Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.     

At the nexus between pattern formation and cell-type specification: the generation of individual neuroblast fates in the Drosophila embryonic central nervous system.

The specification of specific and often unique fates to individual cells as a function of their position within a developing organism is a fundamental process during the development of multicellular organisms. The development of the Drosophila embryonic central nervous system serves as an excellent model system in which to clarify the developmental mechanisms that link pattern formation to cell-type specification. The Drosophila embryonic central nervous system develops from a set of neural stem cells termed neuroblasts. Neuroblasts arise from the ectoderm in an invariant pattern, and each neuroblast acquires a unique fate based on its position within this pattern. Two groups of genes recently have been demonstrated to govern the individual fate specification of neuroblasts. One group, the segment polarity genes, enables neuroblasts that develop in different anteroposterior positions to acquire different fates. The second group, referred to as the columnar genes, ensures that neuroblasts that develop in different dorsoventral domains assume different fates. When integrated, the activities of the segment polarity and columnar genes create a Cartesian coordinate system that bestows unique fates to individual neuroblasts as a function of their position of formation within the ectoderm. BioEssays 1999;21:922-931.     

Geometry and mechanics of teleost gastrulation and the formation of primary embryonic axes

Examination of normal shaping dynamics and immediate and long-term responses to blastoderm cutting in zebrafish and loach embryos prior to the onset of gastrulation and during the course of epiboly revealed that anteroposterior (AP) and dorsoventral (DV) polarity formation is connected with shaping of the blastoderm circumferential region, which stretches along and shrinks across its movement axes and originates the non-isotropic fields of tensile stresses. Based on data from cutting experiments and quantitative morphology, we reconstructed the movement-shaping patterns of epiboly and embryonic shield formation. We revealed that AP and DV axes originate as a mass cell movement subject to the movement-shaping equivalence principle, which means the spatial series of differently shaped areas corresponding to the time succession of the same area shaping. Maintenance of the main body axes in orthogonal orientation depends on the mechanical equilibrium principle allowing for converting shape asymmetry into that of tensile stresses and vice versa. The causal relationship between the main movement-shaping axes and that of embryonic polarity was proved in cutting experiments in which the DV axis direction was subject to rearrangement so as to adjust to the new direction of mass cell movement axes induced by healing the wound in the blastoderm circumferential region.     

Models for the generation of the embryonic body axes: ontogenetic and evolutionary aspects

Coelenterates including hydra are assumed to be close to the last common ancestor before bilaterality evolved. Models based on local self-enhancement and long-range inhibition account for pattern formation and regeneration along this ancestral axis. The body of a hydra-like ancestor evolved into the brain and heart of higher organisms, accounting for the close relationship of both patterning processes. Bilateria require a long-extended organizing region to pattern their dorsoventral axis. Models reveal the difficulties in the generation of such a stripe-like organizer and account for different mechanisms realized in vertebrates and insects. Common pathways involved in hydra budding and in the formation of appendages in higher organisms suggest a possible link.     

Another notch in stem cell biology: Drosophila intestinal stem cells and the specification of cell fates

Previous work has suggested that many stem cells can be found in microanatomic niches, where adjacent somatic cells of the niche control the differentiation and proliferation states of their resident stem cells. Recently published work examining intestinal stem cells (ISCs) in the adult Drosophila midgut suggests a new paradigm where some stem cells actively control the cell fate decisions of their daughters. Here, we review recent literature((1)) demonstrating that, in the absence of a detectable stem cell niche, multipotent Drosophila ISCs modulate the Notch signaling pathway in their adjacent daughter cells in order to specify the differentiated lineages of their descendants. These observations made in Drosophila are challenging and advancing our understanding of stem cell biology.     

HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits

The ultrastructure and biochemical characteristics of HMW-2, the Sertoli cell cytoplasmic dynein isolated from rat testes, were analyzed. Electron microscopic studies revealed a two-headed two-stem structure with dimensions very similar to other dyneins. We found that, like other cytoplasmic dyneins, both heads have an approximately spherical shape with a central cavity. Heavy chain analysis suggested the presence of only one type of heavy chain, a finding that was supported by the simple Michaelis-Menten kinetics displayed by the HMW-2-associated ATPase activity. In addition, dissociation of the HMW-2 complex resulted in a single type of dynein subunit sedimenting at 11.8 S. This fraction contained all the polypeptides present in the undissociated HMW-2. Ultrastructurally the HMW-2 subunits were composed of one globular domain with a tail. The simplest interpretation is that HMW-2 is a dimer of nearly identical subunits, each containing one heavy chain, one 90-kDa intermediate chain, and two light chains.     

Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis

Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. We tested this hypothesis by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. Our data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS.     

Targeted deletion of the tybe IIb Na-dependent Pi-co-transporter, NaPi-IIb, results in early embryonic lethality

NaPi-IIb encodes a Na⁺-dependent Pi co-transporter, which is expressed in various adult tissues and mediates transport of extracellular Pi ions coupling with Na⁺ ion. To define the role of NaPi-IIbin vivo, NaPi-IIb gene deficient mice were generated utilizing targeted mutagenesis, yielding viable, heterozygous NaPi-IIb mice. In contrast, homozygous NaPi-IIb mice died in utero soon after implantation, indicating that NaPi-IIb was essential for early embryonic development. In situ hybridization revealed NaPi-IIb mRNA expression in the parietal endoderm, followed by the visceral endoderm, at a time point prior to establishment of a functioning chorio-allantoic placenta. At the time point of functional placenta development, the main site of NaPi-IIb production resided in the labyrinthine zone, where embryonic and maternal circulations were in closest contact. Expression patterns of NaPi-IIb suggest that NaPi-IIb plays an important role in Pi absorption from maternal circulation.     

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

It has been demonstrated that maternal drinking during pregnancy had serious adverse effects on the health of the newborns. Fetal alcohol syndrome (FAS) is the most important developmental abnormality caused by maternal alcohol abuse during pregnancy. Clinically, it is characterized by head and facial ab-normalities, cardiovascular malformation, and perma-nent nervous system damage[1,2]. A lot of experimental models have been developed to study the ethanol’s effects on embryonic development,…     

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

Exposure to ethanol during human embryonic period has severe teratogenic effects on the cardiovascular system. In our study, we demonstrated that ethanol of gradient concentrations can interfere with the establishment of circulatory system in embryonic zebrafish. The effective concentration to cause 50% malformations (EC₅₀) was 182.5 mmol/L. The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol. It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43% treated embryos. We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish. By employing in situ hybridization with endothelial biomarker (Flk-1), we revealed that ethanol disrupts the establishment of trunk axial vasculature, but has no effect on cranial vessels. Combined with the results of semi-thin histological sections, the in situ hybridization experiments with arterial and venous biomarkers (ephrinB2, ephB4) suggested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein. Further study indicated the negative influence of ethanol on the development of hypochord in zebrafish. The consequent lack of vasculogenic factors including Radar and Ang- 1 partly explains the defects in formation and integrity of dorsal aorta. These results provide important clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures

Mutations in alpha-synuclein, parkin and ubiquitin C-terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin-proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin-mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)-induced impairment of ubiquitin C-terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration-dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of alpha-synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in alpha-synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.     

Stepwise commitment of blast cell fates during the positional specification of the O and P cell lines in the leech embryo

The o and p bandlets of the leech embryo are parallel columns of ectodermal blast cells which are identified by their relative positions, and which during normal embryogenesis follow distinct developmental pathways. A previous study showed that o blast cells are initially capable of following either the O or P pathway, and suggested that commitment to the O pathway depends upon interaction with the adjacent p bandlet. To better understand the nature and timing of this interaction we examined the fate of o blast cells whose p blast cell neighbors had been selectively ablated by photoexcitation of a fluorescent lineage tracer. If an o blast cell has not yet begun its secondary divisions, its normal commitment to the O pathway can be effectively prevented by ablation of the adjacent p bandlet. Comparing the outcome of progressively later lesions reveals that the progeny of the o blast cell become committed to the O pathway in a series of three discrete steps, and that these steps occur around the time of the first three blast cell divisions. Each of the three events affects a different subset of elements within the blast cell clone, and apparently commits those elements to either the O or P pathway depending upon the presence or absence of the other bandlet. These changes in blast cell fate are coextensive with the lesion along the bandlet's length, suggesting that the interaction of the two bandlets is localized to neighboring cells.     

Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development

Analysis of retinal development in Delta (Dl) temperature-sensitive mutants reveals requirements for Delta function in the specification of all retinal cells, including photoreceptors, cone cells, pigment cells and cells that make up interommatidial bristles. In situ hybridization and immunohistochemistry indicate that Delta is expressed dynamically during the specification of different cell types. Comparisons of Delta expression patterns with developmental defects in Dl mutants implies that Delta functions in a cell-nonautonomous manner in the specification of photoreceptors. Delta protein resides predominantly in subcellular vesicles located primarily at the apical ends of developing retinal cells. Localization of Delta protein in Dl and shibire^tsl mutants implies that Delta is targeted to the cell surface, but is efficiently removed via endocytosis, resulting in vesicular accumulation.     

Early events of multiple bud formation and shoot development in soybean embryonic axes treated with the cytokinin, 6-benzylaminopurine

Early events of multiple bud formation and shoot development in germinating soybean embryonic axes treated for 24 hr with the cytokinin, 6-benzylaminopurine (BAP), were compared to the development of untreated control axes using four different techniques: photomicrography, scanning electron microscopy, histology, and autoradiography. Shoot apex development in BAP-treated embryonic axes was delayed by about 9 to 15 hr. A transient inhibition of DNA synthesis in the primary apical meristem and axillary buds was observed with subsequent changes in the timing of cell division patterns in these regions. Meristematic regions (supernumerary vegetative buds) were observed in BAP-treated axes around the perimeter of the apical dome at and above the level of the axillary buds. Cells elongated from some of the BAP-induced meristematic regions to form four to six shoots. In the absence of BAP, excision of the primary apical meristem and/or axillary buds did not result in multiple bud formation. These results suggest that transient exposure to BAP interrupted chromosomal DNA replication and reprogrammed the developmental fate of a large number of cells in the shoot apex. We postulate that interruption of DNA synthesis, either directly, by interfering with DNA replication, or indirectly, by preventing entry into S-phase, effected redetermination of the shoot apex cells.