QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Freeman, M. R. Articles by Doe, C. Q. Search for Related Content PubMed PubMed Citation Articles by Freeman, M. R. Articles by Doe, C. Q. Social Bookmarking                         What's this?

Asymmetric Prospero localization is required to generate mixed neuronal/glial lineages in the Drosophila CNS

HHMI, Institute of Neuroscience, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA

View larger version (54K): [in a new window]   Fig. 1. Gcm protein distribution, spindle orientation and cell migration in neural precursor lineages. (A) NGB 6-4T divides along the apical-basal axis and expresses Gcm before its first division. In the left column are schematic representations; in the right column are paired confocal images (each pair from the same focal plane) corresponding to the diagrammed cells. Ventral view. Anterior is upwards; midline is towards the left. Gcm is expressed in the pre-divisional NGB 6-4T (6-4T). NGB 6-4T divides along the apical-basal axis (n>60 mitotic NGB 6-4T). Both the daughter cell (G) and the post-divisional NGB 6-4T are Gcm positive, and Gcm is localized to the nucleus (a different focal plane is shown for each of these cells). The G cell (arrow) next undergoes a migration to a position just medial to NGB 6-4T (arrowhead; note that NGB 6-4T and G are in the same focal plane at this stage). By the end of this migration, Gcm protein is no longer detectable in NGB 6-4T. The G cell divides twice to generate three glia (one medial cell body glia, m-CBG; and two medial-most cell body glia, mm-CBG), which migrate toward the midline. Subsequent divisions of NGB 6-4T (dotted circle in bottom confocal image) are along the apical-basal axis, and generate neuron-producing daughter cells (n). (B) GB 6-4A divides along the apical-basal axis and expresses Gcm before its first division (oriented as in A). Gcm is expressed in the predivisional GB 6-4A (6-4A). GB 6-4A divides along the apical-basal axis (a different focal plane is shown for each cell), and both daughter cells are Gcm-positive. The G cell next rapidly migrates to a position medial to GB 6-4A (note that these cells are in the same focal plane at this stage); both G and GB 6-4A maintain Gcm expression and differentiate as glia. The gray circle in the diagram and the dotted circle in the confocal image approximate the position of the predivisional GB 6-4A. (C) Glial progeny of NGB 7-4. The position of NGB 7-4 (which is just out of the focal plane) is indicated by the dotted circle in the confocal image. Ventral glia: at the ventral surface of the CNS, two glial progeny of NGB 7-4 migrate to the midline and become channel glia (CG, arrowheads); three glia remain close to NGB 7-4 and become cell body glia (CBG, arrows). Dorsal glia: one to two glia migrate laterally and dorsally and become lateral subperineurial glia (l-SPG, large arrows).

View larger version (49K): [in a new window]   Fig. 2. gcm mRNA is not asymmetrically localized during NGB 6-4T cell division. Ventral view gcm mRNA localization in the NGB 6-4T lineage; oriented as in Fig. 1. NGB 6-4T (6-4T) and the G daughter cell are circled (yellow dots); the adjacent NB 6-2 (6-2), NB 7-2 (7-2), NGB 7-4 (7-4) and the lateral glial precursor (GP) are labeled when visible and were used as landmarks (see Materials and Methods). (A) gcm mRNA is expressed in the predivisional NGB 6-4T (6-4T). (B) Anti-phosphohistone H3 (purple) is a marker for mitotic DNA. gcm mRNA (green) is equally distributed throughout the cell in the mitotic NGB 6-4T. (C) gcm mRNA is present in both the apical NGB 6-4T and its basally positioned G daughter cell after mitosis. (D) After the medial migration of the G daughter cell, gcm mRNA is detected in the G daughter cell but not in the post-divisional NGB 6-4T.

View larger version (49K): [in a new window]   Fig. 3. Asymmetric protein localization along the apical-basal axis in NGB 6-4T and GB 6-4A. (A) Lateral view of stage 10 embryos; apical is upwards; basal is downwards; anterior is towards the left. NGB 6-4T (6-4T) and GB 6-4A (6-4A) were identified as described in Fig. 1 and the Materials and Methods. DNA was labeled with anti-phosphohistone H3. Inscuteable (Insc) forms an apical crescent in mitotic NGB 6-4T (n=7) and GB 6-4A (n=8) and is partitioned to the apical precursor after cytokinesis. Miranda (Mir), Numb and Prospero (Pros) form basal crescents in mitotic NGB 6-4T and GB 6-4A, and are partitioned into the basal G daughter cells in both lineages after cell division (n>13 for Mir and Pros; n>6 for Numb). The arrow in the 6-4T Mir panel shows and example of a mitotic NGB 7-4 during a glia-producing division; we find these divisions to also occur along the apical-basal axis. (B) Summary of Prospero protein (Pros), gcm mRNA and Gcm protein localization in the NGB 6-4T lineage. See text for details.

View larger version (82K): [in a new window]   Fig. 4. prospero and miranda, but not numb or staufen, are required for glial development in the NGB 6-4T lineage. Ventral view of a stage 13 embryos showing Repo-positive glia derived from the NGB 6-4T lineage. Three thoracic segments are shown in all panels; anterior is upwards; n20 for all. (A) Wild-type glial development in the NGB 6-4T lineage. Left: a summary showing the positions of glia (red dots) derived from NGB 6-4T (purple circle) with migration patterns (lines). Right: wild-type embryo stained for Repo, brackets indicate NGB6-4T-derived glial progeny. (B,C) numb and staufen mutants show a wild-type pattern of NGB 6-4T-derived glia. (D) prospero mutants show a loss of NGB 6-4T-derived glia. Repo-positive cells in the region of 6-4T glia in prospero mutants are not derived from NGB 6-4T, based on their lack of expression of the NGB 6-4T marker eagle (data not shown, see also Fig. 5). (E) miranda mutants have a variable phenotype showing reduced, normal or extra NGB 6-4T-derived glia (see text for details).

View larger version (63K): [in a new window]   Fig. 5. Prospero is required to upregulate gcm expression in NGB lineages. (A) Prospero is required for Gcm expression in NGB 6-4T glial progeny. Paired confocal images are from the same focal plane. Ventral view; midline is towards the left; anterior is upwards. Expression of Gcm in the Engrailed-negative lateral glial precursor is seen at the bottom right of most images. (i) prospero mutants show normal levels Gcm in the predivisional NGB 6-4T (arrowhead). (ii) By the time the G daughter cell migrates medially, both the G cell (arrow) and post-divisional NGB 6-4T (arrowhead) have down-regulated Gcm. (iii) Later in development all G cell progeny have severely reduced Gcm levels and they fail to migrate to the midline (arrows). Compare with Fig. 1A. The graph shows a quantitation of the percentage of Gcm-positive cells in the NGB 6-4T lineage in wild type (n=61) and prospero mutants (n=90): wild type Gcm levels, black bar; strongly reduced Gcm levels, gray bar; and lack of detectable Gcm, no bar. G, G alone; G1+G2, the first two G-derived cells; G1+G2+G3, all three G-derived cells. (B) Prospero is required for Gcm expression in the NGB 7-4 lineage (oriented as in A). In the wild type, there are 4-5 Gcm-positive progeny (dotted circle) derived from NGB 7-4 (arrowhead). In prospero mutants, Gcm expression is severely reduced (dotted circle). The graph shows a quantitation of the percentage of Gcm-positive cells in the NGB 7-4 lineage in wild type (n=45) and prospero mutant (n=76) hemisegments. Bars indicate Gcm levels as described in A.

View larger version (61K): [in a new window]   Fig. 6. Miranda is required to prevent gcm upregulation in NGB 6-4T and NGB 7-4 lineages. (A) Wild type and miranda mutant embryos stained for Gcm protein. Anterior is upwards; midline is towards the left. Stage 12 embryos are shown for NGB 6-4T, late stage 11 embryos are shown for NGB 7-4. In wild-type embryos (top row), Gcm is not detected in NGB 6-4T or NGB 7-4 (dotted circles). In miranda mutant embryos (bottom row), Gcm protein is present in NGB 6-4T and NGB 7-4 (dotted circles; 60% of hemisegments scored for both; n=91 hemisegments). (B) miranda mutant embryos can make extra glia from NGB 6-4T. Mutant embryos were stained with antibodies to Repo (to identify glia; red), Eg (to visualize the entire NGB 6-4T lineage, blue) and En (as a second marker for NGB 6-4T, green). Arrowheads, NGB 6-4T; arrows, NGB 6-4T-derived glial progeny; n=38 hemisegments. One segment is shown, the midline is indicated by the vertical bar. To the left of the midline, the NGB 6-4T shows high levels of Repo expression and no neuronal progeny were produced by this NGB. By contrast, to the right of the midline the NGB 6-4T is not expressing Repo and several neuronal progeny have been produced (out of plane of focus).

View larger version (12K): [in a new window]   Fig. 7. Requirements for prospero in separating glial and neuronal cell fates. In wild type, low level gcm mRNA and protein (gray) are evenly segregated to the NGB and the daughter cell. The daughter cell inherits all Prospero, which upregulates gcm expression and induces glial fate (black). In prospero mutants, low level gcm expression (gray) is induced normally in the NGB but fades rapidly and glia are not produced. In miranda mutants, Prospero is evenly segregated into the NGB and the daughter cell resulting in either (1) upregulation of gcm in the NGB, its transformation into a GB or glial cell, and the truncation of the lineage (black); or (2) insufficient Prospero is present in the NGB, low level gcm fades in the NGB, and neuronal progeny are subsequently produced (NB, white).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter