Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages

Cell lineages that give rise to the PNS were studied using the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to visualized DNA replication immunocytochemically. The precursors of the PNS in the body segments of Drosophila embryos replicate their DNA in a spatially and temporally stereotyped pattern. The sequence of DNA replication within developing sensory organs suggests particular lineage relationships of the cells that constitute a sensory organ, i.e., neuron and associated support cells. In embryos that are mutant for the achaete-scute complex or daughterless, in which most or all of the PNS is missing, no BrdU-labeled cells were found in the appropriate regions, suggesting that these PNS precursors either do not form or fail to replicate. Thus, the BrdU technique allows determination as to whether a mutation affects the PNS precursors or terminal differentiation.     

Sulfurtransferases 1 and 2 play essential roles in embryo and seed development in Arabidopsis thaliana

Sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from a donor to a suitable acceptor molecule. The Arabidopsis thaliana genome encodes 20 putative STR proteins. The biological functions of most are unclear. We found that STR1 and STR2 play important roles in embryo/seed development. Mutation of STR1 alone resulted in a shrunken seed phenotype, although growth and development of vegetative and reproductive organs were not affected. The shrunken seed phenotype was associated with the delayed/arrested embryo development, in most cases, at the heart stage. The embryo defect of str1 mutant is not fully penetrant. Approximately 12.5% of embryos developed further and formed normal looking seeds. In severely shrunken seeds, no embryo could be identified after seed collection. Partially shrunken seeds that contained viable embryos could still germinate. However, cotyledons of the seedlings from such seeds were abnormal. An STR1-GUS fusion reporter revealed that the STR1 gene was universally expressed, with high levels of expression in specific tissues/organs including embryos. The incomplete penetrance of str1 embryo/seed phenotype is a result of functional STR2. Single str2 mutant had no phenotype. However, no str1(-/-)/str2(-/-) double mutant embryos were able to develop past the heart stage. Furthermore, STR2 is haplo-insufficient in str1 mutant background, and str1(-/-)/str2(+/-) embryos were 100% lethal. These data provide new insights into the biological functions of the ubiquitous sulfurtransferase in Arabidopsis embryogenesis and seed development.     

Slit-Robo signalling prevents sensory cells from crossing the midline in Drosophila

Maintenance of bilateral symmetry throughout animal development requires that both left and right halves of the body follow nearly identical patterns of cell proliferation, differentiation, death and migration. During formation of the perfectly bilateral Drosophila larval peripheral nervous system (PNS), the sensory precursor cells of the ventral multidendritic neuron vmd1a originating from each hemisegment migrate away from the ventral midline. Our observations indicate that in slit mutant embryos, as well as in robo, robo2 double mutants, sensory precursor cells of the left and right vmd1a neurons aberrantly cluster at the midline and then the pair of vmd1a neurons migrate to their final position on the same side of the embryo. This results in disruption of PNS bilateral symmetry. Expression of slit at the midline rescues the slit mutant vmd1a phenotype, suggesting that midline-secreted Slit activates Robo/Robo2 signalling to control the migration of the vmd1a sensory precursor cells. Our study indicates that midline-secreted Slit prevents vmd1a sensory cells from crossing the midline and thereby maintains PNS bilateral symmetry during development.     

The paired box gene pox neuro: a determinant of poly-innervated sense organs in Drosophila.

This study describes the structure and function of pox neuro (poxn), a gene previously isolated by virtue of a conserved domain, the paired box, which it shares with the segmentation genes paired and gooseberry. Its expression pattern has been analyzed, particularly during development of the PNS. We propose that poxn is a "neuroblast identity" gene acting in both the PNS and the CNS on the basis of the following evidence. Its expression is restricted to four neuronal precursors in each hemisegment: two neuronal stem cells (neuroblasts) in the CNS, and two sensory mother cells (SMCs) in the PNS. The SMCs that express poxn produce the poly-innervated external sense organs of the larva. In poxn- embryos, poly-innervated sense organs are transformed into mono-innervated. Conversely, ectopic expression of poxn in embryos transformed with a heat-inducible poxn gene can switch mono-innervated to poly-innervated sense organs. Expression of poxn in the wing disc is restricted to the SMCs of the poly-innervated sense organs, suggesting that poxn also determines the lineage of poly-innervated adult sense organs.     

Pathfinding by sensory axons in Drosophila: substrates and choice points in early lch5 axon outgrowth

We have examined the pattern of axon growth from the lateral chordotonal (lch5) neurons in the body wall of the Drosophila embryo and identified cellular substrates and choice points involved in early axon pathfinding by these sensory neurons. At the first choice point (TP1), the lch5 growth cones contact the most distal cells of the spiracular branch (SB) of the trachea. The SB provides a substrate along which the axons extend internally to the level of the intersegmental nerve (ISN). In the absence of the SB, the lch5 axons often stall near TP1 or follow aberrant routes towards the CNS. At the second choice point (TP2), the lch5 growth cones make their first contact with other axons and turn ventrally toward the CNS, fasciculating specifically with the motor axons of the ISN.     

The Arabidopsis embryo mutant schlepperless has a defect in the chaperonin-60alpha gene

We identified a T-DNA-generated mutation in the chaperonin-60alpha gene of Arabidopsis that produces a defect in embryo development. The mutation, termed schlepperless (slp), causes retardation of embryo development before the heart stage, even though embryo morphology remains normal. Beyond the heart stage, the slp mutation results in defective embryos with highly reduced cotyledons. slp embryos exhibit a normal apical-basal pattern and radial tissue organization, but they are morphologically retarded. Even though slp embryos are competent to transcribe two late-maturation gene markers, this competence is acquired more slowly as compared with wild-type embryos. slp embryos also exhibit a defect in plastid development-they remain white during maturation in planta and in culture. Hence, the overall developmental phenotype of the slp mutant reflects a lesion in the chloroplast that affects embryo development. The slp phenotype highlights the importance of the chaperonin-60alpha protein for chloroplast development and subsequently for the proper development of the plant embryo and seedling.     

Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm

Mutant alleles at the maternal effect locus dorsal cause a dorsalization of the Drosophila embryo. In extreme mutants, the embryos develop exclusively structures which derive from the dorsal-most region in normal eggs, in less strong phenotypes in addition to dorsal structures, structures normally derived from a dorso-lateral to lateral egg region are formed. Injection of cytoplasm from wild-type embryos into mutant embryos partially restores the dorso-ventral pattern in that injected embryos develop additional structures never formed in uninjected control embryos or embryos injected with mutant cytoplasm. The phenotype of injected embryos resembles that of weaker alleles at the dorsal locus indicating that the wild-type cytoplasm partially rescues the mutant phenotype. The response of the mutant embryos is restricted to the site of injection and occurs only when cytoplasm is injected into the ventral and not into the dorsal side of mutant embryos. The rescuing activity appears to be equally distributed in cleavage stage wild-type embryos, whereas, in syncytial blastoderm embryos, cytoplasm from the ventral side is about twice as effective as that taken from the dorsal side.     

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis

In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next‐generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen‐activated protein kinase 6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single‐mutant plants showed a wrinkled seed coat or a burst‐out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent‐cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin‐like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst‐out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild‐type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal‐mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5–MPK6 cascade is an important player in the maternal control of embryogenesis.     

Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis

To study the roles of intracellular factors in neuronal morphogenesis, we used the mosaic analysis with a repressible cell marker (MARCM) technique to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. We found that individual neurons in the peripheral nervous system (PNS) developed clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster developed a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis revealed that Flamingo did not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limited the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype was partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Our molecular analysis also indicated that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons.     

The role of homeotic genes in determining the segmental pattern of chordotonal organs in Drosophila

The homeotic genes are instrumental in establishing segment-specific characteristics. In Drosophila embryos there is ample evidence that the homeotic genes are involved in establishing the differences in the pattern of sense organs between segments. The chordotonal organs are compound sense organs made up of several stretch receptive sensilla. A set of serially homologous chordotonal organs, lch3 in the 1st thoracic segment, dch3 in the 2nd and 3rd thoracic segments and lch5 in abdominal segments 1 to 7, is composed of different numbers of sensilla with different positions and orientations. Here we examine this set of sense organs and a companion set, vchA/B and veh1, in the wild type and mutants for Sexcombs reduced, Antennapedia, Ultrabithorax, and abdominal-A, using immunostaining. Mutant phenotypes indicate that Ultrabithorax and abdominal-A in particular influence the formation of these sense organs. Differential expression of abdominal-A and Ultrabithorax within compartments of individual parasegments can precisely modulate the types of sense organs that will arise from a segment.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

原位杂交检测pal-1 mRNA在秀丽小杆线虫野生型和突变体早期胚胎中的分布

pal-1是秀丽小杆线虫(Caenorhabditis elegans)早期胚胎发育中决定体细胞命运的重要基因,也是转录因子,调控后续基因的表达,凡含有该基因表达的细胞发育成体细胞。本文通过整体原位杂交技术检测pal-1mRNA在C·elegans野生型和par-1、par-2、par-3、par-4突变体、spn-4突变体、mex-5/mex-6突变体早期胚胎中的分布,探讨这些基因在胚胎发育早期对pal-1mRNA的影响。实验结果表明:par-1、par-3、par-4突变使4细胞胚胎pal-1mRNA完全丧失了野生型不对称分布模式,pal-1mRNA分布在所有卵裂球中;par-2对pal-1mRNA的分布影响较小,在par-2突变体4细胞胚胎中pal-1mRNA分布与野生型相同。spn-4、mex-5、mex-6也能影响pal-1mRNA的分布,使其分布丧失不对称性。在par-1、par-4突变的情况下,pal-1mRNA广泛存在,但PAL-1蛋白也不表达,显示对pal-1mRNA的翻译调控是PAL-1蛋白空间和时序不对称分布的主要原因[动物学报51(5):884-891,2005]。     

Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system

Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb(-/-) embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb(-/-) embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb(-/-) cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo.     

Normal fate and altered function of the cardiac neural crest cell lineage in retinoic acid receptor mutant embryos

Mouse embryos lacking the retinoic acid (RA) receptors RARalpha1 and RARbeta suffer from a failure to properly septate (divide) the early outflow tract of the heart into distinct aortic and pulmonary channels, a phenotype termed persistent truncus arteriosus. This phenotype is associated with a failure in the development of the cardiac neural crest cell lineage, which normally forms the aorticopulmonary septum. In this study, we examined the fate of the neural crest lineage in RARalpha1/RARbeta mutant embryos by crossing with the Wnt1-cre and conditional R26R alleles, which together constitute a genetic lineage marker for the neural crest. We find that the number, migration, and terminal fate of the cardiac neural crest is normal in mutant embryos; however, the specific function of these cells in forming the aorticopulmonary septum is impaired. We furthermore show that the neural crest cells themselves do not utilize retinoid receptors and do not respond to RA during this process, but rather that the phenotype is cell non-autonomous for the neural crest cell lineage. This suggests that an alternative tissue in the vicinity of the outflow tract of the heart responds directly to RA, and thereby induces or permits the neural crest cell lineage to initiate aorticopulmonary septation.