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Context-dependent utilization of Notch activity in Drosophila glial determination

¹ Division of Developmental Genetics,
² National Institute of Genetics,
³ Department of Genetics, Graduate University for Advanced Studies, Mishima, 411-8540, Japan
⁴ CREST, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan

View larger version (84K): [in a new window]   Fig. 1. Developmental dynamics of glial cells in normal embryonic PNS. (A-C) Double labeling using a glial marker REPO (black) and a neuronal marker ELAV (orange) in the PNS (single abdominal segment; dorsal up, anterior to left). Three types of REPO-positive glial cells are present in an abdominal hemisegment of the embryonic PNS; one dbd support glial cell (DBDG), one PG3 cell (arrowhead) and five ligament cells of the lateral chordotonal organ (black asterisk). (A) Stage 12 embryo. (B) Stage 14 embryo. (C) Stage 16 embryo. The dbd neuron (white asterisk) can be identified at the dorsal side of the DBDG in late stage embryos. d, dorsal cluster of sensory neurons. (D) Summary of developmental dynamics of the REPO-positive glial cells (black) (single abdominal segment; dorsal up, anterior to left). PG3 undergoes extensive ventral migration between stage 12 and stage 16. The dorsoventral positions of the PG3 and DBDG are reversed between these two stages. lig., ligament cells.

View larger version (58K): [in a new window]   Fig. 2. Developmental sequence of gene expression patterns in the dbd lineage. (A,B) Double labeling of gcm mRNA (purple) and Nubbin (orange) in the embryonic PNS (single abdominal segment; dorsal up, anterior to left). The dbd lineage is bracketed. The behavior of Nubbin-positive cells located dorsal to the dbd lineage (white circle) acts as a temporal indicator of embryogenesis. The Nubbin expression in this cell is absent by stage 13. We tentatively refer to the processes of the glial fate induction as stages I-IV. (stage I) Nubbin expression is initially detected in both daughter cells at the beginning of stage 12. The smaller daughter cell (arrow), located apicodorsally to the larger daughter cell differentiates as DBDG. The presumptive dbd neuron is indicated by an asterisk. (stage II) Nubbin becomes down-regulated in the presumptive glial cell prior to the onset of the gcm expression. (stage III) The expression of gcm is initiated in the smaller daughter cell where Nubbin expression is low (arrow). (stage IV) After gcm becomes highly activated, Nubbin is re-expressed in the glial daughter cell (arrow). Single labeling of Nubbin in the dbd lineage is shown in inset for stages III and IV. The dda neuron (out of focus) is indicated by an arrowhead. (B) At stage 16, Nubbin expression is again restricted to the dbd neuron. Double labeling of REPO (black) and Nubbin (orange) in the dbd lineage at stage 16 is shown in the inset. Strong REPO expression is detected in DBDG, in which gcm expression has already disappeared (arrow). The dda neuron is located dorsal to the dbd lineage (arrowhead). In this stage, Nubbin expression is detected in the dbd neuron, dda neuron and ligament cells within the PNS. (C) Summary of the expression dynamics of gcm mRNA and Nubbin in the dbd lineage. g, glial cell; n, neuron.

View larger version (80K): [in a new window]   Fig. 3. Visualization of Notch-dependent Su(H)-reporter activity (p12xSu(H)bs-lacZ) in embryos (single abdominal segment; dorsal up, anterior to left). (A-C) The reporter lacZ expression in stage 12 embryos. (A) A normal control embryo. (B) Notch55e11 mutant embryo. (C) sanpodoC55 mutant embryo. The reporter gene activity was dramatically reduced in B and C. (D-G) The lacZ reporter expression in the dbd lineage of stage 16 embryos. Arrows in D, E and G indicate DBDG. (D) In normal embryos strong nuclear staining is observed in DBDG but not in the neuron (encircled by dots). (E) When a constitutively active form of Notch was expressed in neurons (UAS- Notchact/C155-GAL4), ectopic Su(H)-reporter activity was observed in the dbd neuron (dotted circle). (F) In the sanpodo mutation, which produces a double-neuron phenotype at the expense of the glial cell, the reporter activity is undetectable in cells of the dbd lineage cells (dotted circle). (G) Misexpression of gcm in neurons (UAS-gcm/C155-GAL4) does not activate the reporter in the dbd neuron that is transformed to a glial cell (dotted circle).

View larger version (103K): [in a new window]   Fig. 4. Loss of DBDG in Notch mutants. (A,B) Double labeling of repo mRNA (purple) and nubbin mRNA (orange) in stage 12 embryos. (A) Two nubbin-positive cells (dotted circle; one is the dbd neuron and the other is the dda neuron) are associated with a glial cell (arrow) in normal embryos. (B) In Notch55e11 mutant embryos, increased numbers of nubbin-positive neurons (encircled by dotted line) are present, but no DBDG is generated. The Notch mutant shows a neurogenic phenotype, resulting in an excess of repo-positive cells such as PG3 glial cell (asterisk) and ligament cells (bracket) in the PNS. gcm mRNA expression in a normal embryo (C) and a Notch55e11 mutant (D) at the same stage shown in A and B. gcm expression in the dbd lineage (C, arrows) is absent in the Notch embryo (D, arrow). Insets in C and D are higher magnification views of one parasegment. Dorsal is up and anterior to left.

View larger version (162K): [in a new window]   Fig. 5. Roles of the Notch signaling pathway in the dbd and dda lineages. (A,B) The dbd lineage in stage 16 embryos (single abdominal segment; dorsal up, anterior to left). The dorsal cluster of sensory neurons are stained brown (cytoplasm) with mAb 22C10. The dbd neuron is indicated by an arrowhead. DBDG (arrow) is stained black with anti-REPO (nucleus). (A) Normal embryo. (B) Ectopic REPO expression is observed in the nucleus of the dbd neuron when a constitutively active form of Notch is expressed in neurons (UAS-Notchact/C155-GAL4). Note that the morphology of the neuron is also transformed towards that of a glial cell. (C,D) Double labeling of REPO expression (black) and ELAV expression (orange) in the dorsal cluster of PNS. (C) Stage 13 normal embryo. (D) Stage 13 numb1 embryo. The dda neuron (white arrowhead) and the dbd neuron (black arrowhead) ectopically express REPO. ELAV expression is still observed in the cytoplasm of the dda neuron as well as in the dbd neuron, but not in DBDG (arrow). (E-H) Nubbin expression in the dorsal cluster of sensory neurons at stage 16. In normal embryos (E), Nubbin expression is restricted to the dbd neuron (black arrowhead) and the dda neuron (white arrowhead). Both neurons are absent in C155-GAL4/UAS-Notchact embryos (F), and in numb1 embryos (G). Both neurons are duplicated in sanpodoC55 embryos (H). (I-K) gcm mRNA expression in the dbd lineage at stage 12 (single abdominal segment. dorsal up, anterior to left). (I) gcm expression is observed in the presumptive DBDG (arrow) of normal embryos. (J) C155-GAL4/UAS-Notchact gain-of-function embryo. Ectopic gcm expression is observed in the presumptive dbd neuron (arrowhead). (K) numb1 mutant embryo. Ectopic gcm expression is observed in the presumptive dbd neuron (arrowhead). In contrast, gcm expression is absent in the presumptive PG3 and the ligament cells. (L) C155-GAL4/UAS- Notchact embryo at stage 16 (single abdominal segment; dorsal up, anterior to left). Ectopic gcm expression is maintained in the dbd neuron that is transformed into a glial cell (black arrowhead). gcm expression in endogenous DBDG has already disappeared by this stage (arrow). An extra gcm-positive cell (open arrowhead) is also observed in the dorsal cluster of sensory neurons.

View larger version (107K): [in a new window]   Fig. 6. The dda lineage displays similarities with the dbd lineage. (A) Effects of Notch activation in the PNS at stage 14. Confocal image of C155-GAL4/UAS-Notchact embryo. (Left) REPO (green), (middle) Nubbin (purple), (right) merged images. Ectopic REPO expression is observed in the dbd neuron (black arrowhead) and the dda neuron (white arrowhead). Nubbin expression is observed in DBDG (arrow) and in the ligament cells (Dick et al., 1991; Lloyd and Sakonju, 1991), but not in the PG3 (asterisk). lig., ligament cells. (B) Developmental sequence of asymmetric cell division in the dda lineage. Double labeling of gcm mRNA (purple) and Nubbin (orange) in stage 12 PNS (single abdominal segment; dorsal up, anterior to left). The dda lineage is boxed. The position of the dbd lineage (out of focus) is indicated by an asterisk. (stage I) Both daughter cells of the dda SOP express Nubbin. The smaller daughter cell (dotted circle) is located apical to the large daughter cell. (stage II) Two symmetrical daughter cells expressing Nubbin are observed. At this stage there is no detectable expression of gcm in the dda lineage. (stage III) One of the daughter cells transiently accumulates gcm mRNA (arrow). (stage IV) Two cells constituting the dda lineage migrate dorsally and are situated anterior to the dbd lineage. Nubbin expression in the presumptive glial cell becomes significantly down-regulated (arrow). Nubbin is never re-expressed in this cell, unlike the presumptive glial cell in the dbd lineage. (stage IV') repo mRNA (purple) and ELAV (orange) expression in ASC mutant at stage IV. In this genetic background the dorsal cluster contains only dda and dbd lineages (Brewster et al., 2001). Weak expression of ELAV is detected in the presumptive dda neuron (white arrowhead) and the dbd neuron (black arrowhead) at this stage. (C) REPO expression in C155-GAL4/UAS- Notchact, UAS-nub embryo at stage 16. Ectopic REPO expression is observed in the presumptive dbd neuron (black arrowhead), as well as in two cells (black asterisk) that occupy the position dorsal to the glial-transformed dda neuron (white arrowhead). White asterisk indicates PG3 cell; arrow, DBDG. PG3 of the left parasegment is out of focus.

View larger version (44K): [in a new window]   Fig. 7. Comparison of Notch-dependent and Notch-independent gliogenic lineages. Schematic representations of five gliogenic lineages in the CNS (C) and PNS (A,B,D,E). The cells in which Notch is likely to be active are shown in blue. This assignment is based on the expression of the Su(H)-reporter gene (this work), and the mutant phenotypes of Notch and sanpodo embryos. The cells that receive Numb protein upon division, or those whose fate requires numb function, are shown in red. The gcm/repo-positive cell is boxed in yellow. Nubbin expression is shown in orange. (A-C) Three independent lineages in which Notch activates glial fate (this work) (Udolph et al., 2001). In all three lineages Nubbin is expressed in the presumptive glial cell before gcm expression initiates in that cell. (D) Chordotonal organ lineage (Orgogozo et al., 2001). In the chordotonal lineage, the ligament cell neither expresses Su(H)-reporter nor requires Notch activity for its fate specification. It expresses Nubbin only after REPO expression is observed in this cell, contrasting with the situation in three lineages shown in A-C. Whether there is a requirement or localization of Numb in IIIb, the ligament cell and the neuron is not known. (E) Lineage in which Notch represses glial fate (Van De Bor and Giangrande, 2001). Whether Nubbin is expressed in this lineage is unknown. SOP, sensory organ precursor cell; IP, intermediate precursor; IIa, IIb, secondary precursor cells; IIIb, tertiary precursor cell; g, glial cell; n, neuron; C, cap cell; e, ectodermal cell; S, scolopale cell; lig, ligament cell; To, tormogen cell; Tr, trichogen cell; Th, thecogen cell.

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