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Repression of formate dehydrogenase in Solanum tuberosum increases steady-state levels of formate and accelerates the accumulation of proline in response to osmotic stress.

Formate dehydrogenase (FDH, EC 1.2.1.2.) is a soluble mitochondrial enzyme capable of oxidizing formate into CO2 in the presence of NAD+. It is abundant in non-green tissues and scarce in photosynthetic tissues. Under stress, FDH transcripts (and protein) accumulate in leaves, and leaf mitochondria acquire the ability to use formate as a respiratory substrate. In this paper, we describe the analysis of transgenic potato plants under-expressing FDH, obtained in order to understand the physiological function of FDH. Plants expressing low FDH activities were selected and the study was focused on a line (AS23) showing no detectable FDH activity. AS23 plants were morphologically indistinguishable from control plants, and grew normally under standard conditions. However, mitochondria isolated from AS23 tubers could not use formate as a respiratory substrate. Steady-state levels of formate were higher in AS23 leaves and tubers than in control plants. Tubers of untransformed plants oxidized 14C formate into 14CO2 but AS23 tubers accumulated it. In order to reveal a possible phenotype under stress conditions, control and AS23 plants were submitted to drought and cold. These treatments dramatically induced FDH transcripts in control plants but, whatever the growth conditions, no 1.4 kb FDH transcripts were detected in leaves of AS23 plants. Amongst various biochemical and molecular differences between stressed AS23 and control plants, the most striking was a dramatically faster accumulation of proline in the leaves of drought-stressed plants under-expressing FDH.

Amino Acids↗

Coordinate regulation of the Escherichia coli formate dehydrogenase fdnGHI and fdhF genes in response to nitrate, nitrite, and formate: roles for NarL and NarP.

Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons. To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions. Complementary patterns of gene expression were seen. Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression. In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations. Consistent with prior reports, NarL was able to induce fdnG-lacZ expression. However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator). Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP. The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing. Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon.

Anaerobiosis↗

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate.

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H2) when grown on glucose. H2 formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H2 was found to be dependent on Hyd-4 and the F(0)F(1)-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate, H2 production dependent on pH was investigated. In both types of cells, H2 production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N'-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F(0)F(1)-ATPase. The results indicate that Hyd-3 has a major role in H2 production at acidic pH independently on the F(0)F(1)-ATPase.

Biophysics↗

Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens FD-1.

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H2 in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO2 (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, mu = 0.35/h). H2 was formed by a hydrogenase rather than by cleavage of formate, and 13C-NMR and 14C-exchange reaction data indicated that formate was produced by CO2 reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the expense of H2. This shift suggested that reducing equivalents could be balanced through formate or H2 production without affecting the yields of the major carbon-containing fermentation endproducts.

Acetates↗

CO2 reduction to formate by NADH catalysed by formate dehydrogenase from Pseudomonas oxalaticus.

The direct reduction of CO2 to formate is catalysed by formate: NAD oxidoreductase in the presence of substrate amounts of NADH. Proof for this reaction is supplied by the detection of a CO2-dependent NADH oxidation, and by the identification of [14c] formate as the product of a NADH-dependent reduction of [14c]carbonate. The enzyme-catalysed CO2 reduction by NADH attains the equilibrium predicted by thermodynamic considerations, a state which is also reached from the formate side. The Michaelis constant for CO2 is about 40 mM indicating the low affinity of the enzyme for this substrate. The corresponding value for formate is 0.1 mM. Under the special conditions employed the enzyme catalyses the formate oxidation about 30 times faster than the CO2 reduction. That CO2 and not HCO3- is the active species in the reduction was shown by comparing the ph dependency of the velocities of the forward and back reactions and by observing the kinetics of CO2 reduction during the simultaneous attainment of the CO2-HCO3- equilibrium.

Aldehyde Oxidoreductases↗

Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus.

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.

Alcaligenes↗

Relation between colony formation in calcium-deficient medium, colony formation in soft agar, and tumor formation by T51B rat liver cells.

T51B rat liver cells and several carcinogen-treated, carcinogen + saccharin-treated, or spontaneously altered clones of T51B cells were tested for their abilities to form colonies in calcium-deficient medium and soft agar and to produce tumors in athymic nude mice. Most (10 out of 11) of the clones which were derived from colonies in calcium-deficient medium were unable to form colonies in soft agar and 8 out of 11 were non-tumorigenic. Conversely, 6 out of 9 clones derived from colonies in soft agar were unable to multiply significantly in calcium-deficient medium and 5 of these 6 clones were also non-tumorigenic. Two of these 9 soft agar-growing clones were tumorigenic, one of which also proliferated in calcium-deficient medium, and the other of which acquired the ability to proliferate in calcium-deficient medium after it became able to form tumors in athymic nude mice. Thus, T51B rat liver cells gain the ability to grow in calcium-deficient medium and soft agar independently during the process of neoplastic transformation and neither characteristic by itself reliably predicts tumorigenicity.

Animals↗

Cyclic AMP increases endogenous granulocyte colony-stimulating factor formation in monocytes and THP-1 macrophages despite attenuated TNF-alpha formation.

The cytokine granulocyte colony-stimulating factor (G-CSF) is in broad clinical use to treat neutropenia, and trials on its use in immunosuppressed conditions and infections are ongoing. To apply G-CSF effectively, it is crucial to understand the regulation and distribution of its endogenous formation. Since G-CSF release is mediated, at least in part, by TNF-alpha formation, we investigated whether drugs suppressing TNF-alpha also impair G-CSF production. Surprisingly, G-CSF formation was enhanced in lipopolysaccharide (LPS)-stimulated blood from a pentoxifylline-treated patient. In the presence of dibutyryl-cAMP, forskolin, tolafentrine or 3-isobutyl-1-methylxanthine, LPS-induced G-CSF formation was enhanced in THP-1 cells, primary monocytes and whole blood. Correspondingly,rp-8-bromo-cAMP suppressed LPS-induced G-CSF release. Addition of prostaglandin E(2) enhanced and indomethacin suppressed G-CSF formation. Reporter gene studies showed that dibutyryl-cAMP enhanced LPS-induced G-CSF promoter activity, indicating a transcriptional up-regulation. Furthermore, disruption of a newly identified putative cAMP-responsive element (CRE) in the G-CSF promoter demonstrated the regulatory role for G-CSF gene transcription. In conclusion, endogenous G-CSF formation critically depends on both TNF-alpha and cyclooxygenase products, exerting effects via cAMP and the CRE in the G-CSF promoter. This might have bearing for drug side effects, putative G-CSF mimetics and our understanding of G-CSF immunobiology.

1-Methyl-3-isobutylxanthine↗

Cognitive bias in spider fear and control children: assessment of emotional interference by a card format and a single-trial format of the stroop task.

The aim of the study was to clarify whether fear in children is related to a distorted cognitive processing of fear-related information. In anxious children, only a few studies of this bias were performed which yielded inconsistent results. Martin, Horder, and Jones (1992. Cognition and Emotion, 6(6), 479-486) found a bias for spider words in spider-fear children, using a card format of the Stroop task. However, by using a single-trial format of the Stroop task, we previously found that both anxious and control children favored the processing of threatening information (Kindt, Brosschot, & Everaerd, 1997. Journal of Experimental Child Psychology, 64, 79-97). In the present study, we administered both a card format and a single-trial format of the Stroop task to spider-fear and control children. In line with our previous results, a bias for spider words was observed in spider fear but also in control children, regardless of the format used. Furthermore, the processing biases assessed by the two formats did not correlate, which suggests that they measure different mechanisms and/or that one or both mechanisms are unstable. It is speculated that certain cognitive developmental deficits in regulating emotions may be a vulnerability factor in the etiology of anxiety disorders.

Affect↗

Formation factor logging by electrical methods. Comparison of formation factor logs obtained in situ and in the laboratory.

In this paper, a new in situ method for obtaining the formation factor, which is essential for the matrix diffusion, is described and tested in intrusive igneous rock. The method is based on electrical resistivity measurements in rock where the pore water and rock resistivities are essential parameters. The method is based on electromigration instead of diffusion as in traditional diffusion experiments. In previous works, quantitative formation factors of rock have been obtained by electrical methods in the laboratory. Here, a similar approach is used in situ. An in situ logging campaign was performed by SKB during 2000 in the 1700-m-deep borehole KLX02 in Laxemar, Sweden. The rock resistivity was measured with the slimhole Dual Laterolog from Antares. The groundwater resistivity was measured with the Difference Flow Meter from Posiva. A formation factor log was obtained with the maximum vertical resolution of 10 cm. In order to validate the log, 100 rock samples were taken from the bore core, and a formation factor log was obtained by using electrical methods in the laboratory. Both direct current (DC) and alternating current (AC) were used. The measurements on the core confirmed that the in situ log was quantitative, but with a possible systematic error. The in situ formation factors were on average about 1/3 to 1/5 of the laboratory formation factors, depending on depth.

Diffusion↗

Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model.

Expression of parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) and protein was investigated throughout the developmental progression of endochondral bone formation in mouse and intramembranous bone formation in an in vivo model in rabbit, using in situ hybridization and immunohistochemistry. Endochondral bone formation was investigated in a developing embryo, newborn, and adult mouse. In fetal long bones through to newborn (day 7), PTHrP mRNA and protein were consistently expressed in chondrocytes within the proliferative, transitional, and hypertrophic zones. In addition, high levels of PTHrP were also detected in osteoblasts on the surface of trabecular bone surfaces. Similarly, at the adult stage (week 7), PTHrP mRNA and protein were consistently expressed in chondrocytes at epiphyseal ends of the subarticular cartilage, within cortical periosteum, as well as in osteoblasts located at the metaphyseal trabecular bone surfaces. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in preosteoblasts prior to trabecular bone formation (1-week bone harvest). As bone formed (2-, 3-, and 4-week bone tissue harvests), PTHrP mRNA and protein were highly expressed in actively synthesizing osteoblasts and in those osteocytes embedded within the superficial layers of the bone matrix. Lining osteoblasts and osteocytes buried deeply in the bone matrix displayed weak or no signal for PTHrP. The pattern of spatial and temporal expression of PTHrP demonstrated in cartilage cells and osteoblasts in the two systems suggests an important role of PTHrP in both endochondral and intramembranous bone formation.

Animals↗

Triplex formation by oligodeoxyribonucleotides involving the formation of X.U.A triads.

The stabilities of oligodeoxyribonucleotide triplexes containing a single pyrimidine-purine base pair, which interrupts an otherwise purine-pyrimidine base pair motif, were studied by UV melting experiments. The oligomer systems consisted of an oligodeoxyribonucleotide target duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, I.II(Y.Z), or d-GAAGAAAAAGUGAAA/d-TTTCACTTTTTCTTC, IV.V(U.A), where Y.Z is C.G, T.A, or U.A and U is deoxyuridine. The third strand oligodeoxyribopyrimidine was d-CTTCTTTTTTXTTTT, III(X), or d-CTTCTTTTTCXCTTT, VI(X), where C is 5-methyldeoxycytidine. Triplexes were observed in the system III.I.II(X.C.G) when X was T or U. This may involve formation of T. or U.C.G triads in which the 4-carbonyl of T or U serves as a hydrogen bond acceptor for the N4-amino group of C. Triplex formation between III(X) and I.II(T.A) was only observed when X was G. In contrast to T.A or C.G, it appears a U.A base pair in the duplex target is a much more versatile participant in triad formation. Thus, stable triplexes were observed in III.I.II(X.U.A) and in VI.IV.V(X.U.A) when X was C, C, T, or U. The formation of a T.U.A or U.U.A triad can occur if the T or U of III translates approximately 1.4 A into the major groove, thereby allowing the 3-NH of T or U to donate a hydrogen bond to the 4-carbonyl oxygen of U in the duplex. Formation of C. or C.U.A base triads could involve formation of a single hydrogen bond between the third strand N4-amino group of C or C and the 4-carbonyl group of U of the target.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

Pyridine-2, 6-dicarboxylic acid (dipicolinic acid) formation in Bacillus subtilis. II Non-enzymatic and enzymatic formations of dipicolinic acid from alpha, epsilon-diketopimelic acid and ammonia.

Non-enzymatic formation of dipicolinic acid (DPA) from diketopimelic acid and ammonia was clearly demonstrated using a new method for DPA analysis. The reaction rates of DPA formation were almost the same under aerobic and anaerobic conditions. Nearly equimolecular quantities of DPA and tetrahydrodipicolinic acid were detected in spontaneous reaction mixture. The spontaneous reaction seemed to be due to dismutation of dihydrodipicolinic acid, resulting in DPA and tetrahydrodipicolinic acid. The apparent optimum pH of the spontaneous reaction was 8.2 and the maximal rate of DPA formation was observed with a 1 : 4 molar ratio of diketopimelic acid to ammonia. The rate of the spontaneous reaction was stimulated by ferrous sulfate, FMN, and riboflavin. Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate and aspartic beta-semialdehyde, with NADPH as reductant. The reductase was isolated from Bacillus subtilis, and found to stimulate DPA formation from diketopimelic acid and ammonia. The enzymatic DPA formation was absolutely dependent on oxygen, and optimum pH was 6.4. The catalytic action of the enzyme was similar to that of the oxidase. Possible mechanisms of DPA formation from diketopimelic acid and ammonia are proposed.

Aerobiosis↗

Diffusion of the Interspecies Electron Carriers H(2) and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of K(m) for H(2) or Formate Uptake.

We calculated the potential H(2) and formate diffusion between microbes and found that at H(2) concentrations commonly found in nature, H(2) could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H(2) concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H(2) concentration at the cell surface of H(2)-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 mum from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the K(m) for H(2) or formate.

Journal Article↗

Role of ascorbic acid in the osteoclast formation: induction of osteoclast differentiation factor with formation of the extracellular collagen matrix.

Osteoclasts are bone-resorbing multinucleated cells. Tartrate-resistant acid phosphatase-positive (TRAP-positive) mononuclear and multinucleated cells, which are osteoclast-like cells (OCLs), were formed as a result of the coculture of mouse bone marrow cells and clonal stromal ST2 cells in the presence of 1alpha,25-dihydroxy-vitamin D3. Removal of ascorbic acid from the culture medium prevented the formation of TRAP-positive OCLs. Addition of ascorbic acid to the medium formed TRAP-positive OCLs, and the effect of ascorbic acid was dose-dependent. When we examined the level of messenger RNA (mRNA) for osteoclast differentiation factor (RANKL/ODF) in ST2 cells, we found that ascorbic acid caused an approximately 5-fold increase in the level of this mRNA. The half-life of the mRNA was unaffected by ascorbic acid. To characterize the mechanism of action of ascorbic acid, we investigated the relationship between formation of TRAP-positive OCLs and formation of the collagen matrix. Inhibitors of the formation of collagen triple helices blocked both the formation of TRAP-positive OCLs and the expression of the mRNA for RANKL/ODF in response to ascorbic acid. Our findings suggest that ascorbic acid might be essential for osteoclastogenesis and might induce the formation of TRAP-positive OCLs via induction of the synthesis of RANKL/ODF that is somehow mediated by the extracellular matrix.

Animals↗

Dissociation between global markers of bone formation and direct measurement of spinal bone formation in osteoporosis.

UNLABELLED: Regional bone metabolism measured using 18F-fluoride PET was assessed in 72 postmenopausal women classified as normal, osteopenic, or osteoporotic. Lower values of regional bone formation activity at the lumbar spine were seen in osteoporotic women, whereas global markers of bone formation were significantly increased. INTRODUCTION: Evaluations of global bone remodeling have revealed that increased bone turnover is a feature of patients with osteoporosis. The noninvasive functional imaging technique of 18F-fluoride positron emission tomography (PET) allows the direct quantitative assessment of bone metabolism at specific sites in the skeleton, including the clinically important site of the lumbar spine. MATERIALS AND METHODS: The aim of this study was to compare regional skeletal kinetics in 72 postmenopausal women (mean age, 61 years) classified as normal, osteopenic, or osteoporotic according to their BMD T score at the lumbar spine. Each woman had a dynamic PET scan of the lumbar spine after injection of 90 MBq 18F-fluoride ion and measurements of biochemical markers of bone formation and resorption. The arterial plasma input function was derived using aorta arterial activity from the PET image. Time-activity curves were obtained by placing regions of interest over the lumbar vertebrae. A three-compartmental model was used to calculate bone blood flow (K1) and the net plasma clearance of tracer to bone mineral (Ki), reflecting regional osteoblastic activity (ml/minutes/ml). Rate constants k2, k3, and k4, which describe transport between plasma, the extracellular fluid (ECF) compartment, and the bone mineral compartment, were also measured. RESULTS: The net uptake of fluoride to the bone mineral compartment (Ki) was significantly lower in the osteoporotic group compared with both the osteopenic and normal groups, with a mean difference of 0.005 ml/minutes/ml (16.7%). The fraction of the tracer in the extravascular tissue space that underwent specific binding to bone mineral (k3/k2 + k3) was also significantly reduced in the women classified as osteoporotic. In contrast, levels of bone-specific alkaline phosphatase (BSALP) were significantly higher in the osteoporotic group compared with the normal and osteopenic groups by 35% and 27%, respectively. A significant negative correlation (r = -0.41) was observed between levels of BSALP and the fraction of the tracer that underwent specific binding to bone mineral. CONCLUSION: Lower values of Ki, a measurement of regional bone formation activity, were seen in women classified as osteoporotic, whereas levels of BSALP, a measure of global bone formation, were significantly increased. These findings are suggestive of increased global skeletal bone turnover in women with postmenopausal osteoporosis but with relatively reduced regional bone formation at the predominantly trabecular site of the lumbar spine.

Absorptiometry, Photon↗

Cinnabarinate formation in malpighian tubules of the silkworm. Bombyx mori: reaction mechanism of cinnabarinate formation in the presence of catalase and manganese ions.

The formation of cinnabarinate in the presence of manganese ions and catalase was investigated spectrophotometrically. The absorption peak of cinnabarinate at 460 nm appeared only in a reaction system containing manganese ions and catalase. If catalase was omitted, a new absorption peak at 360 nm was observed while the absorption peak of cinnabarinate reached a plateau. Furthermore, in the presence of hydrogen peroxide, the absorption spectrum of cinnabarinate changed; catalase suppressed this absorption change. We conclude that in the course of cinnabarinate formation in the presence of manganese ions hydrogen peroxide is produced, which decomposes cinnabarinate. Catalase prevents the accumulation of hydrogen peroxide, which results in the steady increase of cinnabarinate. Cinnabarinate formation by manganese ions shows an initial lag phase. This lag phase disappeared by preincubating 3-hydroxyanthranilate under aerobic conditions. Incubation of 3-hydroxyanthranilate resulted in the generation of superoxide anions. When both manganese ions and superoxide anions were present, the lag phase of cinnabarinate formation disappeared. In the process of cinnabarinate formation, manganese ions serve to dismutate superoxide anions, as does superoxide dismutase; manganese (II) ions were oxidized to manganese (III) ions by superoxide anions. From these results we have proposed a mechanism of cinnabarinate formation catalysed by manganese ions.

3-Hydroxyanthranilic Acid↗

Mechanisms for pattern formation leading to axis formation and lineage allocation in mammals: a marsupial perspective.

Developing patterns in early embryogenesis are analysed in conceptuses from several families, including Dasyuridae, Phalangeridae, Macropodidae and Didelphidae, in which cleavage has been examined in some detail. Features common to cleavage and blastocyst formation, and in some cases to hypoblast formation, are used to develop an outline of possible mechanisms leading to axis formation and lineage allocation. Relevant features that have been described only in some species are also included. It is suggested that certain features of marsupial cleavage establish patterns in the developing blastocyst epithelia, pluriblast, trophoblast and hypoblast that contribute to axis formation and lineage allocation. All marsupials examined had a polarized oocyte or conceptus, the polarity of which was related to the conceptus embryonic-abembryonic axis and, eventually, the conceptus dorsal-ventral axis and the formation of the pluriblast (future embryo) and trophoblast. The embryonic dorsal-ventral and anterior-posterior axes were established after the allocation of hypoblast and epiblast. Mechanisms that appear to result in patterning of the developing epithelia leading to axis formation and lineage allocation are discussed, and include sperm entry point, gravity, conceptus polarity, differentials in cell-zona, cell-cell and cell-type (boundary effects) contacts, cell division order during cleavage and signals external to the conceptus. A model of the patterning effects is included. The applicability of these mechanisms to other amniotes, including eutherian mammals, is also examined.

Animals↗