Cellular Blastoderm Stage Research Articles

Two coacting shadow enhancers regulate twin of eyeless expression during early Drosophila development.

The Drosophila PAX6 homolog twin of eyeless (toy) sits at the pinnacle of the genetic pathway controlling eye development, the retinal determination network. Expression of toy in the embryo is first detectable at cellular blastoderm stage 5 in an anterior-dorsal band in the presumptive procephalic neuroectoderm, which gives rise to the primordia of the visual system and brain. Although several maternal and gap transcription factors that generate positional information in the embryo have been implicated in controlling toy, the regulation of toy expression in the early embryo is currently not well characterized. In this study, we adopt an integrated experimental approach utilizing bioinformatics, molecular genetic testing of putative enhancers in transgenic reporter gene assays and quantitative analysis of expression patterns in the early embryo, to identify 2 novel coacting enhancers at the toy gene. In addition, we apply mathematical modeling to dissect the regulatory landscape for toy. We demonstrate that relatively simple thermodynamic-based models, incorporating only 5 TF binding sites, can accurately predict gene expression from the 2 coacting enhancers and that the HUNCHBACK TF plays a critical regulatory role through a dual-modality function as an activator and repressor. Our analysis also reveals that the molecular architecture of the 2 enhancers is very different, indicating that the underlying regulatory logic they employ is distinct.

The ftz upstream element drives late ftz stripes but is not required for regulation of ftz target genes

The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. Here, we asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS7S homozygotes, ftz stripes appear in the same order as wildtype and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz striped expression, with all stripes decaying earlier than wildtype in ftzΔUPS7S homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. We propose that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, our results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development.

Dynamics expression of DmFKBP12/Calstabin during embryonic early development of Drosophila melanogaster

BackgroundCalcium signaling are conserved from invertebrates to vertebrates and plays critical roles in many molecular mechanisms of embryogenesis and postnatal development. As a critical component of the signaling pathway, the RyR medicated calcium-induced calcium release signaling system, has been well studied along with their regulator FK506-binding protein 12 (FKBP12/Calstabin). Lack of FKBP12 is known to result in lethal cardiac dysfunction in mouse. However, precisely how FKBP12 is regulated and effects calcium signaling in Drosophila melanogaster remains largely unknown.ResultsIn this study, we identified both temporal and localization changes in expression of DmFKBP12, a translational and transcriptional regulator of Drosophila RyR (DmRyR) and FKBP12, through embryonic development. DmFKBP12 is first expressed at the syncytial blastoderm stage and undergoes increased expression during the cellular blastoderm and early gastrulation stages. At late gastrulation, DmFKBP12 expression begins to decline until it reaches homeostasis, which it then maintains throughout the rest of development. Throughout these described changes in expression, DmFKBP12 mRNA remain stable, which indicates that protein dynamics are attributed to regulation at the mRNA to protein translation level. In addition to temporal changes in expression, dynamic expression profiles during Drosophila development also revealed DmFKBP12 localization. Although DmFKBP12 is distributed evenly between the anterior to posterior poles of the blastoderm egg, the protein is expressed more strongly in the cortex of the early Drosophila gastrula with the highest concentration found in the basement membrane of the cellular blastoderm. Fertilized egg, through the profile as under-membrane cortex distribution concentering onto basement at cellular blastoderm, to the profile as three-gem layer localization in primitive neuronal and digestion architecture of early Drosophila gastrula. By late gastrulation, DmFKBP12 is no longer identified in the yolk or lumen of duct structures and has relocated to the future brain (suboesophageal and supraesophageal ganglions), ventral nervous system, and muscular system. Throughout these changes in distribution, in situ DmFKBP12 mRNA monitoring detected equal distribution of DmFKBP12 mRNA, once again indicating that regulation of DmFKBP12 occurs at the translational level in Drosophila development.ConclusionAs a critical regulator of the DmRyR-FKBP complex, DmFKBP12 expression in Drosophila fluctuates temporally and geographically with the formation of organ systems. These finding indicate that DmFKBP12 and RyR associated calcium signaling plays an essential role in the successful development of Drosophila melanogaster. Further study on the differences between mammalian RyR-FKBP12 and Drosophila DmRyR-FKBP12 can be exploited to develop safe pesticides.

Buchnera aphidicola of the birch blister aphid, Hamamelistes betulinus (Horváth, 1896) (Insecta, Hemiptera, Aphididae: Hormaphidinae): molecular characterization, transmission between generations and its geographic significance

AbstractThe birch blister aphid Hamamelistes betulinus, like most aphids, is host to obligate symbiotic bacterium Buchnera aphidicola. Ultrastructural and molecular analyses did not reveal the presence of secondary symbionts in the body of H. betulinus. The bacteria Buchnera aphidicola are transmitted to the next generation vertically (maternally). The bacteria released from the cytoplasm of the bacteriocyte to the haemolymph migrate to the embryo at the cellular blastoderm stage, through the opening at its posterior pole. Next, the bacteria enter the cytoplasm of newly formed bacteriocytes. The concept of the relationship between the geographic distribution of Hormaphidini aphids and the presence/absence of bacterium Buchnera aphidicola is discussed.

Natural variation of the expression pattern of the segmentation gene even-skipped in melanogaster

The evolution of canalized traits is a central question in evolutionary biology. Natural variation in highly conserved traits can provide clues about their evolutionary potential. Here we investigate natural variation in a conserved trait—even-skipped (eve) expression at the cellular blastoderm stage of embryonic development in Drosophila melanogaster. Expression of the pair-rule gene eve was quantitatively measured in three inbred lines derived from a natural population of D. melanogaster. One line showed marked differences in the spacing, amplitude and timing of formation of the characteristic seven-striped pattern over a 50-min period prior to the onset of gastrulation. Stripe 5 amplitude and the width of the interstripe between stripes 4 and 5 were both reduced in this line, while the interstripe distance between stripes 3 and 4 was increased. Engrailed expression in stage 10 embryos revealed a statistically significant increase in the length of parasegment 6 and a decrease in the length of parasegments 8 and 9. These changes are larger than those previously reported between D. melanogaster and D. pseudoobscura, two species that are thought to have diverged from a common ancestor over 25 million years ago. This line harbors a rare 448 bp deletion in the first intron of knirps (kni). This finding suggested that reduced Kni levels caused the deviant eve expression, and indeed we observed lower levels of Kni protein at early cycle 14A in L2 compared to the other two lines. A second of the three lines displayed an approximately 20% greater level of expression for all seven eve stripes. The three lines are each viable and fertile, and none display a segmentation defect as adults, suggesting that early-acting variation in eve expression is ameliorated by developmental buffering mechanisms acting later in development. Canalization of the segmentation pathway may reduce the fitness consequences of genetic variation, thus allowing the persistence of mutations with unexpectedly strong gene expression phenotypes.

Cloning of cDNA encoding a Bombyx mori homolog of human oxidation resistance 1 (OXR1) protein from diapause eggs, and analyses of its expression and function

To better understand the molecular mechanisms of diapause initiation, we used the sensitive cDNA subtraction (selective amplification via biotin- and restriction-mediated enrichment) method and isolated a novel gene expressed abundantly in diapause eggs of the silkworm, Bombyx mori, which encodes a homolog of the human oxidation resistance 1 (OXR1) protein. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses confirmed that BmOXR1 mRNA and its 140-kDa protein were differentially expressed in diapause eggs compared to non-diapause eggs. OXR1 double-stranded RNA (dsRNA) was injected into diapause-destined eggs before the cellular blastoderm stage, and 4days later, when untreated eggs reached the diapause stage, the OXR1 protein disappeared; however, these eggs remained in diapause, suggesting that BmOXR1 is not essential for diapause initiation and/or maintenance. To further investigate the in vivo function of BmOXR1 apart from its role in diapause, we overexpressed BmOXR1 in Drosophila melanogaster. The fruit fly male adult life-span was significantly extended in the 50%-survival time when adults were reared on diets both with and without H2O2 solution under 25°C incubation. These results suggest that BmOXR1 functions in D. melanogaster via a possible antioxidant effect. As BmOXR1 was expressed mainly in the nuclei of D. melanogaster cells, the mechanism underlying its antioxidation effect appears to be different from that in humans where it is expressed mainly in the mitochondria. Taken together, these results suggest that BmOXR1 might serve as an antioxidant regulator during the early diapause stage.

Temporal pattern of the posterior expression of Wingless in Drosophila blastoderm

In most animals, the antero-posterior (A-P) axis requires a gradient of Wnt signaling. Wnts are expressed posteriorly in many vertebrate and invertebrate embryos, forming a gradient of canonical Wnt/β-Catenin activity that is highest in the posterior and lowest in the anterior. One notable exception to this evolutionary conservation is in the Drosophila embryo, in which the A-P axis is established by early transcription factors of maternal origin. Despite this initial axial establishment, Drosophila still expresses Wingless (Wg), the main Drosophila Wnt homologue, in a strong posterior band early in embryogenesis. Since its discovery 30 years ago this posterior band of Wg has been largely ignored. In this study, we re-examined the onset of expression of the Wg posterior band in relation to the expression of Wg in other segments, and compared the timing of its expression to that of axial regulators such as gap and pair-rule genes. It was found that the posterior band of Wg is first detected in blastoderm at mid nuclear cycle 14, before the segment-polarity stripes of Wg are formed in other segments. The onset of the posterior band of Wg expression was preceded by that of the gap gene products Hunchback (hb) and Krüppel (Kr), and the pair-rule protein Even-skipped (Eve). Although the function of the posterior band of Wg was not analyzed in this study, we note that in temperature-sensitive Wg mutants, in which Wg is not properly secreted, the posterior band of Wg expression is diminished in strength, indicating a positive feedback loop required for Wg robust expression at the cellular blastoderm stage. We propose that this early posterior expression could play a role in the refinement of A-P patterning.

Artificial selection on egg size perturbs early pattern formation in Drosophila melanogaster.

Pattern formation in Drosophila embryogenesis has been widely investigated as a developmental and evolutionary model of robustness. To ask whether genetic variation for pattern formation is suppressed in this system, artificial selection for divergent egg size was used to challenge the scaling of even-skipped (eve) pattern formation in mitotic cycle 14 (stage 5) embryos of Drosophila melanogaster. Three-dimensional confocal imaging revealed shifts in the allometry of eve pair-rule stripes along both anterior–posterior (A–P) and dorsoventral (D–V) axes as a correlated response to egg size selection, indicating the availability of genetic variation for this buffered trait. Environmental perturbation was not required for the manifestation of this variation. The number of nuclei at the cellular blastoderm stage also changed in response to selection, with large-egg selected lines having more than 1000 additional nuclei relative to small-egg lines. This increase in nuclear number in larger eggs does not scale with egg size, however, as nuclear density is inversely correlated with egg length. Nuclear density varies along the A–P axis but does not correlate with the shift in eve stripe allometry between the selection treatments. Despite its macroevolutionary conservation, both eve stripe patterning and blastoderm cell number vary genetically both within and between closely related species.

Asymmetric mitosis: Unequal segregation of proteins destined for degradation

Mitotic cell division ensures that two daughter somatic cells inherit identical genetic material. Previous work has shown that signaling by the Smad1 transcription factor is terminated by polyubiquitinylation and proteasomal degradation after essential phosphorylations by MAPK and glycogen synthase kinase 3 (GSK3). Here, we show that, unexpectedly, proteins specifically targeted for proteasomal degradation are inherited preferentially by one mitotic daughter during somatic cell division. Experiments with dividing human embryonic stem cells and other mammalian cultured cell lines demonstrated that in many supposedly equal mitoses the segregation of proteins destined for degradation (Smad1 phosphorylated by MAPK and GSK3, phospho-beta-catenin, and total polyubiquitinylated proteins) was asymmetric. Transport of pSmad1 targeted for degradation to the centrosome required functional microtubules. In vivo, an antibody specific for Mad phosphorylated by MAPK showed that this antigen was associated preferentially with one of the two centrosomes in Drosophila embryos at cellular blastoderm stage. We propose that this remarkable cellular property may be explained by the asymmetric inheritance of peripheral centrosomal proteins when centrioles separate and migrate to opposite poles of the cell, so that one mitotic daughter remains pristine. We conclude that many mitotic divisions are unequal, unlike what was previously thought.

Male or Female? The Answer Depends on When You Ask

Male or Female? The Answer Depends on When You Ask

Establishment of cell fate during early Drosophila embryogenesis requires transcriptional Mediator subunit dMED31

During early Drosophila embryogenesis, formation of the anterior–posterior (A/P) axis depends on spatial gradients of maternal morphogens. It is well recognized that positional information is transmitted from these morphogens to the gap genes. However, how this information is being transmitted is largely unknown. The transcriptional Mediator complex is involved in the fine tuning of the signaling between chromatin status, transcription factors and the RNA polymerase II transcription machinery. We found that a mutation in the conserved subunit of the Mediator complex, dMED31, hampers embryogenesis prior to gastrulation and leads to aberrant expression of the gap genes knirps and Krüppel and the pair-rule genes fusi tarazu and even-skipped along the A/P axis. Expression of the maternal morphogens dorsal and hunchback was not affected in dMED31 mutants. mRNA expression of dMED31 exactly peaks between the highest expression levels of the maternal genes and the gap genes. Together, our results point to a role for dMED31 in guiding maternal morphogen directed zygotic gap gene expression and provide the first in vivo evidence for a role of the Mediator complex in the establishment of cell fate during the cellular blastoderm stage of Drosophila melanogaster.

TFIIH trafficking and its nuclear assembly during early Drosophila embryo development

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.

TFIIH trafficking and its nuclear assembly during earlyDrosophilaembryo development

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.

Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide

The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT-rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X-chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA-binding ligands. P9 fed to larvae suppresses the position-effect variegation phenotype of white-mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene.

Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknullt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55 degrees C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Fat Facets Interacts with Vasa in the Drosophila Pole Plasm and Protects It from Degradation

Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm [1, 2]. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm [1, 2]. Vasa (VAS) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and VAS is a candidate translational activator in the pole plasm [3, 4]. VAS is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage [5]. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism [6], is another example. Here, we used a biochemical approach to identify proteins that copurify with VAS in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (FAF), a pole plasm component [7], but one whose roles in posterior patterning and germ line specification have remained unclear. We present evidence that FAF interacts with VAS physically and reverses VAS ubiquitination, thereby stabilizing VAS in the pole plasm.

Radiosurgery using heavy ion microbeams for biological study: Fate mapping of the cellular blastoderm-stage egg of the silkworm, Bombyx mori

We investigated the effects of heavy ions on embryogenesis of the silkworm, Bombyx mori using a collimated heavy ion microbeam from the vertical beam line of an AVF-cyclotron. Eggs were exposed to carbon ions at the cellular blastoderm stage. Microbeams were found to be extremely useful for radio-microsurgical inactivation of nuclei or cells in the target site. Spot irradiation caused abnormal embryos, which showed localized defects such as deletion, duplication and fusion, depending on dose, beam size and site of irradiation. The location and frequency of defects on the resultant embryos were closely correlated to the irradiation site. Based on this correlation, a fate map was established for the Bombyx egg at the cellular blastoderm stage.

Stage-specific chromosomal association of Drosophila dMBD2/3 during genome activation

The Drosophila gene dMBD2/3 encodes a protein with significant homologies to the mammalian methyl-DNA binding proteins MBD2 and MBD3. These proteins are essential components of chromatin complexes involved in epigenetic gene regulation. Because the available in vitro data on dMBD2/3 are conflicting we have started an in vivo characterization of dMBD2/3. We detected expression of two isoforms specifically during embryonic development. Staining of whole embryos combined with high-resolution confocal microscopy revealed a highly regulated spatial distribution. During the syncytial blastoderm stage, dMBD2/3 formed speckles that localized to the cytoplasm. Shortly after, during the cellular blastoderm stage, the protein entered the nucleus and formed bright foci that associated with DNA. This rapid transition coincided with the activation of the embryonic genome. A similar observation was made during activation of the spermatocyte genome as dMBD2/3 formed distinct foci associated with the activated Y chromosome. Our results indicate that dMBD2/3 forms specialized nuclear compartments to keep certain genes epigenetically silenced during genome activation.

Biphasic activation of the BMP pathway patterns the Drosophila embryonic dorsal region.

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), we followed signaling directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp.

Techniques update: Crossreactivity of anti-dual-phosphorylated antibodies with actin

Mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs) are components of protein kinase cascades that permit eukaryotic cells to respond to many extracellular signals[1xMarshall, C.J. Curr. Opin. Genet. Dev. 1994; 4: 82–89Crossref | PubMed | Scopus (798)See all References[1]. To monitor the activity of these signalling pathways under physiological conditions, several companies have recently developed specific antibodies directed against phosphopeptides corresponding to the TXY motif essential for MAPK and SAPK activation. These products specifically recognize single- or dual-phosphorylated forms of ERK MAPKs and the SAPKs Jun N-terminal kinase (JNK) and P38 kinase.These antibodies have been used to analyse processes ranging from the effects of growth hormone stimulation in different cell types[2xLove, D.W. et al. Endocrinology. 1998; 139: 1965–1971Crossref | PubMedSee all References[2]to the role of MAPKs in macrophage migration induced by integrins[3xWei, J. et al. J. Biol. Chem. 1998; 273: 5903–5907Crossref | PubMed | Scopus (62)See all References[3]. They have also been applied to biochemical studies, such as the elucidation of the nonprocessive mechanism of activation of MAPKs by MEK[4xBurack, W.R. and Sturgill, T.W. Biochemistry. 1997; 36: 5929–5933Crossref | PubMed | Scopus (128)See all References[4]. Two recent publications have made a new, even more exciting, use of these tools. Shilo and his colleagues have studied in situ the activation of MAPK during Drosophila development[5xGabay, L. et al. Science. 1997; 277: 1103–1106Crossref | PubMed | Scopus (297)See all References, 6xGabay, L. et al. Development. 1997; 124: 3535–3541PubMedSee all References], and the work was highlighted in this journal[7xHafen, E. Trends Cell Biol. 1997; 7: 436See all References[7].Using a similar approach, we examined the pattern of activation of the P38 kinase during Drosophila embryogenesis. In this study, we used the Anti-Active P38 antibody developed by Promega against a phosphopeptide containing the TGY motif of the mouse P38 kinase. The epitope recognized by this antibody is well conserved in the two recently isolated Drosophila homologues of the P38 kinase (VPQESEMVATRWYRAPE—DMPK2A; RPTENEMVATRWYRAPE—DMPK2B)[8xHan, S.J. et al. J. Biol. Chem. 1998; 273: 369–374Crossref | PubMed | Scopus (78)See all References, 9xHan, Z.Q.S. et al. Mol. Cell. Biol. 1998; 18: 3527–3539Crossref | PubMedSee all References].An Anti-Active P38 signal was detected from very early stages of Drosophila development, decorating all cell membranes from cellular blastoderm stages (Fig. 1Fig. 1a). At late stages, Anti-Active signal appeared associated mainly with cuticle structures (denticle belts; Fig. 1Fig. 1b) and the neuropile in the central nervous system (CNS; Fig. 1Fig. 1c). These patterns of activation correlated closely with the distribution of actin during development, and the remarkable similarity between the actin and active P38 expression led us to re-analyse the specificity of the antibody. We found that Anti-Active P38 associated with actin filaments polymerized in vitro from Drosophila embryo extracts (E. Martin-Blanco and A. Martinez-Arias, unpublished). More definitively, we found by western analysis that the antibody recognized rabbit skeletal actin as well as the dual-phosphorylated form of P38 (Fig. 1Fig. 1d). Immunoadsorption of the antibody eliminated this crossreactivity but maintained the specific binding to dual-phosphorylated P38 (Fig. 1Fig. 1e). The association of the signal with actin at early stages and in the embryo cuticle was eliminated (E. Martin-Blanco and A. Martinez-Arias, unpublished), whereas specific staining in the CNS (as in Fig. 1Fig. 1c) and several other conspicuous locations was reproducibly detected (a detailed analysis will be described elsewhere).Fig. 1Old, contaminated antibody detected phosphorylation of the stress-activated kinase P38 (arrowheads) during embryonic development of Drosophila. (a) In the cellular blastoderm, Anti-Active P38 staining associates with cell membranes. (b) In a stage 15 embryo, denticle structures accumulate Anti-Active P38 signal. (c) In a stage 16 embryo, Anti-Active P38 signal occurs in commissures and connectives of the embryonic central nervous system (CNS). In most cases, these signals correlated with actin accumulation. The anti-actin component of Promega Anti-Active P38 antibody was eliminated by adsorption to rabbit skeletal actin [compare (d) and (e)], but the antibody still retains its ability to recognize dual-phosphorylated P38 (e). Using new, clean batches, P38 phosphorylation is still detected in the Drosophila CNS [data not shown, but similar to (c)].View Large Image | Download PowerPoint SlideProduction and sale of the antibody by Promega were halted, all customers were informed, technical support has been provided and new uncontaminated batches have now been supplied. This type of in vivo analysis is one approach that can be used to verify antibody specificity. Users of this or similar antibodies should nevertheless take extreme care in the interpretation of their results and investigate carefully if any ambiguity arises.