Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster

Both Proboscipedia (PB) and Sex Combs Reduced (SCR) activities are required for determination of proboscis identity. Here we show that simultaneous removal of PB and SCR activity results in a proboscis-to-antenna transformation. Dominant negative PB molecules inhibit the activity of SCR indicating that PB and SCR interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of PB and SCR and the ability of PB and SCR to interact in a multimeric complex control the determination of four adult structures. The absence of PB and SCR expression leads to antennal identity; expression of only PB leads to maxillary palp identity; expression of only SCR leads to tarsus identity; and expression of both PB and SCR, which results in the formation of a PB-SCR-containing complex, leads to proboscis identity. However, the PB-SCR interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the PB-SCR interaction may be regulated and indirect. This regulation may also explain why ectopic expression of SCR(Q50K) and SCR do not result in the expected transformation of the maxillary palp to an antennae and proboscis, respectively. Previous analysis of the requirements of SCR activity for adult pattern formation has shown that ectopic expression of SCR results in an antenna-to-tarsus transformation, but removal of SCR activity in a clone of cells does not result in a tarsus-to-arista transformation. Here we show in five independent assays the reason for this apparent contradictory requirement of SCR activity in tarsus determination. SCR activity is required cell nonautonomously for tarsus determination. Specifically, we propose that SCR activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity. This study characterizes a combinatorial interaction between two HOX proteins; a mechanism that may have a major role in patterning the anterior-posterior axis of other animals.

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Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis

Development ◽

10.1242/dev.104.4.713 ◽

1988 ◽

Vol 104 (4) ◽

pp. 713-720 ◽

Cited By ~ 19

Author(s):

A. Busturia ◽

G. Morata

Keyword(s):

Ectopic Expression ◽

Homeotic Genes ◽

Sex Combs Reduced ◽

Hierarchical Order ◽

Sex Combs ◽

Morphological Patterns

The morphological patterns in the adult cuticle of Drosophila are determined principally by the homeotic genes of the bithorax and Antennapedia complexes. We find that many of these genes become indiscriminately active in the adult epidermis when the Pc gene is eliminated. By using the Pc3 mutation and various BX-C mutant combinations, we have generated clones of imaginal cells possessing different combinations of active homeotic genes. We find that, in the absence of BX-C genes, Pc- clones develop prothoracic patterns; this is probably due to the activity of Sex combs reduced which overrules Antennapedia. Adding contributions of Ultrabithorax, abdominal-A and Abdominal-B results in thoracic or abdominal patterns. We have established a hierarchical order among these genes: Antp less than Scr less than Ubx less than abd-A less than Abd-B. In addition, we show that the engrailed gene is ectopically active in Pc- imaginal cells.

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Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

Development ◽

10.1242/dev.126.22.5137 ◽

1999 ◽

Vol 126 (22) ◽

pp. 5137-5148 ◽

Cited By ~ 8

Author(s):

H.D. Ryoo ◽

T. Marty ◽

F. Casares ◽

M. Affolter ◽

R.S. Mann

Keyword(s):

Nuclear Localization ◽

Target Genes ◽

Dominant Negative ◽

Homeodomain Protein ◽

Hox Proteins ◽

Trimeric Complex ◽

Binding Residue ◽

Dominant Negative Form

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

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Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling

Development ◽

10.1242/dev.126.16.3607 ◽

1999 ◽

Vol 126 (16) ◽

pp. 3607-3616 ◽

Cited By ~ 3

Author(s):

Y. Chen ◽

J.R. Cardinaux ◽

R.H. Goodman ◽

S.M. Smolik

Keyword(s):

Gene Expression ◽

Target Gene ◽

Ectopic Expression ◽

Proteolytic Processing ◽

Dominant Negative ◽

Cubitus Interruptus ◽

Target Gene Expression ◽

Exogenous Expression

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

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Developmental role of endogenous retinoids in the determination of morphallactic field in budding tunicates

Development ◽

10.1242/dev.117.3.835 ◽

1993 ◽

Vol 117 (3) ◽

pp. 835-845 ◽

Cited By ~ 1

Author(s):

K. Kawamura ◽

K. Hara ◽

S. Fujiwara

Keyword(s):

Retinoic Acid ◽

Aldehyde Dehydrogenase ◽

Ectopic Expression ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Proximal Extremity ◽

Developmental Role

We have extracted retinoids from the budding tunicate Polyandrocarpa misakiensis and, using HPLC, identified some major peaks as cis-retinal, all-trans-retinal and all-trans-retinoic acid, of which cis-retinal was most abundant (~2 micromolar). In developing buds, the amount of cis-retinal was about one-fifth that of the adult animals. In those buds, aldehyde dehydrogenase, which could metabolize retinal in vitro, was expressed in epithelial cells and then in mesenchymal cells at the proximal extremity, that is, the future developmental field of the bud. Exogenous retinoic acid comparable to the endogenous level could induce an additional field at the distal end of the bud, resulting in a double monster. The induction always accompanied an ectopic expression of aldehyde dehydrogenase. The results of this work suggest that retinoic acid or related molecule(s) act as an endogenous trigger of morphallactic development of Polyandrocarpa buds.

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Role of the esc+ gene product in ensuring the selective expression of segment-specific homeotic genes in Drosophila

Development ◽

10.1242/dev.76.1.297 ◽

1983 ◽

Vol 76 (1) ◽

pp. 297-331

Author(s):

Gary Struhl

Keyword(s):

Gene Product ◽

The Body ◽

Homeotic Genes ◽

Bithorax Complex ◽

Sex Combs Reduced ◽

Sex Combs ◽

Correct Determination ◽

Correct Expression

The product of the extra sex combs+ (esc+) gene is required during embryogenesis for the correct determination of segments in Drosophila. If this product is absent, most segments develop like the normal eighth abdominal segment. Here, I extend previous results (Struhl, 1981a) showing that this phenotype results in large part from indiscriminate expression of the bithorax-complex genes which are normally active only in particular segments of the thorax and abdomen. In addition, I test whether the esc+ gene product is required for the correct expression of other homeotic genes. First, I have examined two genes of the Antennapedia-complex (Sex combs reduced+ and Antennapedia+): I find that both genes are normally required in only some of the body segments, but that in the absence of the esc+ gene product, both appear to function adventitiously in other segments. Second, comparing esc+ and esc− embryos lacking both these genes as well as the bithorax-complex, I find that additional homeotic genes (possibly those normally involved in specifying head segments) appear to be expressed indiscriminately when the esc+ gene product is absent. Finally, I present evidence that the products of the esc+ gene and the Polycomb+ gene (a second gene required for the correct regulation of the bithorax-complex) act independently. On the basis of these results, I propose a tentative outline of the roles and realms of action of all of these genes.

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The Transcriptional Repressor ZEB Regulates p73 Expression at the Crossroad between Proliferation and Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8461-8470.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8461-8470 ◽

Cited By ~ 86

Author(s):

Giulia Fontemaggi ◽

Aymone Gurtner ◽

Sabrina Strano ◽

Yujiro Higashi ◽

Ada Sacchi ◽

...

Keyword(s):

Biological Activities ◽

Ectopic Expression ◽

Transcriptional Repressor ◽

Dominant Negative ◽

C2c12 Cells ◽

Monocytic Differentiation ◽

Hl60 Cells ◽

Proliferation And Differentiation

ABSTRACT The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53+/+ and p53−/− cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73−/−-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

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Analysis of Drosophila proboscipedia mutant alleles

Genome ◽

10.1139/g03-133 ◽

2004 ◽

Vol 47 (3) ◽

pp. 600-609 ◽

Cited By ~ 6

Author(s):

I Tayyab ◽

H M Hallahan ◽

A Percival-Smith

Keyword(s):

Dna Sequence ◽

Hox Genes ◽

Genetic Interaction ◽

Direct Interaction ◽

Protein Product ◽

Maxillary Palp ◽

Phenotypic Analysis ◽

Sex Combs Reduced ◽

Important Region ◽

Sex Combs

Proboscipedia (PB) is a HOX protein required for adult maxillary palp and proboscis formation. To identify domains of PB important for function, 21 pb point mutant alleles were sequenced. Twelve pb alleles had DNA sequence changes that encode an altered PB protein product. The DNA sequence changes of these 12 alleles fell into 2 categories: missense alleles that effect the PB homeodomain (HD), and nonsense or frameshift alleles that result in C-terminal truncations of the PB protein. The phenotypic analysis of the pb homeobox missense alleles suggests that the PB HD is required for maxillary palp and proboscis development and pb – Sex combs reduced (Scr) genetic interaction. The phenotypic analysis of the pb nonsense or frameshift alleles suggests that the C-terminus is an important region required for maxillary palp and proboscis development and pb–Scr genetic interaction. PB and SCR do not interact directly with one another in a co-immunoprecipitation assay and in a yeast two-hybrid analysis, which suggests the pb–Scr genetic interaction is not mediated by a direct interaction between PB and SCR.Key words: proboscipedia, Sex combs reduced, Hox genes, mutant analysis, Drosophila body plan, appendage development.

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Ectopic expression and function of the Antp and Scr homeotic genes: the N terminus of the homeodomain is critical to functional specificity

Development ◽

10.1242/dev.118.2.339 ◽

1993 ◽

Vol 118 (2) ◽

pp. 339-352 ◽

Cited By ~ 11

Author(s):

W. Zeng ◽

D.J. Andrew ◽

L.D. Mathies ◽

M.A. Horner ◽

M.P. Scott

Keyword(s):

Dna Binding ◽

Target Genes ◽

Ectopic Expression ◽

Homeotic Genes ◽

Cell Fates ◽

Nmr Studies ◽

Sex Combs Reduced ◽

Sex Combs ◽

N Terminus

The transcription factors encoded by homeotic genes determine cell fates during development. Each homeotic protein causes cells to follow a distinct pathway, presumably by differentially regulating downstream ‘target’ genes. The homeodomain, the DNA-binding part of homeotic proteins, is necessary for conferring the specificity of each homeotic protein's action. The two Drosophila homeotic proteins encoded by Antennapedia and Sex combs reduced determine cell fates in the epidermis and internal tissues of the posterior head and thorax. Genes encoding chimeric Antp/Scr proteins were introduced into flies and their effects on morphology and target gene regulation observed. We find that the N terminus of the homeodomain is critical for determining the specific effects of these homeotic proteins in vivo, but other parts of the proteins have some influence as well. The N-terminal part of the homeodomain has been observed, in crystal structures and in NMR studies in solution, to contact the minor groove of the DNA. The different effects of Antennapedia and Sex combs reduced proteins in vivo may depend on differences in DNA binding, protein-protein interactions, or both.

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Normal and ectopic domains of the homeotic gene Sex combs reduced of Drosophila

Development ◽

10.1242/dev.117.3.917 ◽

1993 ◽

Vol 117 (3) ◽

pp. 917-923 ◽

Cited By ~ 1

Author(s):

S. Pelaz ◽

N. Urquia ◽

G. Morata

Keyword(s):

Ectopic Expression ◽

Secondary Wave ◽

Homeotic Gene ◽

Bithorax Complex ◽

Germ Band ◽

Sex Combs Reduced ◽

Normal Functions ◽

Sex Combs ◽

Normal Expression ◽

Thoracic And Abdominal

The normal expression of the homeotic gene Sex combs reduced (Scr) is initially restricted to parasegment 2, later extends to 3, and by germ band retraction extends further to part of parasegment 4 (T1p). We find that in the absence of the bithorax complex (BX-C) genes there is Scr expression in the epidermis of the posterior compartments of the thoracic and abdominal parasegments. This ectopic expression appears at the same time as the normal one in T1p and requires the normal functions of the genes Antennapedia (Antp) and engrailed (en). In particular, en appears to play an important role in the activation of Scr because the expansion of en expression in naked mutants produces a corresponding expansion of the ectopic Scr stripes. We also find that in the epidermis Antp can have opposite effects on Scr expression; moderate levels of Antp product enhance Scr expression, whereas high levels suppress it. We propose the existence of a secondary wave of Scr activation, which takes place during germ band retraction, is triggered by en and requires Antp expression. It is repressed by the BX-C genes in the meso-, metathoracic and the abdominal segments.

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Differential Re-Programming of Myeloid Stem Cell Development by Activator Versus Repressor Isoforms of Human CCAAT/Enhancer Binding Protein Epsilon (C/EBPε).

Blood ◽

10.1182/blood.v108.11.1177.1177 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1177-1177

Author(s):

Richa Bedi ◽

Steven J. Ackerman

Keyword(s):

Suspension Culture ◽

Rna Splicing ◽

Ectopic Expression ◽

Dominant Negative ◽

Transactivation Domain ◽

Retroviral Transduction ◽

Cell Counts ◽

Eosinophil Colonies

Abstract Human C/EBPε is expressed as four isoforms (32, 30, 27, 14kD) through alternative promoter usage, RNA splicing, and translational start sites. We are studying the C/EBPε isoforms in vivo in cord blood (CB) CD34+ progenitors to define their roles in granulocyte differentiation and hematopoietic lineage specification. The C/EBPε32/30 isoforms function as transcriptional activators. We reported that C/EBPε27 interacts with and is a repressor of GATA-1 transactivation of eosinophil-specific genes (Du J et al. JBC2002; 277:43481). C/EBPε14, which contains DNA binding and bZIP domains but lacks a transactivation domain, may function as a dominant negative repressor of C/EBPε32,30 or other C/EBPs through heterodimerization or competition for C/EBP binding sites. We assessed mRNA expression of the C/EBPε isoforms during eosinophil differentiation of CB CD34+ progenitors to determine their temporal patterns of expression. CD34+ progenitors (>95% pure) induced to differentiate into eosinophils by SCF, IL-3, and IL-5 showed differentiation to >90% eosinophils by 17–19 days. Semi-quantitative RT-PCR using C/EBPε isoform-specific primers showed that CD34+ progenitors initially express only the repressor C/EBPε14 isoform, with induction of all isoforms by day 3 to different peak levels of expression by days 9–11 during the promyelocyte to myelocyte transition. These results define temporal changes in the expression ratios of the C/EBPε activator vs. repressor isoforms during eosinophil development that may differentially regulate gene transcription in this process. We subcloned the C/EBPε isoform cDNAs into an MSCV-based bicistronic retroviral vector (pGCDNsam IRES-EGFP) and ectopic expression was induced in CB CD34+ progenitors by retroviral transduction for 72hrs. The CD34+/GFP+ cells were sorted by FACS and plated in Collagen Cult™ media containing SCF, IL-3 and a lineage-specific cytokine (i.e. EPO, G-CSF, or IL-5), or in suspension culture containing SCF, IL-3 and IL-5 to drive eosinophil differentiation. Total and differential colony and cell counts were performed after 15–17 days based on colony morphology, histochemical and enzyme staining of the cells. The activator C/EBPε32 isoform significantly altered myeloid development, favoring eosinophil over neutrophil or erythroid development. Even in cultures containing EPO, cells transduced with C/EBPε32 failed to develop into erythroid colonies (BFU-E). C/EBPε27, a potent repressor of GATA-1 in vitro, inhibited erythroid colony growth by ~50%, and doubled the numbers of granulocyte-macrophage colonies compared to C/EBPε14 or empty vector control. C/EBPε32 strongly induced eosinophil colony formation at the expense of neutrophil other myeloid lineages regardless of the cytokines used, inducing ~90% eosinophil colonies. C/EBPε27 reduced eosinophil colonies by >50%. Likewise, >90% of cells transduced with C/EBPε32 and grown in suspension culture were eosinophils, whereas C/EBPε27 and C/EBPε14 inhibited eosinophil differentiation by ~50% and >98%, respectively. Thus, the C/EBPε isoforms: (1) are differentially expressed during eosinophil development, (2) have the capacity to reprogram stem cells to different myeloid lineages consistent with their predicted activator vs. repressor activities and interactions with hematopoietic transcription factors such as GATA-1 or other C/EBPs, and (3) may be useful in their ability to reprogram myeloid terminal differentiation for the development of novel approaches to treat myeloid or other leukemias.

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