Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system.

The mouse midbrain-hindbrain constriction is centrally involved in patterning of the midbrain and anterior hindbrain (cerebellum), as revealed by recent genetic studies using mice and embryological studies in chick (reviewed in [1,2]). This region can act as an organizer region to induce midbrain and cerebellar development. Genes such as Engrailed-1, Pax-2 and Pax-5, which are expressed in the embryonic cells that will form the midbrain and the cerebellum, are required for development of these regions. Fate-mapping experiments at early somite stages in chick have revealed that the cerebellar primordium is located both anterior and posterior to the midbrain-hindbrain constriction, whereas midbrain precursors lie more anteriorly. Fate mapping in mice has been complicated by the inaccessibility of the postimplantation embryo. Here, we report the use of a new in vivo approach involving the Cre-IoxP site-specific recombination system [3] to map the fate of cells in the mouse midbrain-hindbrain constriction. We show that cells originating in the mouse dorsal midbrain-hindbrain constriction during embryonic days 9-12 contribute significantly to the medial cerebellum and colliculi. Our data demonstrate the feasibility of using a recombinase-based lineage-tracing system for fate mapping in the mouse.

Subregion- and cell type-restricted gene knockout in mouse brain.

Using the phage P1-derived Cre/loxP recombination system, we have developed a method to create mice in which the deletion (knockout) of virtually any gene of interest is restricted to a subregion or a specific cell type in the brain such as the pyramidal cells of the hippocampal CA1 region. The Cre/loxP recombination-based gene deletion appears to require a certain level of Cre protein expression. The brain subregional restricted gene knockout should allow a more precise analysis of the impact of a gene mutation on animal behaviors.

Cell-specific expression from the human dopamine beta-hydroxylase promoter in transgenic mice is controlled via a combination of positive and negative regulatory elements.

The promoter region of the human dopamine beta-hydroxylase (DBH) gene was analyzed in transgenic mice to identify DNA sequences responsible for the tissue- and cell-specific expression of the gene. Transgenic mice were generated that carried the Escherichia coli lacZ gene under control of DBH promoter fragments between 0.6 and 5.8 kilobases (kb) in length. Sequences required for expression in adult and fetal noradrenergic neurons were located between 0.6 and 1.1 kb 5' to the DBH transcriptional start site. Sequences in this region and farther upstream also directed expression to dopaminergic and noncatecholaminergic brain neurons that was repressed by negative elements elsewhere in the gene. The results indicate that the neuron-specific expression of the DBH gene is mediated by positive regulatory elements but that negative elements are required to restrict expression to the proper subset of neurons.

Expression of NGF in sympathetic neurons leads to excessive axon outgrowth from ganglia but decreased terminal innervation within tissues.

The effects of nerve growth factor (NGF) on sympathetic axon growth were investigated by generating transgenic mice in which the beta subunit of NGF was expressed in sympathetic neurons using the human dopamine beta-hydroxylase (DBH) promoter. In DBH-NGF mice, the sympathetic trunk and nerves growing to peripheral tissues were enlarged and contained an increased number of sympathetic fibers. Although sympathetic axons reached peripheral tissues, terminal sympathetic innervation within tissues was decreased in DBH-NGF mice. This effect could be reversed in the pancreas by overexpression of NGF in pancreatic islets. The observations are consistent with a model in which NGF gradients are not required to guide sympathetic axons to their targets, but are required for the establishment of the normal density and pattern of sympathetic innervation within target tissues.

The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice.

Dopamine beta-hydroxylase (DBH) catalyzes the final step in the biosynthesis of norepinephrine, the principal classic neurotransmitter of peripheral sympathetic neurons. We have shown that 5.8 kb of 5' upstream region from a cloned human DBH gene promoter is sufficient to direct expression of the E. coli lacZ gene in transgenic mice to neurons of the locus ceruleus and other classic noradrenergic brain stem nuclei, sympathetic ganglion neurons, and adrenal chromaffin cells. lacZ expression was also observed in neurons of the enteric system, the retina, some sensory and all cranial parasympathetic ganglia, and some diencephalic and telencephalic brain nuclei. The expression pattern of the transgene in DBH-immunonegative sites overlapped with many sites where expression of tyrosine hydroxylase or phenylethanolamine N-methyltransferase, two other catecholamine biosynthetic enzymes, has been reported.

Some neuronal cell populations express human dopamine beta-hydroxylase-lacZ transgenes transiently during embryonic development.

The 5' flanking region from the human dopamine beta-hydroxylase gene directs expression of bacterial beta-galactosidase reporter genes to a subset of adult neurons and adrenal chromaffin cells of transgenic mice. In this paper, we examine the spatial and temporal patterns of expression of these transgenes during embryogenesis. Expression begins at embryonic day 9 in the developing central and peripheral nervous systems and persists in cell populations in which expression is observed in adult transgenic mice. However, transient embryonic expression occurs in presumptive neuroblasts in developing sensory ganglia and ventrolateral neural tube that are destined to synthesize neurotransmitters other than catecholamines. These observations support the concept that some cells fated to become "non-catecholaminergic" neurons exhibit transient catecholaminergic features during their differentiation.

Endosymbiosis and cellular tolerance in the Hawaiian soft coral Sarcothelia edmondsoni verrill.

The relationship between the soft coral Sarcothelia edmondsoni Verrill and its symbiotic algae is considered as an early instance of cellular tolerance which can be disturbed by a variety of adverse conditions. The algal cells lie in vesicles deep within the endodermal cells of the host and are not subject to digestion. Their expulsion appears to be a reverse translocation to the distal end of the host cell and escape by a form of reversed phagocytosis resembling secretion. The cellular mechanisms involved are not clear.

Ultrastructure of the obligate halophilic bacterium Halobacterium halobium.

The fine structure of Halobacterium halobium was studied by means of a modified double-fixation technique. The cell envelope is shown to consist of both a "wall" and a plasma membrane. Some electron-dense strands were seen inside the cytoplasm running parallel to the cell envelope. An unusual organelle (or organelles) appeared inside the cytoplasm in the form of parallel striated strands.

Cocoon surrounding desert-dwelling frogs.

A cocoon formed from a single cell layer of shed stratum corneum may reduce water loss from the skin of desert-dwelling frogs while these aestivate in soil-filled burrows. In several Australian examples, the cocoon is a single layer of cells, and thus differs from the multilayered structure obtained from an American species, Scaphiopus couchi.

Antibody formation by single cells from lymph nodes and efferent lymph of sheep.

The antibody-forming cells which appear in the popliteal lymph node and efferent lymph of the sheep following immunization with boiled Salmonella have been studied by light and electron microscopy. Cells were incubated in monolayers with target erythrocytes sensitized with bacterial lipopolysaccharide. Three types of interaction between a proportion of the lymph cells and the erythrocytes surrounding them have been shown to indicate antibody formation: plaque-formation, immuno-cyto-adherence, and localized agglutination. At the peak of the response, 4 days after antigenic stimulation approximately 1 cell in every 200 from lymph node suspensions produces detectable specific antibody, while up to 1 cell in 20 in the lymph is active. For light microscope examination, individual antibody-forming cells were smeared in serum and stained with Leishman's stain. For electron microscopy, a number of active cells were clumped with antiserum to form a specimen of convenient size, then sectioned. Most of the active cells from efferent lymph are large and basophilic, while a small proportion are blastlike. These cells contain abundant free ribosomes and very little endoplasmic reticulum. In the node only, an additional class of antibody-forming plasma cells is found which have considerable amounts of endoplasmic reticulum in their cytoplasm.