The role of maternal axin in patterning the Xenopus embryo.

Regulation of the stability of beta catenin protein is a critical role of Wnt signaling cascades. In early Xenopus development, dorsal axis specification depends on regulation of beta catenin by both cytoplasmic and nuclear mechanisms. While the cytoplasmic protein axin is known as a key component of the cytoplasmic beta catenin degradation complex, loss-of-function studies are needed to establish whether it is required for dorso-ventral patterning in the embryo, and to test where in the embryo it carries out its function. Here, we show that embryos lacking maternal axin protein have increased levels of soluble beta catenin protein and increased nuclear localization of beta catenin in ventral nuclei at the blastula stage. These embryos gastrulate abnormally and develop with excessive notochord and head structures, and reduced tail and ventral components. They show increased expression of dorsal markers, including siamois, Xnr3, chordin, gsc, Xhex, and Otx2, decreased expression of Xwnt 8 and Xbra, and little alteration of BMP4 and Xvent1 and -2 mRNA levels. The ventral halves of axin-depleted embryos at the gastrula stage have dramatically increased levels of chordin expression, and severely decreased levels of Xwnt 8 mRNA expression, while BMP4 transcript levels are only slightly reduced. This dorso-anterior phenotype is rescued by axin mRNA injected into the vegetal pole of axin-depleted oocytes before fertilization. Interestingly, the phenotype was rescued by ventral but not dorsal injection of axin mRNA, at the 4-cell stage, although dorsal injection into wild-type embryos does cause ventralization. These results show directly that the localized ventral activity of maternal axin is critical for the correct patterning of the early Xenopus embryo.

Time-lapse analysis of living mouse germ cell migration.

In mouse embryos, the primordial germ cells arise during gastrulation prior to, and distant from, the prospective gonads. Observations of PGCs in culture, and in fixed sections, have suggested, but not proved, that they migrate to the gonad by a process of active migration. The opaque nature of the early mouse embryo has precluded direct observation. Using confocal microscopy, we have filmed living PGCs expressing eGFP in tissue slices from mouse embryos at different stages of development. We find four clearly distinct phases of PGC migration. First, until E9.0-E9.5, PGCs are already highly motile, but do not leave the gut. Second, in the E9.0-E9.5 period, before the mesentery forms, PGCs very rapidly exit the gut, but do not migrate towards the genital ridges. Third, during the E10.0-E10.5 period, PGCs migrate directionally from the dorsal body wall into the genital ridges. In contrast to the prevailing model of germ cell migration, very few, if any, PGCs found in the gut mesentery at E10.5 migrate into the genital ridges. Finally, at E11.5, PGCs are slowing and the direction of movement is dependent on the sex of the embryo. This allows, for the first time, a formal description of the events of PGC migration in the mouse.

Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads.

Primordial germ cells are the founder cells of the gametes. They are set aside at the initial stages of gastrulation in mammals, become embedded in the hind-gut endoderm, then actively migrate to the sites of gonad formation. The molecular basis of this migration is poorly understood. Here we sought to determine if members of the integrin family of cell surface receptors are required for primordial germ cell migration, as integrins have been implicated in the migration of several other motile cell types. We have established a line of mice which express green fluorescent protein in germline cells that has enabled us to efficiently purify primordial germ cells at different stages by flow cytometry. We have catalogued the spectrum of integrin subunit expression by primordial germ cells during and after migration, using flow cytometry, immunocytochemistry and RT-PCR. Through analysis of integrin beta1(-/-)-->wild-type chimeras, we show that embryonic cells lacking beta1 integrins can enter the germline. However, integrin beta1(-/-) primordial germ cells do not colonize the gonad efficiently. Embryos with targeted deletion of integrin subunit alpha3, alpha6, or alphaV show no major defects in primordial germ cell migration. These results demonstrate a role for beta1-containing integrins in the development of the germline, although an equivalent role for * integrin subunit(s) has yet to be established.

Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line.

During normal embryonic development, mammalian germ cells use both cell migration and aggregation to form the primitive sex cords. Germ cells must be able to interact with their environment and each other to accomplish this; however, the molecular basis of early germ cell adhesion is not well characterized. Differential adhesion is also thought to occur in the adult seminiferous tubules, since germ cells move from the periphery to the lumen as they differentiate. In a screen for additional adhesion molecules expressed by the germ line, expression of the homophilic adhesion molecule, Ep-CAM, was identified in embryonic, neonatal and adult germ cells using immunocytochemistry and flow cytometry with an Ep-CAM-specific monoclonal antibody. At embryonic stages, germ cells were found to express Ep-CAM during migration at embryonic day 10.5 and early gonad assembly at embryonic day 12.5. Expression of Ep-CAM was also found on neonatal male and female germ cells. In the adult testis, Ep-CAM was detected only on spermatogonia, and was absent from more differentiated cells. Finally, embryonic stem cells were shown to express this receptor. It is proposed that Ep-CAM plays a role in the development of the germ line and the behaviour of totipotent cells.

Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction.

The heme in prostaglandin endoperoxide synthase (PGH synthase) was substituted with Mn(III)-protoporphyrin IX. The resulting enzyme, Mn-PGH synthase, showed full cyclooxygenase activity but only 0.9% of the peroxidase activity of the native iron enzyme. During the reaction with exogenous or endogenously produced hydroperoxides, a spectral intermediate of Mn-PGH synthase was observed. The electronic absorption bands of the resting enzyme at 376, 472, and 561 nm decreased, and the intermediate's bands at 417, around 513, and 625 nm appeared. The rate constant of the formation of the intermediate was about 10(4) M-1.s-1 at 22 degrees C, three orders of magnitude lower than with the iron enzyme. Spectral properties, conditions of formation, and the suppressed formation in the presence of electron donors provide evidence for a higher oxidation state of Mn-PGH synthase, tentatively a Mn(IV) species. This species was assigned to an intermediate in the peroxidase reaction of Mn-PGH synthase, the low activity of which was explained by the rate-limiting slow reaction of Mn-PGH synthase with hydroperoxides. The findings and interpretation are consistent with the published properties of other manganese-substituted peroxidases. Although the cyclooxygenase activity was similar to that of Fe-PGH synthase, the cyclooxygenase reaction of Mn-PGH synthase showed distinct differences in comparison with Fe-PGH synthase. A longer activation phase was observed which resembled the time course of the formation of the higher oxidation state. Glutathione peroxidase with glutathione, a hydroperoxide-scavenging system, inhibited the cyclooxygenase of Mn-PGH synthase at concentrations where the activity of Fe-PGH synthase was not affected. It is demonstrated that Mn-PGH synthase requires higher concentrations of hydroperoxides for the activation of the cyclooxygenase. These findings suggest that the substitution of iron with manganese in PGH synthase does not change the mechanism of the enzyme. The main difference is the much lower rate of the reaction with hydroperoxides which affects both the peroxidase activity and the hydroperoxide-dependent activation of the cyclooxygenase. A reaction scheme for Mn-PGH synthase is proposed analogous to that suggested for Fe-PGH synthase (Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 313-320).

The effect of fetal hypothalamus grafts on weight gain resulting from lesions of the ventromedial hypothalamus.

Bilateral ventromedial hypothalamic lesions in female adult rats which resulted in hyperphagia and rapid weight gain were followed by placement of fetal brain tissue in the anterior third ventricle. The treatment group received fetal hypothalamus grafts, and fetal cortical tissue of identical age was grafted into the control group. A significant reduction in average daily weight gain was noted from 4 to 12 weeks following transplantation in the treatment group. At 12 weeks posttransplantation, the animals were sacrificed for histological analysis. Examination of the hypothalamus grafts revealed neurons, ependymal clusters, and axonal processes which appeared to infiltrate the surrounding hypothalamic parenchyma.

Examination of the utility of the rat as an animal model for human anticoagulation.

The feasibility of employing the rat as an experimental model for investigation of full-dose heparin anticoagulation was assessed. Striking similarities were found to exist between rats and humans regarding baseline-activated partial thromboplastin time (APTT) values, and dosage per kilogram of heparin required to produce an APTT value of 1 1/2-3 times normal, the clinical definition of full-dose heparinization. Based upon these similarities, it appears that the rat can effectively serve as an experimental model for investigating the effects of heparin in humans.

Evaluation of the risks of anticoagulation therapy following experimental craniotomy in the rat.

The risk of hemorrhagic complications with anticoagulation therapy in patients following intracranial surgery has prevented investigation of the potential use of heparin in the early postoperative period. The authors have evaluated the safety of anticoagulation therapy following experimental craniotomy in male Holtzman rats. The dose and schedule of heparin administration, which elevated and maintained the activated partial thromboplastin time (APTT) within the therapeutic range of 1 1/2 to 3 X control APTT, was alternating doses of 400 and 500 IU/kg injected subcutaneously every 6 hours. This schedule was initiated 2, 4, 7, 10, and 14 days after craniotomy and was continued for 72 hours thereafter. The results demonstrated that the incidence of intracerebral hemorrhage declined as the postoperative interval prior to initiation of anticoagulation increased. If anticoagulation therapy was initiated during the first 7 postoperative days, the risk of intracerebral hemorrhage was high (mean 14.7%): however, if an additional 3 to 7 days elapsed prior to initiation of anticoagulation, the incidence of intracerebral hemorrhage dropped significantly (mean 0%) (p less than 0.05). These results suggest that anticoagulation therapy can be safely initiated 10 to 14 days after craniotomy.

Ultrasound-guided periventricular stereotaxis.

Intraoperative ultrasound can aid the biopsy of deep intracranial lesions. It is, perhaps, less clear whether ultrasound could be useful in functional neurosurgery, where the target is not abnormal in echogenicity. As an example, we chose to investigate in a dog model the periventricular gray target, which is frequently the choice for the placement of electrodes to control intractable pain. Autopsies showed the placement of our electrodes with less than 1 mm of error in four of five brains and a 1.5-mm error in the fifth brain. The largest error was seen to occur on the video screen and was due to our failure to tighten the guide properly. The potential advantages of this technique over conventional stereotaxis include the avoidance of: ventricular catheterization, the injection of contrast agent into the ventricles, the necessity for a stereotactic frame, and multiple x-ray exposures. Also, with real time scanning the surgeon has instant visual confirmation of electrode placement and can observe quickly any significant hematoma formation.