Tubulin and actin synthesis during brain development

Chick brain proteins from 5- through 13-day embryos were labeled with l-[³⁵S]methionine for 30 min in vitro and analyzed by two-dimensional gel electrophoresis. Autoradiographs of the gels were scanned with a computer-coupled densitometer to measure the relative rates of protein synthesis. The actins and the tubulins were the most abundant proteins and had the highest rates of synthesis. β and γ actin were synthesized at constant rates throughout this period of development, but the rate of tubulin synthesis increased fourfold. Six α tubulins and two β tubulins were distinguished, and they were all synthesized at all times. The relative rates of synthesis of these forms changed with development in a complex pattern, but the stoichiometry of α:β remained 1:1.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

Tubulin isotypes in maize endosperm. Alterations during development and water deficit

Maize ( Zea mays L. cv . Pioneer 3925) endosperm development is sensitive to water deficit during rapid cell division and nuclear DNA endoreduplication. To gain insight into effects of water deficit on gene-products that are involved in these processes, we examined the accumulation of β-tubulin, a 50-kDa subunit of microtubules. Proteins extracted from endosperms were separated by SDS-PAGE and immunoblotted with antibodies to β-tubulin. In addition to the expected 50-kDa β-tubulin protein, monoclonal antibodies recognized a 35-kDa protein that predominated at early stages of development and progressively disappeared coincident with the appearance of 50-kDa β-tubulin. Various tests demonstrated that the cross-reacting 35-kDa protein was not a post-harvest artifact, but represented a group of in situ tubulin isotypes preferentially detected by the monoclonal antibodies we used. The pattern of appearance of the fragment suggested that differential expression or degradation of tubulin isotypes normally occurs during development. This expression pattern is prologed or altered during water deficit, which may affect cell division.     

Tubulin Sorting during Dimerization In Vivo

We demonstrate sorting of beta-tubulins during dimerization in the Drosophila male germ line. Different beta-tubulin isoforms exhibit distinct affinities for alpha-tubulin during dimerization. Our data suggest that differences in dimerization properties are important in determining isoform-specific microtubule functions. The differential use of beta-tubulin during dimerization reveals structural parameters of the tubulin heterodimer not discernible in the resolved three-dimensional structure. We show that the variable beta-tubulin carboxyl terminus, a surface feature in the heterodimer and in microtubules, and which is disordered in the crystallographic structure, is of key importance in forming a stable alpha-beta heterodimer. If the availability of alpha-tubulin is limiting, alpha-beta dimers preferentially incorporate intact beta-tubulins rather than a beta-tubulin missing the carboxyl terminus (beta 2 Delta C). When alpha-tubulin is not limiting, beta 2 Delta C forms stable alpha-beta heterodimers. Once dimers are formed, no further sorting occurs during microtubule assembly: alpha-beta 2 Delta C dimers are incorporated into axonemes in proportion to their contribution to the total dimer pool. Co-incorporation of beta 2 Delta C and wild-type beta 2-tubulin results in nonmotile axonemes because of a disruption of the periodicity of nontubulin axonemal elements. Our data show that the beta-tubulin carboxyl terminus has two distinct roles: 1) forming the alpha-beta heterodimer, important for all microtubules and 2) providing contacts for nontubulin components required for specific microtubule structures, such as axonemes.     

Specific endonucleolytic cleavages of mouse albumin mRNA and their modulation during liver development.

We have detected specific endonucleolytic cleavages of mouse albumin mRNA by S1 nuclease protection analysis of total RNA from fetal mouse liver using a cDNA probe spanning the middle, coding region of albumin mRNA. With the use of probe labeled at its 5' end, three prominent cleavages were detected which were confirmed and their endonucleolytic nature was established by further analysis using 3' end-labeled probe. The latter probe also revealed one more cleavage which was not detected with the 5' end-labeled probe. These cleavages mapped to positions on the mRNA which included a unique sequence motif CCAN1-3CUGN0-1UGAU. Degradation intermediates corresponding to these cleavages were consistently observed, specifically in fetal liver but not in normal or regenerating adult liver and appeared to have originated in vivo. Their levels decreased progressively from 18th day of gestation and became undetectable by 20 days after birth. No detectable changes in the levels of any of the prominent degradation products of alpha-fetoprotein (a homologue of albumin) mRNA could be observed during this period of development. Since accumulation of degradation intermediates is known to correlate with higher rate of mRNA turnover, our observations raise the possibility that the stability of albumin mRNA may be lower in fetal than in adult mouse liver.     

Spindle-pole organization during early mouse development

Spindle-pole organization during early mouse development was examined using a variety of immunological reagents that recognize centrosomal components. Spindle poles of unfertilized eggs and blastocysts were found to react positively with two antisera (centrin and NRS-01), whereas poles of activated eggs and early cleavage-stage embryos were negative when treated with the same sera. In contrast, a third antiserum (5051) showed positive spindle-pole staining throughout the preimplantation stages of development. Two monoclonal antibodies (MPM-1 and MPM-2) that are known to react with mitotic phosphoproteins were also used in this study. Both antibodies stained the cytoplasm of mitotic cells with extremely high intensity. In addition, MPM-2 was found to stain spindle poles. These results suggest that organizational changes in the spindle pole are occurring during early mouse development. Embryos homozygous for a recessive lethal mutation known as oligosyndactyly (Os) were also treated with the reagents described above. This mutation results in a metaphase arrest at the blastocyst stage with intact spindles being present. Spindle poles were observed in Os homozygous mutants stained with centrin, NRS-01, and 5051. However, when Os mutants were stained with the MPM monoclonal antibodies, about half of the mitotic cells completely lacked the dramatic cytoplasmic staining. This observation is in contrast to that observed for wild-type embryos, where greater than 95% of mitotic cells showed positive cytoplasmic staining.     

Application of high-performance liquid chromatography for the study of the microheterogeneity changes of mouse alpha-fetoprotein in fetal development

Changes in the microheterogeneity of mouse alpha-fetoprotein (MAFP) during fetal development were investigated by high-performance liquid chromatography (HPLC). A total of six distinct isoforms (Iso-1, Iso-2, Iso-3, Iso-4, Iso-5, Iso-6) of the heterogeneous MAFP were resolved from mouse amniotic fluid (MAF). Analysis of MAF collected at various times revealed that these isoforms were accumulated at different stages of the fetal development.     

CTGF expression during mouse embryonic development

Connective tissue growth factor (CTGF) is a potent fibroblast mitogen and angiogenic factor which plays an important role in wound healing, cancerogenesis and fibrotic and vascular disease. Here we explored the regulation and the cellular site of the mRNA synthesis for this growth factor in the developing mouse embryo by in situ hybridisation. Strong and persistent CTGF gene expression was limited to three types of tissue: the vascular endothelium, particularly the high-pressure part of the cardiovascular system, condensed connective tissue around bone and cartilage, and maturing layer VII neurons in the cerebral cortex. With few exceptions (late tooth bud, neuroepithelium) epithelial tissue was negative. Very transient but strong expression was observed early during formation of cartilage, in late stages during perichondral ossification, on cerebral neuroepithelium, and in several discrete stages of tooth formation, on mesenchymal precursors of odontoblasts condensing on inner dental epithelium, and later on apposing regions of ameloblast and odontoblast epithelium. Altogether, the current study suggests that CTGF performs a dual role: a continuous function in the cardiovascular system, bone and cartilage-associated mesenchyme and maturing layer VII neurons, but also a more transient function associated with the formation of cartilage, bone, tooth and cerebral nerve cells.     

Immunolocalization of hepatocyte growth factor and its receptor (c-Met) during mouse liver development

Although hepatocyte growth factor (HGF) was discovered as a potent hepatotrophic factor responsible for liver regeneration and may involve some organ development in embryogenesis, it remains to be revealed what roles HGF plays in liver development. The present study was undertaken to determine which cells express HGF and its receptor c-Met and when c-Met is activated in mouse liver development by using immunoblotting and immunohistochemical techniques. HGF was detected in hepatocytes and non-parenchymal cells, including biliary epithelial cells, periportal connective tissue cells, megakaryocytes, endothelial cells, and sinusoidal cells, throughout liver development. Positive HGF immunostaining in hepatocytes increased during postnatal development, and reached the maximal level in the adult stage. c-Met protein was also expressed in hepatocytes throughout liver development, but maximal staining was obtained in 1- or 2-week-old livers. Phosphorylation of tyrosine residues in the c-Met beta chain also occurred in these stages. These results suggest that HGF signaling is implicated in hepatocyte growth during postnatal liver development, and its action could be in a paracrine mode; HGF produced by non-parenchymal cells such as sinusoidal cells acts on hepatocytes expressing c-Met receptors. Positive immunostaining in adult and postnatal hepatocytes may be derived from their blood clearance of HGF.     

Rat liver hexokinases during development.

The four glucose-phosphorylating isozymes from rat liver were separated by DEAE-cellulose column chromatography at several ages during development. The isozymes exhibited a sequential mode of appearance. The isozymes A, B, and C reached maximal values of activity at days +1, +3, and +7, respectively, decaying afterwards to the low characteristic adult value. Isozyme D activity was detected in very low levels as early as day +3, rising from day +18 to reach constant adult values at day +30. A fraction of the glucose phosphorylating activity was found associated with particulate material in prenatal and newborn rats. From day +15 onwards, no particulate activity could be detected. The particulate activity was found to be composed of three low-Km isozymes as of the corresponding soluble fraction.     

Drapc1 expression during mouse embryonic development

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.     

Expression of Apc2 during mouse development

APC2 (previously known as APCL), a molecule closely related to the adenomatous polyposis coli (APC) tumor suppressor, can deplete cytoplasmic beta-catenin, like APC itself. Recently, it has been shown that APC2 may regulate the localization of p53 and the microtubule stability and/or extension. Although it has been reported that APC2 mRNA is expressed in human brain, the anatomical and ontogenic expression patterns remain unclear. The purpose of this study was to investigate the distribution of mouse Apc2 during mouse development. In the adult brain, Apc2 is expressed predominantly in neurons and throughout the brain. Northern blot analysis demonstrated a high level of Apc2 expression in embryonic and early postnatal brain. Ontogenic analysis has indicated that Apc2 is expressed in neural tissue, including the peripheral nervous system. During development of cortex, retina and cerebellum, Apc2 is expressed in post-mitotic cells. These findings suggest that Apc2 may contribute to the development of neuronal cells.     

Proteome profile changes during mouse testis development

Spermatogenesis is a process of terminal differentiation that results in the formation of mature sperm. In the first wave of this differentiation in the mouse testis, different cell types appear in the seminiferous epithelium at specific times. These cytological changes must be accompanied by changes in protein expression patterns. The aim of the present study was the comparative analysis of proteomic profiles of the soluble proteins expressed at different stages of mouse testis development (8, 18 and 45 postnatal days). Conspicuous variations in their accumulation (representing up or downregulation) were detected over the course of development. Using mass spectrometry (MALDI-TOF), 44 proteins or variant forms were identified. Proteins with redox or antioxidant activity were identified in high proportions; others involved in lipid and carbohydrate metabolic pathways, as well as a number of proteins or isoforms not previously characterized in testis were also detected. These results contribute to identify changes in soluble protein associated to the complex process of male germ cell differentiation.