valve connecting the right atrium to the right ventricle and both an atrial septal defect (ASD) and a ventricular septal defect (VSD). Some patients also have pulmonic stenosis, persistence of a left-sided superior vena cava or transposition of the great arteries. Most cases of TA are sporadic, but familial occurrences with disease in multiple siblings have been reported. Gata4 is a zinc-finger transcription factor with a role in early cardiac development. Gata4-deficient mice fail to form a ventral heart tube and die of circulatory failure at embryonic day (E) 8.5. Zfpm2 (also known as Fog-2) is a multi-zinc-finger protein that is co-expressed with Gata4 in the developing heart beginning at E8.5. Zfpm2 interacts specifically with the N-terminal zinc finger of Gata4 and represses Gata4-dependent transcription. Targeted mutagenesis was used to explore the role of Zfpm2 in normal cardiac development. Zfpm2-deficient mice die of congestive heart failure at E13 with a syndrome of tricuspid atresia that includes an absent tricuspid valve, a large ASD, a VSD, an elongated left ventricular outflow tract, rightward displacement of the aortic valve and pulmonic stenosis. These mice also display hypoplasia of the compact zone of the left ventricle. These findings indicate the importance of Zfpm2 in the normal looping and septation of the heart and suggest a genetic basis for the syndrome of tricuspid atresia (Svensson, 2000a).
GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 a functional analysis of the FOG-2 protein was performed. The results demonstrate that (1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins; (2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and (3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, a novel N-terminal domain of FOG-2 (amino acids 1-247) has been identifed that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development (Svensson, 2000b).
Members of the GATA family of zinc-finger transcription factors have critical roles in a variety of cell types. GATA-1, GATA-2 and GATA-3 are required for proliferation and differentiation of several hematopoietic lineages, whereas GATA-4, GATA-5 and GATA-6 activate cardiac and endoderm gene expression programs. Two GATA cofactors have recently been identified. Friend of GATA-1 (FOG-1) interacts with GATA-1 and is expressed principally in hematopoietic lineages, whereas FOG-2 is expressed predominantly in heart and brain. Although gene targeting experiments are consistent with an essential role for FOG-1 as an activator of GATA-1 function, reporter assays in transfected cells indicate that FOG-1 and FOG-2 can act as repressors. A Xenopus laevis homolog of FOG has been cloned that is structurally most similar to FOG-1, but is expressed predominantly in heart and brain, as well as the ventral blood island and adult spleen. Ectopic expression and explant assays demonstrate that FOG proteins can act as repressors in vivo, in part through interaction with the transcriptional co-repressor, C-terminal Binding Protein (CtBP). FOG may regulate the differentiation of red blood cells by modulating expression and activity of GATA-1 and GATA-2. It is proposed that the FOG proteins participate in the switch from progenitor proliferation to red blood cell maturation and differentiation (Deconinck, 2000).
The commitment of multipotent cells to particular developmental pathways requires specific changes in their transcription factor complement to generate the patterns of gene expression characteristic of specialized cell types. The role of the GATA cofactor Friend of GATA in the differentiation of avian multipotent hematopoietic progenitors has been examined. Multipotent cells express high levels of FOG mRNA, which are rapidly down-regulated upon their C/EBP-mediated commitment to the eosinophil lineage. Expression of FOG in eosinophils leads to a loss of eosinophil markers and the acquisition of a multipotent phenotype, and constitutive expression of FOG in multipotent progenitors blocks activation of eosinophil-specific gene expression by C/EBPbeta. These results show that FOG is a repressor of the eosinophil lineage, and that C/EBPbeta-mediated down-regulation of FOG is a critical step in eosinophil lineage commitment. Furthermore, the results indicate that maintenance of a multipotent state in hematopoiesis is achieved through cooperation between FOG and GATA-1. A model is presented in which C/EBPbeta induces eosinophil differentiation by the coordinate direct activation of eosinophil-specific promoters and the removal of FOG, a promoter of multipotency as well as a repressor of eosinophil gene expression (Querfurth, 2000).
GATA-4 has been implicated in formation of the vertebrate heart. Since the mouse Gata-4 knock-out is early embryonic lethal because of a defect in ventral morphogenesis, the in vivo function of this factor in heart development remains unresolved. To search for a requirement for Gata4 in heart development, mice were created harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2, a homolog of Drosophila U-shaped. Gata4ki/ki mice die just after embryonic day 12.5, exhibiting features in common with Fog2-/- embryos as well as additional semilunar cardiac valve defects and a double-outlet right ventricle. These findings establish an intrinsic requirement for GATA-4 in heart development. It is inferred that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation (Crispino, 2001).
The power of this analysis rests on the exquisite specificity of the knock-in mutation within the N finger of GATA-4. The residue that was modified is required for physical interaction with FOG-like proteins and does not influence the DNA-binding specificity of the GATA-protein. Although displaying many similar features, Gata4ki/ki hearts are distinguished from Fog2-/- hearts, however, by the presence of a double-outlet right ventricle and defects in the semilunar valves and outflow tracts. Since immunostaining confirms that GATA-4 is expressed at wild-type levels in the semilunar valves of the Gata4 mutant heart, it is likely that another FOG, or FOG-like protein, that functions as a cofactor for GATA-4 in transcription, is expressed in these valve cells. Though high-level expression of the only other known vertebrate FOG-like factor, FOG-1, has not previously been observed by in situ RNA hybridization, FOG-1 transcripts are present at low levels in Northern blots of total heart RNA. Thus, it is quite possible that disruption of the physical interaction between GATA-4 and FOG-1, or a novel, undefined FOG protein, is responsible for impaired development of the semilunar valves and the appearance of a double-outlet right ventricle (Crispino, 2001).
Given the profound effects of mutation of either GATA-4 or FOG-2 proteins on heart morphogenesis in mice, it is worth considering their potential relevance to human congenital heart defects, such as the Tetralogy of Fallot or the double-outlet right ventricle. Whereas mutation of several genes, such as Jmj (Jumanji), Sox4, and Egfr/Shp2, give rise to the double-outlet right ventricle defect in mice, and defects in other genes for transcription factors, such as FOG-2, NF1, neurotrophin 3, and RXR, result in all or a subset of the Tetralogy of Fallot, the consistent and combined phenotype seen in the Gata4ki/ki mice is unique. Although it is sometimes difficult to distinguish between double-outlet right ventricle with associated pulmonary stenosis and the Tetralogy of Fallot, it is clear that the defects in the Gata4ki/ki hearts are different in the outflow tracts than those observed in Fog2-deficient embryos (Crispino, 2001).
Through the use of an altered specificity mutant, it has been demonstrated that GATA-4 very likely requires both FOG-2 and an additional FOG, or FOG-like protein, as cofactors for distinct aspects of heart development. Interaction with FOG-2 is essential for the initiation of coronary vasculature and for some morphogenetic events, whereas interaction with a distinct FOG protein appears to be required for formation of cardiac valves. The results are surprising in that two other GATA-factors, GATA-5 and GATA-6, are also expressed in myocardium, and indirect data have suggested that they might compensate for the absence of GATA-4. For example, previous studies show that GATA-4 is dispensable for terminal differentiation of cardiomyocytes and that Gata4-/- ES cells contribute to all layers of the heart. In these experiments, it has been suggested that GATA-5 or GATA-6 functionally replace GATA-4. It is possible that proper expression of the GATA-4ki/ki protein, as distinguished from the absence of GATA-4 in the knock-out situation, precludes compensation by other GATA factors. Indeed, immunostaining with an alpha-GATA-6 antibody has demonstrated that GATA-6 expression, though similar to that of GATA-4, is not up-regulated in the Gata4ki/ki hearts. In addition, staining with alpha-GATA-5 antibody reveals a normal pattern in Gata4ki/ki hearts. Since GATA-5 is no longer expressed within the ventricles of the heart at E12.5, it is unlikely that it would compensate for the absence of functional GATA-4 (Crispino, 2001).
These findings implicate GATA-4 as the principal GATA factor relevant to heart morphogenesis and coronary vasculature development and as the primary partner for FOG proteins in the heart. This represents the second example of transcriptional regulation involving GATA-FOG protein complexes and argues for their broad involvement as key regulators of multiple developmental pathways (Crispino, 2001).
The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. The molecular basis for the megakaryocyte-restricted activation of the aIIb gene has been examined. FOG-1 stimulates GATA-1-dependent aIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the aIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. These studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes (Wang, 2002).
GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, the three-dimensional structure of a complex comprising the interaction domains of these proteins has been determined. The structure reveals how zinc fingers can act as protein recognition motifs. Notably, none of the FOG ZnFs that contact GATA-1 are part of tandem arrays of ZnFs. Thousands of such 'isolated' ZnFs exist, and it is likely that many serve as protein recognition motifs. The surface used by FOG ZnFs to recognize GATA-1 overlaps with the surface normally used by classical ZnFs to bind to DNA, indicating that the classical ZnF has acted throughout evolution as a versatile structural scaffold, onto which different binding functions have been 'grafted'. In line with this idea, the third classical ZnF from FOG has been shown to mediates a specific interaction with the coiled-coil protein TACC3. Indeed, given that a single classical ZnF is capable of mediating protein-protein interactions, and that an array of such domains is necessary for high affinity DNA binding, it is likely that the latter function arose later as a consequence of gene duplication events (Liew, 2004).
Inderstanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the ascidian mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal β-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ciona friend of GATA gene (Ci-fog) is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. This study has identified a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program (Rothbacher, 2007).
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 (see Drosophila Beadex) 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).
Search PubMed for articles about Drosophila u-shaped
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Crispino, J. D., et al. (2001). Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes Dev. 15: 839-844. 11297508
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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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date revised: 11 June 2022
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