SNR1 is an essential subunit in a subset of Drosophila brm complexes,targeting specific functions during development

The snr1 gene of Drosophila melanogaster encodes a conserved component of the multiprotein Brahma (Brm) complex, a counterpart to the SWI/SNF complexes that participate in ATP-dependent chromatin remodeling. Loss-of-function and null mutations in the snr1 gene reveal its essential role in Drosophila development. We identified new mutant alleles and ectopically expressed deleted forms to dissect the specific functions of SNR1. Somatic and germ cell clone analyses confirmed its requirement in a continuous and widespread fashion for proper cell fate determination and oogenesis. Expression of SNR1 transgenes revealed unexpected roles in wing patterning, abdomen development, oogenesis, and sustained adult viability. A widespread distribution of SNR1 and BRM on the salivary gland polytene chromosomes showed that the Brm complex associated with many genes, but not always at transcribed loci, consistent with genetic data suggesting roles in both gene activation and repression. Despite essential Brm complex functions in leg development, genetic and protein localization studies revealed that snr1 was not required or expressed in all tissues dependent on Brm complex activities. Thus, SNR1 is essential for some, but not all Brm functions, and it likely serves as an optional subunit, directing Brm complex activity to specific gene loci or cellular processes.     

LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues

The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.     

Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.     

Xfin: an embryonic gene encoding a multifingered protein in Xenopus.

The Xenopus laevis genome was screened for putative DNA-binding gene products by using the 'finger' region of the Drosophila gene Krüppel as a probe. The one gene detected, named Xfin, codes for a protein with 37 finger domains that comprise nearly 90% of the protein. In the light of studies by Rhodes and Klug (Cell, 46, 123-132, 1986), these data suggest that the Xfin protein has the capacity to bind an unusually large stretch (185 bases) of DNA. The Xfin gene is expressed as a maternal and zygotic mRNA that undergoes extensive polyadenylation changes during early development. The Xfin mRNA expression pattern and the potential DNA binding activity of the protein point to the possibility that the Xfin gene may have a role in controlling gene activity during early embryonic development.     

Extensive nonmuscle expression and epithelial apicobasal localization of the Drosophila ALP/Enigma family protein,Zasp52

This study describes the broad tissue distribution and subcellular localization of Drosophila Zasp52, which is related to the large family of ALP (α-actinin associated protein)/Enigma PDLIM (PDZ and LIM domain) proteins of vertebrates. Results demonstrate that ZCL423 is a protein trap insertion in the Zasp52 locus tagging multiple endogenous splice isoforms with GFP. While Zasp52 has been previously characterized in muscle tissues primarily, visualization of GFP fluorescence in Zasp52 protein trap lines revealed expression in many nonmuscle tissues including the central nervous system, secretory glands, and epithelial tissues constituting the embryonic epidermis, the somatic follicle cell layer encapsulating the germline during oogenesis, and imaginal disc precursors to the adult body. In epithelial cells, Zasp52 typically accumulated basally, adjacent to integrin adhesion sites, and apically along adherens junctions, particularly enriched near junctional vertices of multicellular interfaces. Also Zasp52 showed polarized accumulation at the leading edge of migrating cell populations and morphogenetic boundaries similarly enriched for myosin. As such, Zasp52 GFP protein traps may be useful molecular markers for dynamic epithelial rearrangements. Moreover, the pattern of Zasp52 expression within nonmuscle tissues reveals potential functional roles in cell–cell and cell–matrix adhesion, specifically at sites of increased actomyosin contractile tension. In these contexts, the investigation of Zasp52 may provide insights into the functions of numerous PDLIM proteins of the metazoan lineages.     

Stage-specific localization of the small heat shock protein Hsp27 during oogenesis inDrosophila melanogaster

The developmental and heat shock-induced expression of the small heat shock protein Hsp27 was investigated by confocal microscopy of whole-mount immunostained preparations of ovarioles during oogenesis inDrosophila melanogaster. In unstressed flies, Hsp27 was mainly associated with germline nurse cells throughout egg development. A small group of somatic follicle cells also expressed Hsp27 specifically at stages 8 to 10 of oogenesis. Interestingly, this Hsp showed a different intracellular localization depending on the stages of egg chamber development. Thus Hsp27 was localized in the nucleus of nurse cells during the first stages of oogenesis (from germarium to stage 6) whereas it showed a perinuclear and cytoplasmic localization from stage 8. After a heat shock, Hsp27 accumulated in somatic follicle cells surrounding the egg chamber whereas the expression of this small Hsp did not seem to be enhanced in nurse cells. The stage-dependent pattern of intracellular localization of Hsp27 observed in nurse cells of unstressed flies was also observed following heat shock. At late stages of oogenesis, Hsp27 also showed a perinuclear distribution in follicle and nurse cells after heat stress. These observations suggest that different factors may modulate the expression and intracellular distribution of Hsp27. This modulation may be associated with the specific activities occurring in each particular cell type throughout oogenesis during both normal development and under heat shock conditions. Edited by: E.R. Schmidt     

Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.     

Expression of ras oncogene p21 during human fetal development as determined by monoclonal antibodies RAP-5, Y13-259, and DWP

In this report we describe the expression of the ras proto-oncogene p21 protein in various tissues during normal fetal development. Conventional, formalin fixed and paraffin-embedded sections of normal organs were examined from fetuses ranging 9 to 42 weeks of gestation. Immunohistochemical localization of ras p21 was accomplished using the broadly reactive, mouse monoclonal antibodies RAP-5 and Y13-259. The monoclonal antibody DWP, which is specific for a mutated form of ras p21 having a valine/cysteine at amino acid position 12, was also used. Detectable expression of the p21 protein was seen at different time periods during fetal development depending on the tissue. The expression of ras p21 (as detected by RAP-5 and Y13-259) was noted in a wide range of cell types and tissues; intense immunostaining was noted in epithelial cells of the gastrointestinal tract, exocrine and endocrine pancreas, renal tubules and transitional urotheliem, as well as in other tissues. This immunostaining generally, but not invariably, corresponded with patterns previously reported in benign and/or malignant neoplasms of adult tissues. In most instances ras p21 expression, when present, occurred during periods of rapid growth in given organ systems. However, some actively proliferating fetal tissues such as thymus and spleen, failed to express detectable ras p21 suggesting that factors other than cell cycle may influence its expression. No reactivity with DWP was noted in any of the tissues, suggesting that the mutated forms detected by this monoclonal antibody are not expressed during normal human embryogenesis. These data show that there is regulated expression, and broad distribution of this gene product in normal developing human fetal tissue.     

Importin-alpha3 is required at multiple stages of Drosophila development and has a role in the completion of oogenesis

The Drosophila importin-alpha3 gene was isolated through its interaction with the large subunit of the DNA polymerase alpha in a two-hybrid screen. The predicted protein sequence of Importin-alpha3 is 65-66% identical to those of the human and mouse importin-alpha3 and alpha4 and 42.7% identical to that of Importin-alpha2 (Oho31/Pendulin), the previously reported Drosophila homologue. Both Importin-alpha3 and Importin-alpha2 interact with similar subsets of proteins in vitro, one of which is Ketel, the importin-beta homologue of Drosophila. importin-alpha3 is an essential gene, whose encoded protein is expressed throughout development. During early embryogenesis, Importin-alpha3 accumulates at the nuclear membrane of cleavage nuclei, whereas after blastoderm formation it is characteristically found within the interphase nuclei. Nuclear localisation is seen in several tissues throughout subsequent development. During oogenesis its concentration within the nurse cell nuclei increases during stages 7-10, concomitant with a decline in levels in the oocyte nucleus. Mutation of importin-alpha3 results in lethality throughout pupal development. Surviving females are sterile and show arrest of oogenesis at stages 7-10. Thus, Importin-alpha3-mediated nuclear transport is essential for completion of oogenesis and becomes limiting during pupal development. Since they have different expression patterns and subcellular localisation profiles, we suggest that the two importin-alpha homologues are not redundant in the context of normal Drosophila development.     

The cytoplasmic dynein and kinesin motors have interdependent roles in patterning the Drosophila oocyte

BACKGROUND: Motor proteins of the minus end-directed cytoplasmic dynein and plus end-directed kinesin families provide the principal means for microtubule-based transport in eukaryotic cells. Despite their opposing polarity, these two classes of motors may cooperate in vivo. In Drosophila circumstantial evidence suggests that dynein acts in the localization of determinants and signaling factors during oogenesis. However, the pleiotropic requirement for dynein throughout development has made it difficult to establish its specific role. RESULTS: We analyzed dynein function in the oocyte by disrupting motor activity through temporally restricted expression of the dynactin subunit, dynamitin. Our results indicate that dynein is required for several processes that impact patterning; such processes include localization of bicoid (bcd) and gurken (grk) mRNAs and anchoring of the oocyte nucleus to the cell cortex. Surprisingly, dynein function is sensitive to reduction in kinesin levels, and germ line clones lacking kinesin show defects in dorsal follicle cell fate, grk mRNA localization, and nuclear attachment that are similar to those resulting from the loss of dynein. Significantly, dynein and dynactin localization is perturbed in these animals. Conversely, kinesin localization also depends on dynein activity. CONCLUSIONS: We demonstrate that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein.     

The lysyl hydroxylase isoforms are widely expressed during mouse embryogenesis, but obtain tissue- and cell-specific patterns in the adult

Lysyl hydroxylase catalyzes the hydroxylation of lysine residues in collagenous sequences. Three isoforms (LH1, LH2 and LH3) of lysyl hydroxylase have been characterized, and LH2 is present as two alternatively spliced forms. In order to better understand the functional differences between the isoforms in vivo, the expression of the different isoforms was studied in mouse embryos and adult tissues. Our data indicate a widespread expression of all isoforms during embryogenesis, whereas the expression profiles become more specialized in adult tissues. The expression of LH2 was more tissue-specific, whereas a uniform and housekeeping like behavior was observed for LH3. Some cells express both LH2 and LH3, while a clear cell specificity was seen in some tissues. Moreover, immunoelectron microscopy revealed differences in the localization of LH2 and LH3. LH2 was localized intracellularly in the ER in all tissues studied, whereas the localization of LH3 was either intracellular or extracellular or both, depending on the tissue. Furthermore, our data indicate that the alternative splicing of LH2 is developmentally regulated. The short form of LH2 (LH2a) is the predominant form until E11.5; the long form (LH2b) dominates thereafter and is the major form in many adult tissues. Interestingly, however, adult mouse kidney and testis express exclusively the short form, LH2a. The results reveal a specific regulation for the expression of LH isoforms as well as for alternative splicing of LH2 during embryogenesis and in different tissues.     

Drosophila tubulin-binding cofactor B is required for microtubule network formation and for cell polarity

Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-β-tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and β-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division.     

Polarizing genetic information in the egg: RNA localization in the frog oocyte

RNA localization is a powerful strategy used by cells to localize proteins to subcellular domains and to control protein synthesis regionally. In germ cells, RNA targeting has profound implications for development, setting up polarities in genetic information that drive cell fate during embryogenesis. The frog oocyte offers a useful system for studying the mechanism of RNA localization. Here, we discuss critically the process of RNA localization during frog oogenesis. Three major pathways have been identified that are temporally and spatially separated in oogenesis. Each pathway uses a different mechanism to effect RNA localization. In some cases, localization elements within the 3' untranslated region have been identified and have provided unique insights into the localization process. This important field is still in its infancy, however, and much remains to be learned. BioEssays 21:546–557, 1999. © 1999 John Wiley & Sons, Inc.     

The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.