Axial mesendoderm refines rostrocaudal pattern in the chick nervous system.

There has long been controversy concerning the role of the axial mesoderm in the induction and rostrocaudal patterning of the vertebrate nervous system. Here we investigate the neural inducing and regionalising properties of defined rostrocaudal regions of head process/prospective notochord in the chick embryo by juxtaposing these tissues with extraembryonic epiblast or neural plate explants. We localise neural inducing signals to the emerging head process and using a large panel of region-specific neural markers, show that different rostrocaudal levels of the head process derived from headfold stage embryos can induce discrete regions of the central nervous system. However, we also find that rostral and caudal head process do not induce expression of any of these molecular markers in explants of the neural plate. During normal development the head process emerges beneath previously induced neural plate, which we show has already acquired some rostrocaudal character. Our findings therefore indicate that discrete regions of axial mesendoderm at headfold stages are not normally responsible for the establishment of rostrocaudal pattern in the neural plate. Strikingly however, we do find that caudal head process inhibits expression of rostral genes in neural plate explants. These findings indicate that despite the ability to induce specific rostrocaudal regions of the CNS de novo, signals provided by the discrete regions of axial mesendoderm do not appear to establish regional differences, but rather refine the rostrocaudal character of overlying neuroepithelium.     

Sox-9 and cDachsund-2 expression in the developing chick telencephalon

We have created a transgenic mouse line that expresses Cre recombinase under the control of the novel mouse promoter/enhancer D6. We describe the expression pattern of D6-Cre in a Gtrosa26 reporter background as assayed by LacZ activity. The enhancer activity starts at 10.5 days post-coitum in the telencephalon and is at the later embryonic stages highly restricted to the hippocampus and the neocortex. In adult mice D6-derived cells are found in cortical layers II–VI, in the granular cells of the dentate gyrus and in hippocampal fields CA1–CA3. D6-Cre activity is also detected in the ependymal and subependymal zone of the lateral ventricles which is known to harbor neural stem cells.     

A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication

The centrosome plays a fundamental role in organizing the interphase cytoskeleton and the mitotic spindle, and its protein complexity is modulated to support these functions. The centrosome must also duplicate itself once during each cell cycle, thus ensuring the formation of a bipolar spindle and its continuity through successive cell divisions. In this study, we have used a battery of antibodies directed against centrosomal components to study the general organization of the centrosome during the cell cycle and during the centrosome duplication process. We demonstrate that a subset of centrosomal proteins are arranged together to form a tubular pattern within the centrosome. The tubular conformation defined by these proteins has a polarity and is closed at one end. The centriole complement of the centrosome is normally placed near this end. We show that the "wall" of the tube is enriched in proteins such as CDC2, ninein, and pericentrin as well as gamma-tubulin. In addition, a subset of gamma-tubulin is localized to the "lumen" of the tube. We also demonstrate, for the first time, that antibody staining can be used to detect centrosome duplication allowing the identification of duplication intermediates. We show that one product of centrosome duplication is the replication of the tubular structure found within the centrosome. The position of the centriole duplexes prior to and during centrosome duplication is documented and a model of the morphogenesis of the centrosome during the duplication process is proposed.     

Role of the telencephalon in regulating the electrical activity of skeletal muscles in chick embryos

On 18-day chick embryos, studies have been made of functional properties of hyperstriatum accesorium (HA) of the telencephalon and that of neck muscles. To investigate the relationship between the HA structure and the EMG in muscles, the former was subjected to unilateral coagulation. Specific levels were established of the energy of oscilatory processes--EEG in the HA and EMG of neck muscles--as well as the relationship between them and possible role of HA structure in regulation of fast movements.     

A monoclonal antibody that defines rostrocaudal gradients in the mammalian nervous system

Spinal cord axons display a rostrocaudal, positional bias in their innervation of sympathetic ganglia and intercostal skeletal muscles. In an effort to examine the molecular basis of this positional specificity, we used the cyclophosphamide immunosuppression method to produce monoclonal antibodies that bind preferentially to rostral ganglia. The staining distribution of one of these antibodies, ROCA1, has been analyzed using a novel histological method. A graded decline in binding is observed along the chain of adult rat sympathetic ganglia, as well as in the nerves innervating intercostal muscles. The antigen is identified on immunoblots as a 65 kd protein, whose distribution corresponds to the pattern found histologically. Surprisingly, ROCA1 appears to bind to glial cells, implying rostrocaudal, molecular differences in their surfaces.     

Early chick embryonic cells can form clones in agarose cultures

Early chick embryonic cells prior to the formation of the primitive streak, have been cultured in a two-layer soft-agarose system. Single, primary cells when grown in this system were capable of producing colonies ranging in size from 30 to 100 cells. The plating efficiency varied between 1 and 5% and the colonies remained viable for about 2 weeks. We believe this is the first report of normal, non-passaged cells which show anchorage-independent growth properties by forming colonies in a standard agarose culture in the absence of additional factors. The importance of being able to use normal monoclonal embryonic cell populations in studying early developmental processes is also discussed.     

Biphasic dispersion of clones containing Purkinje cells and glia in the developing chick cerebellum.

The cerebellum is a highly conserved structure which exhibits patterns of gene expression and axonal connections that are organized into parasagittal domains. These aspects of the mature cerebellum are presaged during embryonic development by the expression patterns of vertebrate homologs of Drosophila segmentation genes. We wished to determine whether the parasagittal domains of gene expression are compartments of lineage restriction. To this end, a clonal analysis of the chick cerebellum was conducted with a complex retroviral library. From embryonic day (E) 8 to E12, clones derived from the more medial portion of the cerebellar ventricular zone (VZ) were observed to spread preferentially in the mediolateral direction, crossing the boundaries of the parasagittal domains of gene expression. In late embryonic and posthatch periods, VZ clones were found to comprise Purkinje cells, glial cells, or both types of cells. At these later times, clonally related glial cells formed tight parasagittal clusters, while clonally related Purkinje cells were scattered extensively in the anteroposterior direction. We propose that a subset of the cerebellar VZ clones, those with medial origins, undergoes a biphasic dispersion: an early phase of mediolateral dispersion and a late phase of anteroposterior dispersion. This novel pattern of clonal dispersion suggests that the cerebellar VZ is not partitioned into parasagittal domains of lineage restriction. It leaves open the possibility that the later dispersion along the anteroposterior axis results from the parasagittal patterns of gene expression in the developing cerebellar cortex.     

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K⁺ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.     

Zinc accumulation in the telencephalon of lizards

The zinc concentration in the brains of two species of lizard was determined by atomic-absorption spectrophotometry. The zinc concentration was found to be highest in the telencephalon of Lacerta galloti (21.1 micrograms/g fresh weight) and Podarcis hispanica (16.77 +/- 0.8 micrograms/g) while the mesencephalon and brain stem exhibited lower zinc concentrations, i.e., 7.0 micrograms/g in Lacerta galloti and 6.08 +/- 0.4 micrograms/g in Podarcis hispanica. This high telencephalic concentration of zinc is paralleled by intense and well-defined Timm reactivity used for demonstrating the presence of zinc-containing boutons at the light-microscope level. Volumetric-densitometric studies of these Timm-reactive zones were performed using serial transverse sections of the same lizard brains.     

In vitro locomotion of allosensitized T lymphocyte clones in response to metabolites of arachidonic acid is subset specific

The effects of the arachidonic acid metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) on the in vitro random migration of cloned murine T lymphocytes (derived from limiting dilution analysis of a C57BL/6 anti-DBA/2 mixed leukocyte culture) were examined. Experiments were also performed to study the effects of the cyclooxygenase inhibitor indomethacin on both random lymphocyte migration and lymphocyte migration in the presence of PGE2. The responses of cloned lymphocytes to PGE2 and LTB4 were compared with those of unsensitized lymph node lymphocytes. PGE2 at 100 ng/ml significantly inhibited (p less than 0.001) the in vitro migration of helper clones of T lymphocytes, but had no effect on random migration of cytotoxic T cells or helper independent cytotoxic (HIT) cloned cells. In contrast, LTB4 significantly (p less than 0.001) enhanced the random locomotion of helper, cytotoxic, and HIT cloned cells at 0.1 and 0.3 ng/ml. The effects of both PGE2 and LTB4 were found to be completely reversible by cell washing. Indomethacin (10(-7) M) did not alter random migration of any of the clones, and in particular, did not affect the inhibition of helper lymphocyte migration induced by PGE2. Unsensitized bulk lymph node lymphocyte migration was not affected by either PGE2 or LTB4. The results suggest that modulation of lymphocyte locomotor function by environmental stimuli may depend on cellular activation, and the locomotor responses of activated lymphocytes to arachidonic acid metabolites may be subset specific.     

Perlecan controls neurogenesis in the developing telencephalon

Background  

Perlecan is a proteoglycan expressed in the basal lamina of the neuroepithelium during development. Perlecan absence does not impair basal lamina assembly, although in the 55% of the mutants early disruptions of this lamina conducts to exencephaly, impairing brain development. The rest of perlecan-null brains complete its prenatal development, maintain basal lamina continuity interrupted by some isolated ectopias, and are microcephalic. Microcephaly consists of thinner cerebral walls and underdeveloped ganglionic eminences. We have studied the mechanisms that generate brain atrophy in telencephalic areas where basal lamina is intact.