Expression of SRG3, a chromatin-remodelling factor, in the mouse oocyte and early preimplantation embryos.

SRG3 (Smarcc1) is a core subunit of the SWI/SNF complex. In the absence of SRG3, embryonic development ceases during peri-implantation stages, indicating that SRG3, as well as the chromatin-remodelling process, plays an essential role in early mouse development. To gain a better understanding of chromatin remodelling during the early stages of development, we examined SRG3 expression during oogenesis and preimplantation stages using immunofluorescence and western blot assays. SRG3 was detected in nuclei of oocytes during growth and maturation. Following fertilization, SRG3 was detected in pronuclei shortly after their formation. Nuclear concentrations of SRG3 increased in a time-dependent fashion and were found to be greater in the male pronucleus than in the female pronucleus. The increase in nuclear SRG3 was partially inhibited by a protein synthesis inhibitor, but not by a transcriptional inhibitor. Expression of SRG3 is accompanied by expression of Brg1 and Ini1, two other core subunits of the SWI/SNF complex. The expression of these three remodelling factors parallels that of SP1 and TBP, both spatially and temporally, in the mouse embryo, suggesting a role for remodelling factors in chromatin structure and function during early development.

Analysis of ingredient and heating value of municipal solid waste.

Great differences between municipal solid wastes (MSW) produced at different places and different times in terms of such parameters as physical ingredient and heating value lead to difficulty in effective handling of MSW. In this paper, ingredient, heating value and their temporal varying trends of typical MSW in Beijing were continuously measured and analyzed. With consideration of the process in pyrolysis and incineration, correlation between physical ingredients and heating values was induced, favorable for evaluation of heating value needed in handling of MSW from simple analysis of physical ingredients of it.

Internal circulating fluidized bed incineration system and design algorithm.

The internal circulating fluidized bed (ICFB) system is characterized with fast combustion, low emission, uniformity of bed temperature and controllability of combustion process. It is a kind of novel clean combustion system, especially for the low-grade fuels, such as municipal solid waste (MSW). The experimental systems of ICFB with and without combustion were designed and set up in this paper. A series of experiments were carried out for further understanding combustion process and characteristics of several design parameters for MSW. Based on the results, a design routine for the ICFB system was suggested for the calculation of energy balance, airflow rate, heat transfer rate, and geometry arrangement. A test system with ICFB combustor has been set up and the test results show that the design of the ICFB system is successful.

Early steps in pituitary organogenesis.

Significant advances have been made in defining the transcription cascade that is responsible for the early steps of pituitary formation and the environmental signals that induce, pattern and specify the pituitary gland and its cell types. It is now possible to outline the molecular mechanisms underlying the formation of the pituitary gland, as well as the initial development of organ-specific cell types.

Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5.

The mammalian hippocampus contains the neural circuitry that is crucial for cognitive functions such as learning and memory. The development of such circuitry is dependent on the generation and correct placement of the appropriate number and types of neurons. Mice lacking function of the LIM homeobox gene Lhx5 showed a defect in hippocampus development. Hippocampal neural precursor cells were specified and proliferated, but many of them failed to either exit the cell cycle or to differentiate and migrate properly. Lhx5 is therefore essential for the regulation of precursor cell proliferation and the control of neuronal differentiation and migration during hippocampal development.

LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons.

The circuits that control movement are comprised of discrete subtypes of motor neurons. How motor neuron subclasses develop and extend axons to their correct targets is still poorly understood. We show that LIM homeodomain factors Lhx3 and Lhx4 are expressed transiently in motor neurons whose axons emerge ventrally from the neural tube (v-MN). Motor neurons develop in embryos deficient in both Lhx3 and Lhx4, but v-MN cells switch their subclass identity to become motor neurons that extend axons dorsally from the neural tube (d-MN). Conversely, the misexpression of Lhx3 in dorsal-exiting motor neurons is sufficient to reorient their axonal projections ventrally. Thus, Lhx3 and Lhx4 act in a binary fashion during a brief period in development to specify the trajectory of motor axons from the neural tube.

Formation of Rathke's pouch requires dual induction from the diencephalon.

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke's pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke's pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.

Multistep control of pituitary organogenesis.

Lhx3 and Lhx4 (Gsh4), two closely related LIM homeobox genes, determine formation of the pituitary gland in mice. Rathke's pouch is formed in two steps-first as a rudiment and later as a definitive pouch. Lhx3 and Lhx4 have redundant control over formation of the definitive pouch. Lhx3 controls a subsequent step of pituitary fate commitment. Thereafter, Lhx3 and Lhx4 together regulate proliferation and differentiation of pituitary-specific cell lineages. Thus, Lhx3 and Lhx4 dictate pituitary organ identity by controlling developmental decisions at multiple stages of organogenesis.

Lhx4, a LIM homeobox gene.

LIM homeobox genes are well conserved in evolution and play important roles as transcriptional regulators of embryonic development. Here we report on the structure of LIM domains of the mouse Lhx4 (Gsh4) gene. The cDNA was generated by modified reverse transcription-PCR from midgestation embryo templates, using a degenerate consensus primer. The deduced amino acid sequence of the first LIM domain reveals 77% identity and that of the second domain reveals 86% identity with the corresponding sequences of the closely related Lhx3 gene. In addition, there is 38-56% similarity to other members of the Lhx gene family. The LIM consensus sequence is well conserved in Lhx4.

Expression of murine Lhx5 suggests a role in specifying the forebrain.

A LIM homeobox gene, Lim5, is known to be expressed in the forebrain of Xenopus and zebrafish (Toyama et al. [1995] Dev. Biol. 170:583-593). Results from developmental and comparative studies of its mouse ortholog, Lhx5, indicate that this gene may play important roles in forebrain development. Lhx5 expression is detected in the most anterior portion of the neural tube at the headfold stage, overlapping partially with Otx2 expression domain. After neural tube closure, Lhx5 is expressed as a transverse stripe, covering most of the diencephalic primordium. This expression recedes to restricted areas as Dlx gene expression occurs. By midgestation, both genes, Lhx5 and Dlx5, are expressed in the diencephalon and ventral telencephalon in an alternating complementary pattern. It may be that Dlx inhibits Lhx5, and this may represent a step of early regionalization of the forebrain. Lhx5 is also expressed in midbrain, hindbrain, and spinal cord, overlapping extensively with Lhx1 starting from day E10.5 of gestation. The early, persistent, and dynamic expression of Lhx5 suggests a regulatory function in forebrain formation.

Molecular cloning, structure, and chromosomal localization of the mouse LIM/homeobox gene Lhx5.

Lhx5, the mouse ortholog of the Xenopus Xlim-5, is a LIM/homeobox gene expressed in the central nervous system during both embryonic development and adulthood. During development its domain of expression is mainly localized at the most anterior portion of the neural tube, and it precedes the morphological differentiation of the forebrain; for this reason we believe that Lhx5 could play an important role in forebrain patterning. Here we present the structural organization and the chromosomal localization of the Lhx5 gene. The gene is composed of five exons spanning more than 10 kb of genomic sequence. The first and second LIM domains are encoded by the first and second exon, while the codons of the homeobox are split between the third and the fourth exons. The structure of Lhx5 is similar to that of other LIM/homeodomain proteins, Lhx1/lim1 and Lhx3/lim3, but differs from that of other LIM genes, such as mec3 and LMO1/Rbtn1, in which the codons for the LIM domains are interrupted by introns. We have mapped Lhx5 to the central region of mouse chromosome 5.

Defects in enteric innervation and kidney development in mice lacking GDNF.

Glial-lial-cell-line-derived neurotrophic factor (GDNF) has been isolated as neurotrophic factor for midbrain dopaminergic neurons. Because of its neurotrophic activity on a wide range of neuronal populations in vitro and in vivo, GDNF is being considered as a potential therapeutic agent for neuronal disorders. During mammalian development, it is expressed not only in the nervous system, but also very prominently in the metanephric kidney and the gastrointestinal tract, suggesting possible functions during organogenesis. We have investigated the role of GDNF during development by generating a null mutation in the murine GDNF locus, and found that mutant mice show kidney agenesis or dysgenesis and defective enteric innervation. We demonstrate that GDNF induces ureter bud formation and branching during metanephros development, and is essential for proper innervation of the gastrointestinal tract.

Specification of pituitary cell lineages by the LIM homeobox gene Lhx3.

During pituitary organogenesis, the progressive differentiation of distinct pituitary-specific cell lineages from a common primordium involves a series of developmental decisions and inductive interactions. Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages. In mice homozygous for the Lhx3 mutation, Rathke's pouch formed but failed to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary. The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage.

Combinatorial expression of immediate early genes in single neurons.

To address the question how relatively small numbers of immediate early gene (IEG) could specifically couple a wide range of stimulus-response cascades, we examined the possibility that IEG could be expressed heterogeneously in individual neurons. Analysis of multiple IEG in single neurons revealed that many individual DRG neurons express several IEGs. The combinatorial expression of IEGs by individual DRG displays substantial heterogeneity. Analysis of mRNA species encoding AP-1 composition in single cells also revealed coordinated change of mRNAs coding for AP-1 factors after membrane depolarization. Our results indicate that differential expression of IEG in individual cells, and the possible interaction among them may represent a mechanism by which the specificity in stimulation-response coupling may be achieved by IEGs.

Analysis of multiple heterogeneous mRNAs in single cells.

We describe a two-stage amplification procedure designed to analyze multiple heterogeneous mRNA from single cells. Using an oligo(dT) primer with an attached phage promoter, the whole mRNA pool from a single cell was first transcriptionally amplified. This step brings the weak signal from one cell to a range in which it can be reliably picked up by the polymerase chain reaction (PCR) procedure. The cDNA was then divided for separate PCR amplification to obtain an unambiguous signal for each gene product. The linear amplification of phage transcription increased the convenience and reliability of detecting multiple messengers in the second stage. The procedure is extremely sensitive because it combines the amplification generated by both phage transcription and PCR. Using a pAW109 artificial RNA, we demonstrated that this procedure detects 10 copies of pAW109 RNA per original sample with 90% confidence and 50 copies per sample with > 95% confidence. This procedure of multiple mRNA analysis allows "phenotyping" of any cell for its mRNA composition. Examples involving several immediate early genes and subunits of the gamma-aminobutyric acidA receptor genes are given. The method should greatly facilitate the analysis of combinatorial expression of various regulatory or channel molecules in their native environments. The procedure should also provide a direct and efficient way of decoding the developmental instruction coded through combinatorial transcriptional regulation.

Specific regulation of immediate early genes by patterned neuronal activity.

Electrical activity shapes development of the nervous system, presumably in part by regulating gene expression. A set of regulatory genes, immediate early genes (IEGs), which are responsive to a number of extrinsic cellular stimuli have been proposed to play a role in coupling such activity to gene expression. Using a semiquantitative polymerase chain reaction assay, we show that in dissociated mouse dorsal root ganglion neurons the expression of two IEGs, c-fos and nur/77, is differentially sensitive to patterns of electrical stimulation. Differences in c-fos activation did not correlate with the peak intracellular calcium [Ca++]i produced by the different stimulation patterns or with residual [Ca++]i following stimulation. However, the net increase in [Ca++]i (calcium time integral) was greater for the pulsed stimulus that activated c-fos (6 impulses/min), compared to the ineffective stimulus (12 impulses/2 min). This system of genes seems suited to mediating the coupling between electrical activity and other functional genes.

A rapid method to quantify neurons in mixed cultures based on the specific binding of [3H]ouabain to neuronal Na+,K(+)-ATPase.

The high-affinity binding of [3H]ouabain to Na+,K(+)-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [3H]ouabain was found to be time-dependent, high-affinity (apparent Kd = 8.5 nM), saturable (Bmax = 20.6 pmol/mg protein), dependent upon the presence of ATP, inhibited by K+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins N-methyl-D-aspartate (NMDA) and kainate, reduced the amount of [3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [3H]ouabain binding to the neuronal form of Na+,K(+)-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.

Epidermal growth factor inhibits the expression of myelin basic protein in oligodendrocytes.

The major function of the oligodendrocyte is to myelinate axons in the central nervous system (CNS). Two of the components of myelin, galactocerebroside (galc) and myelin basic protein (MBP), have been used as markers of oligodendrocyte maturation in the developing CNS, and it has been found that galc+ cells arise initially, which then mature into MBP+ oligodendrocytes several days later. We have been interested in the control of expression of MBP and have followed its appearance in cultures of brain cells isolated from 4 day-old mice. In low serum (0.5% foetal bovine serum), approximately 330 MBP+ cells arise per 2 x 10(5) brain cells after 3 days incubation. We have examined the ability of several growth factors to influence the expression of MBP in these cultures, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and the fibroblast growth factors (acidic and basic FGF). EGF was found to suppress strongly the developmental expression of MBP in these cultures, but the suppression was reversible, since the number of MBP+ cells approached control numbers 3 days after removal of EGF from the cultures. It was also found that MBP could be down-regulated in mature MBP+ oligodendrocytes. The action of EGF in these cultures could be mimicked by transforming growth factor-alpha (TGF alpha). The effects of EGF appear to be associated primarily with MBP production in oligodendrocytes since expression of galc is unaffected by EGF.