41 research outputs found
Wingless activity in the precursor cells specifies neuronal migratory behavior in the Drosophila nerve cord.
Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection
Neuralized mediates asymmetric division of neural precursors by two distinct and sequential events: Promoting asymmetric localization of Numb and enhancing activation of Notch-signaling
AbstractIn the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1→RP2/sib lineage, we found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. We further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. Our results tie Numb and Notch-signaling through a single player, Neur, thus giving us a more complete picture of the events surrounding asymmetric division of precursor cells. We also show that Neur and Numb are interdependent for their asymmetric-localizations
Incorporation of plasma proteins during oocyte growth and its hormonal control in winter flounder, Pseudopleuronectes americanus (Walbaum)
Direct evidence is provided for ovarian incorporation of plasma proteins other than vitellogenin in winter flounder Pseudopleuronectes americanus. Two major polypeptides of molecular weights 70 K and 28 K from Peak A protein(s) of the plasma were demonstrated to be incorporated into ovarian proteins. Both the polypeptides were structurally different from vitellogenin and were found to exist in the oocyte as polypeptide fragments of molecular weights 28 K, 70 K and 76 K. Vitellogenin was shown to occur as 96 K and 86 K fragments in the oocyte. The ovarian polypeptides of molecular weight 96 K fragment from vitellogenin, and 28 K fragment from Peak A protein(s) appear to be completed together and are classified here as lipovitellin 1 and lipo-vitellin II. This is similar to the molecular organization of yolk in Xenopus laevis [Berridge, M.V., and Lane, C.D. (1976). Cell, 8, 283-297]. In addition, the presence of the 70 K and 28 K polypeptides of Peak A protein(s) in the testicular proteins has been established. -- Carbohydrate-poor pituitary proteins stimulated the ovarian incorporation of vitellogenin and the non-vitellogenin plasma proteins, Peak A protein(s) and Peak E protein(s). A biologically active peptide (Rf 0.72 protein) was obtained from the carbohydrate-poor proteins; this has a molecular weight of 14.3 K by polyacrylamide gel electrophoresis containing sodium dodecyl sulphate and elutes in the region of 28 K on Ultrogel AcA 44 together with prolactin and growth hormone. A regulatory role of this gonadotropin in the ovarian uptake of Peak A protein(s) was established. These findings were in agreement with the earlier studies on the duality of gonadotropins in many species of teleosts, reported from this laboratory (see Idler and Ng, 1983)
Cloning and analysis of mouse chromosomal loci specifically active in embryonal carcinoma stem cells
Chromosomal loci that are specifically active in the mouse embryonal carcinoma stem cells were cloned by using a functional selection procedure. The pluripotent P19 embryonal carcinoma cells were transfected with an enhancer-trap plasmid containing an enhancerless, inactive neomycin resistance gene and NEO⁺-transformant cell lines were isolated. When the cells were induced to differentiate, most of the cell lines continued to express the neomycin resistance gene, however, in some cell lines, the neomycin resistance gene because repressed. From the later group of cell lines, eight in total, the integrated transgene plus the flanking cellular DNA sequences were cloned. Three of the cloned fragments from the above eight cell lines possessed a high NEO⁺-transforming enhancer activity in the undifferentiated P19 cells. Among these three, two were inactive in differentiated P19 cells in NIH 3T3 cells, while the remaining one was active in both these differentiated cell types. Further analysis of these stem cell specific enhancers revealed that they were derived from the stem-cell specific Early Transposon-like genes. -- In order to search for the presence of genes in the above stem cell specific loci, a P19 genomic library was constructed and the preinsertion regions at the neomycin resistance gene-integration sites were cloned from these cell lines. The cloned DNA was analyzed for the presence of genes by Northern blotting analysis. Messages were detected in the Northern blots against some of the loci, however, their identity as functional genes is yet to be established. -- During the course of this investigation, I observed the presence of Early Transposon-like genes in three of the above loci. Restriction mapping of the preinsertion loci and the Southern blot analysis of the DNA from mouse testis, parent P19 cells, and the three NEO⁺ cell lines with the locus-specific probes, provided direct evidence that the transposon was inserted into these loci during the experimental time-frame and therefore was movable in the mouse genome. Analysis of the cell extracts from the three embryonal carcinoma cell lines, P19, F9, and PCC3 with a transposon-specific probe detected extrachromosomal copies of this transposon only in the P19 cells. Southern blot analysis of the DNA from mouse germ cell and various somatic cell lineages with the ends-specific transposon probes indicated that there were no apparent differences in the transposon integration sites between the germ line and the soma, suggesting that transposition of these ETn-like genes is strictly stem cell specific and ceases to occur before allocation of founder cells to the germ cell lineage and somatic lineages during mouse embryogenesis. These results demonstrate that the early transposon-like genes can act as a powerful insertion mutagen in the founder cells of the mouse embryo
Post-guidance signaling by extracellular matrix-associated Slit/Slit-N maintains fasciculation and position of axon tracts in the nerve cord.
Axon-guidance by Slit-Roundabout (Robo) signaling at the midline initially guides growth cones to synaptic targets and positions longitudinal axon tracts in discrete bundles on either side of the midline. Following the formation of commissural tracts, Slit is found also in tracts of the commissures and longitudinal connectives, the purpose of which is not clear. The Slit protein is processed into a larger N-terminal peptide and a smaller C-terminal peptide. Here, I show that Slit and Slit-N in tracts interact with Robo to maintain the fasciculation, the inter-tract spacing between tracts and their position relative to the midline. Thus, in the absence of Slit in post-guidance tracts, tracts de-fasciculate, merge with one another and shift their position towards the midline. The Slit protein is proposed to function as a gradient. However, I show that Slit and Slit-N are not freely present in the extracellular milieu but associated with the extracellular matrix (ECM) and both interact with Robo1. Slit-C is tightly associated with the ECM requiring collagenase treatment to release it, and it does not interact with Robo1. These results define a role for Slit and Slit-N in tracts for the maintenance and fasciculation of tracts, thus the maintenance of the hardwiring of the CNS
Slit-Roundabout Signaling Neutralizes Netrin-Frazzled-Mediated Attractant Cue to Specify the Lateral Positioning of Longitudinal Axon Pathways
An extending axon growth cone is subjected to attractant and repellent cues. It is not clear how these growth cones discriminate the two opposing forces and select their projection paths. Here, we report that in the Drosophila nerve cord the growth cones of longitudinal tracts are subjected to attraction by the Netrin-Frazzled pathway. However, the midline Slit neutralizes this pathway in a Robo-dependent manner and prevents Netrin-Frazzled-mediated attraction of longitudinal tracts. Our results suggest that the loss of a neutralizing effect on the Netrin-mediated attraction is responsible for the longitudinal tracts entering the midline in slit mutants as opposed to a loss of repulsion as is currently believed. This effect is not via a direct inhibition of Frazzled by Robo; instead, it is at a level downstream of Frazzled. Thus, the growth cones of longitudinal tracts subjected to two opposing forces are able to block one with the other and specify their correct lateral positioning along the midline
Distance between longitudinal tracts and connectives in the anterior and posterior ends in wild-type, <i>ptc</i>, and <i>comm</i> mutant embryos.
<p>Distance between longitudinal tracts and connectives in the anterior and posterior ends in wild-type, <i>ptc</i>, and <i>comm</i> mutant embryos.</p
Full-length Slit and Slit-N are loosely associated with the ECM, whereas Slit-C is tightly bound to the ECM.
<p>Extracts were derived from ~16 hours old embryos and were prepared under identical conditions unless otherwise noted. Tubulin was used as a loading control. (A): Full-length Slit is detected in the media only with >4 mechanical strokes of embryonic cells in a Dounce homogenizer. Slit-C is not detected in these samples either in the cells-pellet extracts or in the media. (B, C): The Slit-N peptide is readily detected in total embryo extracts with anti-Slit-N (B), whereas the Slit-C fragment is rarely detected in total embryo extracts with anti-Slit-C (C). Both antibodies readily detect full-length Slit. (D): Slit-C is readily detected in the supernatant of embryonic cells treated with collagenase but is not detected without the collagenase treatment. (E): Slit-C is also present in axon tracts as indicated by its presence in the collagenase-treated supernatant in <i>ptc</i> mutant embryos.</p
The expression of Slit is progressively lost from the midline and tracts in an anterior-posterior direction in <i>ptc</i> mutant embryos.
<p>(A, B):Wild-type embryos were examined for <i>slit</i> transcription by RNA whole mount in situ (alkaline phosphatase, AP) and Slit protein (anti-Slit-C) by DAB-histochemistry. The image analysis was done using the ImageJ software and shown as expression profile plot. The yellow-rectangle in the inset photomicrograph marks the area of ImageJ analysis. The <i>slit</i> transcription is restricted to the midline glia, and the Slit protein is present in midline glia as well as in axon tracts of commissures and longitudinal connectives. Scale bar: Panel A, 10 μm; panel B, 8 μm. (C, D): Wild-type embryos were stained with anti-Slit-C and the signals were detected using a fluorescently labeled secondary antibody and confocal microscopy (C, single section-plane), or stained with anti-Slit-N and detected with DAB-histochemistry (D). Note the presence of Slit in commissures and connectives. AC, anterior commissure; PC, posterior commissure; LC, longitudinal connectives. Scale bar: 8 μm. (E): The expression of <i>slit</i> RNA in 10 hpf wild-type and <i>ptc</i> mutant embryos. No loss of <i>slit</i> expression is detected at this age. Scale bar: 8 μm. (F): The expression of <i>slit</i> mRNA and protein in 14 hpf wild-type and 14.5 hpf old <i>ptc</i> mutant embryos. The expression (both the mRNA and the protein) is progressively lost from the midline as well as from the tracts in an anterior-posterior direction (compare panel F to panel B). Scale bar: 8 μm. (G): In a minority of <i>ptc</i> embryos, loss of Slit expression was less organized, although the posterior region was more affected. In such embryos as well, the narrowing of tracts phenotype correlated with the loss of abundance of Slit in tracts. Scale bar: 8 μm.</p
Slit-Robo signaling during early and late embryogenesis and within axon tracts.
<p>(A): The Slit-Robo signaling during early neurogenesis specifies the initial position of longitudinal tracts. Robo1 is present in growth cones that give rise to the M-tract, Robo1, and Robo3 in growth cones of the I-tract, and Robo1, Robo2, and Robo3 in growth cones of the L-tract. These growth cones project towards the midline where they encounter Slit. The combined strength of Robo-Slit interaction specifies the lateral position of each tract: the L-tract growth cones, because of the highest amounts of Robo proteins and, the strongest Robo-Slit effect, occupy the lateral-most position. The M-tract growth cones, because of the lowest levels of Robo (only Robo1), occupy a position closest to the midline. The I-tract growth cones, with Robo greater than the M but less than the L, occupy a middle position. (B): The Slit-Robo signaling maintains axon tracts in their position from the midline and between tracts after their guidance. The secreted Slit from the midline is transported along the commissural tracts to the longitudinal connectives (which are composed of both the longitudinal tracts and commissural tracts). Slit then gets distributed along or adjacent to the M, I, and L-tracts. The Slit and Slit-N are bound loosely to the ECM of commissural tracts and interacts with Robo in the adjacent longitudinal tracts. This Robo-Slit interaction in tracts maintains the fasciculation of individual axons within tracts and the position of tracts between each other and from the midline. By maintaining inter-tract position within the connectives, Slit-Robo also maintains their position away from the midline as tracts are unable to move from their position. These results suggest that the tracts have a default position and that default position is at the midline.</p