Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.     

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.     

Localization of insulin-like growth factor-1 mRNA in murine central nervous system during postnatal development

Insulin-like growth factor-1 (IGF-1) is believed to play a role in the regulation of brain growth. The identity of cells responsible for its synthesis in the immature brain, however, has not been established. To identify potential sites of IGF-1 synthesis, in situ hybridization has been utilized to localize IGF-1 mRNA in the murine brain during the first postnatal month. Although IGF-1 mRNA was detected in all regions of the neonatal brain, there was considerable regional variation in the level of expression. Neurons were the principle sites IGF-1 mRNA expression and expression was typically restricted to one or two neuronal cell types within each region. In the cerebellar cortex, for example, only Purkinje cells hybridized to the IGF-1 probe. In contrast to gray matter, IGF-1 labeled cells were rarely found in presumptive white matter tracts of the forebrain. The hybridization signal was most prominent in regions where neurogenesis persisted after birth, including the cerebellum, olfactory bulb, and hippocampal complex. The timing of IGF-1 mRNA expression appeared to be temporally related to local neuronal proliferation. The number of labeled cells and intensity of hybridization signal was greatest during the first 2 postnatal weeks, a period of rapid neuronal proliferation in these regions. At the end of the first month, when neurogenesis had essentially ceased, IGF-1 signal strength had declined to background levels. The temporal and spatial pattern IGF-1 mRNA expression in the immature CNS was consistent with a role for locally produced IGF-1 in the regulation of brain development.     

Cell cycle-dependent change of proteasome distribution during embryonic development of the ascidian Halocynthia roretzi.

The proteasome is a multicatalytic proteinase complex composed of nonidentical subunits. By immunocytochemical analysis using monoclonal antibody raised against the egg proteasome, we demonstrate that the proteasome undergoes changes in its subcellular distribution, depending on the cell division cycle during embryonic development of the ascidian Halocynthia roretzi. During interphase, the proteasome is localized in the nucleus, i.e., in the nucleoplasm and along the nuclear membrane. The proteasome disappears from the nucleoplasm in prophase and from the nuclear envelope in prometaphase. During early metaphase, the proteasome is detectable in the chromosomes and, at late stages of metaphase, the immunoreactivity also occurs in the peripheral region of each spindle pole and at the mitotic spindle. In anaphase, however, the staining disappears in the mitotic apparatus. In telophase, the proteasome is again localized in the newly formed nucleus. In addition to the localization in the nucleus and around the mitotic apparatus, the proteasome shows cytoplasmic localization throughout the cell division cycle. Such a change of subcellular distribution of the proteasome is clearly demonstrated in the synchronously dividing blastomeres and also is believed to occur in the postcleavage embryos. These observations suggest that the proteasome may play a key role in the progression of cell division cycle.     

Localization of HB9 homeobox gene mRNA and protein during the early stages of chick feather development

We performed in situ hybridization and immunohistochemical analysis of HB9 homeobox gene mRNA and protein, respectively, during chick feather development. HB9 mRNA was highly expressed in epidermal basal cells and dermal cells of the placodes and feather buds, but not in those of the interplacodes and interbud regions. HB9 protein was predominantly expressed in dermal cells of the symmetric short buds and decreased after the asymmetric bud stage when the feather bud had become elongated along the anterior-posterior (A-P) and proximal-distal (P-D) axis. These results suggest that HB9 gene is regulated in a spatiotemporal manner during feather development, and may be involved in early feather bud morphogenesis.     

Segregation during cleavage of a factor determining endodermal alkaline phosphatase development in ascidian embryos.

Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.     

Distinct tubulin genes are differentially expressed during barley grain development

Tubulins, as major components involved in the organization of microtubules, play an important role in plant development. We describe here the expression profiles of all known α-tubulin (TUA), β-tubulin (TUB) and γ-tubulin (TUG) genes of barley ( Hordeum vulgare ), involving eight newly identified TUB sequences, five established TUA genes and one TUG gene. Macroarray and Northern blot-based expression patterns in the pericarp, endosperm and embryo were obtained over the course of the development of the grain between anthesis and maturation. These revealed that the various tubulin genes differed in their levels of expression, and to some extent were tissue specific. Two expression peaks were detected in the developing endosperm. The first and more prominent peak, at 2 days after flowering, included expression of almost all the tubulin genes. These tubulins are thought to be involved in mitoses during the formation of the syncytial endosperm. The second, less pronounced but more extended, peak included only some of the tubulin genes ( HvTUA3 , HvTUB1 and HvTUG ) and might be associated with the cell wall organization in aleurone and starchy endosperm. The HvTUA5 gene is expressed only in embryo of the developing grain and may be associated with shoot establishment. The expression profiles of the tubulin folding cofactors HvTFC A and HvTFC B as well as small G-protein HvArl2 genes were almost perfectly correlated with the global levels of tubulin mRNA, implying that they have a role in the control of the polymerization of α/β-tubulin heterodimers.     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Evolution of tubulin isoforms during the sporophytic development of Allomyces arbuscula

Abstract Tubulins extracted from the sporophytic developmental stages of Allomyces arbuscula have been characterised by one- and two-dimensional SDS-PAGE gels immunoblotted with monoclonal antibodies as α-, acetylated α- ( M _r 57 kDa both) and β- ( M _r 55 kDa) isoforms. The zoosporangial isoforms could be characterised only when precautions were taken to inhibit the strong tubulin proteolytic activity at this stage. The zoospores and zoosporangia contained greater amounts of the acetylated α- and β-isoforms than the mycelium, while the non acetylated α-isoform was present in greater quantity in the mycelium than in the zoospores or zoosporangia.