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First published online February 18, 2004
doi: 10.1242/10.1242/dev.01014

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Upregulation of Mitimere and Nubbin acts through Cyclin E to confer self-renewing asymmetric division potential to neural precursor cells

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA

View larger version (69K): [in a new window]   Fig. 1. The GMC-1 division pattern in mitiP embryos. (A-F) Embryos stained for Eve; (G,H) embryos stained for Eve (red) and Zfh1 (green; yellow indicates co-localization); (I,J) embryos stained for Eve (red) and 22C10 (green). Embryos in A-H were analyzed by light microscopy, those in I and J were analyzed by confocal microscopy. Anterior end is up, vertical lines indicate the midline. Arrow indicates an RP2, small arrow indicates a sib. (A,B) Wild-type embryos, note the smaller sib (with fading Eve expression) and the larger RP2 (A); by 13 hours of age, the sib loses Eve expression and is no longer visible (B). (C-F) mitiP embryos. Multi-cell clusters are seen in the younger stage embryo (C, arrowheads), whereas in older stage mitiP embryos (D-F) two to three RP2s were observed per hemisegment. (E) Three RP2s and two sibs can be seen (sibs are barely visible). (G) Wild-type embryo stained for Eve and Zfh1. Note that the RP2 expresses Zfh1 at high levels. (H) mitiP embryo, all three RP2s co-express Eve and Zfh1. (I,J) Wild-type (I) and mitiP (J) embryos double stained for Eve and 22C10. The two Eve-positive RP2s also express 22C10 and project normally.

View larger version (89K): [in a new window]   Fig. 2. The GMC-1 adopts a reiterating division pattern in embryos overexpressing miti or nub. Embryos were stained for Eve (red) and Zfh1 (green). Only merged images are shown, yellow indicates co-expression. The line drawings (C,E,G,I) are interpretations of the data from panels A,B,D,F,H. The boxed area in each line drawing indicates cells observed in the corresponding panels. (A,B) Wild-type embryos. The GMC-1 has terminally divided to generate a sib (Eve positive, Zfh1 negative) and an RP2 (Eve and Zfh1 positive) with no additional cells formed. (D,F) miti transgenic embryos in which the gene was induced at 6.75 hours of development for 30 minutes. At the 7.5-hour stage, the GMC-1 appears to have self-renewed (larger of the two cells) and generated an RP2 (D) or sib (F). By the 8.5-hour stage, the GMC-1 is dividing to generate a sib, an RP2 has already been generated (H). (J) An 10.5-hour stage mitiP embryo in which the transgene was induced at 6.75 hours of development for 90 minutes. A multi-cell cluster of several RP2s, one sib and a GMC-1 can be seen.

View larger version (86K): [in a new window]   Fig. 3. Self-renewing asymmetric division of GMC-1 in nub gain-of-function embryos. (A-D) Embryos stained for Eve; (E-J) embryos double stained for Eve and Spectrin. Arrowhead indicates a GMC-1, arrow indicates an RP2, small arrow indicates a sib. Thin, long arrows (E,G,J) denote indentations indicative of the cell undergoing cytokinesis. (A) 7.75-hour-old wild-type embryo. The GMC-1 has divided to generate a sib and an RP2. (B) 9-hour-old wild-type embryo. Note the fading Eve expression in sib. (C) 7.75-hour-old UAS-nub; ftz-GAL4 embryo. The GMC-1 has divided to generate two cells. (D) 9-hour-old UAS-nub; ftz-GAL4 embryo with a 3-cell phenotype: two sibs and an RP2. (E,F) Wild-type embryos. Both nuclear division and cytokinesis of GMC-1 is asymmetric (E; 7.5-hours old) and this gives rise to the smaller sib and the larger RP2 (F; 8-hours old). (G) 7.5-hour-old UAS-nub; ftz-GAL4 embryo. The GMC-1 is undergoing a nearly equal nuclear division and cytokinesis to self-renew and to generate an RP2. (H) 8.5-hourold UAS-nub; ftz-GAL4 embryo. The GMC-1 is in the process of dividing to generate a smaller sib; an Eve-positive RP2 has already been generated in this hemisegment (the top cell). (I,J) Two different focal planes of the same area of an 8.5-hour-old UAS-nub; ftz-GAL4 embryo, showing a sib cell generated earlier (I) and the GMC-1 dividing to generate two RP2s (J). The long arrow marks the position of the sib. Scale bars: 7.5µm for A-D; 5µm for E-J.

View larger version (86K): [in a new window]   Fig. 4. Summary of the observed GMC-1 division phenotypes due to overexpression of miti and or nub. WT, wild type; mitiGOF or nubGOF, overexpression of miti or nub, respectively, for 20 minutes (blue bar) or 90 minutes (green bar). The phenotypes that are specific to 20-minute or 90-minute induction times are marked with same colored short bars. Arrows indicate times at which embryos were analyzed. The unmarked phenotypes were observed in both inductions. Note that a 20-minute induction of miti or nub only in a late GMC-1 (7.25 hours) induces a symmetrical division of GMC-1 to generate two RP2s (see Mehta and Bhat, 2001). In the UAS-nub; ftz-GAL4 embryo, the UAS-nub transgene was induced with ftz-GAL4 (the ftz promoter is active in GMC-1, see text for details).

View larger version (160K): [in a new window]   Fig. 5. Cyclin E protein is upregulated in late GMC-1 in embryos overexpressing miti. Embryos are double stained for CycE (green) and Eve (red). Only CycE and merged images of the two are shown. Arrowhead indicates a GMC-1, arrow indicates an RP2, small arrow indicates a sib. Only hemisegments are shown. (A,B) A late GMC-1 in a wild-type embryo (7.25 hours old) with very little CycE. (C,D) A newly formed RP2 and a sib in a wild-type embryo (7.75 hours old) with undetectable levels of CycE. (E,F) A late GMC-1 in a hs-miti embryo (7.25 hours old) without any induction. (G,H) A newly formed RP2 and a sib in a hs-miti embryo (7.75 hours old) without any induction. (I,J) A late GMC-1 in a hs-miti embryo induced for 20 minutes. Note the high levels of CycE. (K,L) A newly formed larger cell (presumably the self-renewed GMC-1, see text) and a sib in a hs-miti embryo where the transgene was induced for 20 minutes at the GMC-1 stage. The larger cell has high levels of CycE; CycE is undetectable in the smaller cell. (M,N) Two sibs and an RP2 in a hs-miti embryo (9.5 hours old) in which the transgene was induced for 20 minutes at the GMC-1 stage. (O,P) Three RP2 in a hsmiti embryo (9 hours old) in which the transgene was induced for 20 minutes at the GMC-1 stage. In panels M-P, CycE has been downregulated after the additional round of division. Scale bar: 7.5 µm.

View larger version (80K): [in a new window]   Fig. 6. The upregulation of CycE induces GMCs to undergo a self-renewing asymmetric division. Embryos in panels A-F are stained for Eve (red). Embryos in panels G-L are stained for Eve (red) and Zfh1 (green); co-localization of Eve and Zfh1 is indicated by yellow. Embryos in panels M and N are stained for Eve (green) and Vnd (red); yellow indicates co-localization, only the merged images are shown. Arrow indicates an RP2, small arrow indicates a sib, arrow with a star indicates a multi-cell cluster, arrowhead indicates an aCC, small arrowhead indicates a pCC, arrowhead with a star indicates friend of pCC (fpCC). (A-C) 9-hour-old UAS-CycE; ftz-GAL4 embryos showing the 3-cell phenotypes with two sibs and an RP2 (A,B) or two RP2s and a sib (C). (D) A 9-hour-old hs-CycE embryo in which the transgene was induced for 25 minutes at the GMC-1 stage. Note the 3-cell phenotype (2 sibs and an RP2). (E,F) A 10.5-hour-old hs-CycE embryo in which the transgene was induced for 90 minutes at the GMC-1 stage. (G) 9-hour-old wild-type embryo. (H,I) 9-hour-old hs-CycE embryos in which the embryos were heat shocked for 30 minutes at 6.75 hours of age. The GMC-1 has self-renewed once to generate either a sib (H) or an RP2 (I), and then terminally divided to generate an RP2 and a sib. (J) Wild-type embryo, showing an aCC (Eve and Zfh1 positive), a pCC (only Eve positive) and a fpCC (Eve and Zfh1 positive). (K,L) 9-hour-old hs-CycE embryos in which the embryos were heat shocked for 30 minutes at 6 hours of age. The GMC-1 has self-renewed to generate either two pCCs and an aCC (K) or two aCCs and a pCC (L). fpCC is formed later than aCC/pCC, therefore this particular induction timing does not affect its division pattern; a later induction affects fpCC as well (data not shown). (M) Wild-type embryo, showing an aCC (only Eve-positive), a pCC (Eve and Vnd positive) and a fpCC (Eve-positive). (N) 9-hour-old hs-CycE embryos in which the embryos were heat shocked for 30 minutes at 6 hours of age. The GMC-1 has self-renewed to generate two pCCs and an aCC.

View larger version (136K): [in a new window]   Fig. 7. Self-renewing asymmetric division in ago mutant embryos. (A-C) Embryos stained for Eve. (D-I) Merged images of embryos stained for Eve (red) and CycE (green). Arrow indicates an RP2, small arrow indicates a sib, arrowhead indicates a GMC-1. Vertical lines mark the midline. (A) An 9.5-hour-old ago mutant embryo. The hemisegment on the left has the 3-cell phenotype (two sibs and one RP2). (B,C) An 11-hour-old ago mutant embryo with the same segment shown in two different focal planes. The hemisegment on the left has the 3-cell phenotype, two RP2s (B) and one sib with fading Eve expression (C). (D,E) A late GMC-1 in an ago mutant embryo (7.25 hours old) with high levels of CycE protein. (F,G) An 7.75-hour-old ago mutant embryo in which the GMC-1 appears to have nearly completed its division to generate two cells of more or less equal sizes. One of these two cells, presumably the self-renewed GMC-1, has high levels of CycE and the other cell, presumably the RP2, has very low levels of CycE. (H,I) A hemisegment showing a 3-cell phenotype in an 9-hour-old ago mutant embryo. All three cells have undetectable levels of CycE. Scale bars: 10µm for A-C; 7.5µm for D-I.

View larger version (93K): [in a new window]   Fig. 8. The cell fate of the differentiating progeny of a GMC-1 self-renewing asymmetric division is Insc and Nb dependent. (B,D,F) Eve is red, Zfh1 is green; (J-O) Eve is green and Insc is red. Anterior end is up, vertical lines mark the midline. In wild type, there is only one Eve- (A) and Eve- and Zfh1 (B)-positive RP2 per hemisegment, whereas in insc mutants, the RP2 is duplicated, both being Eve- (B) and Zfh1 (D)-positive. (E,F) In mitiP; insc embryos, the GMC-1 generates an RP2 in each of its self-renewing divisions (the right hemisegments). (G) Wild-type embryo with an RP2 and a sib. (H) nb mutant. The GMC-1 has symmetrically divided to generate two sibs. (I) mitiP; nb embryo. In the right hemisegment the sib cells have lost Eve expression (arrowhead), whereas in the left hemisegment several sib cells can be still seen. (J-O) Localization of Insc in GMC-1 is affected in mitiP embryos. Only the merged images are shown. Insc is asymmetrically distributed in wild-type GMC-1 (J), but its distribution is non-asymmetric in mitiP embryos (K). Localization of Insc is also affected in several other unidentified GMCs (e.g. a row 4 GMC; L). Localization of Insc is not affected in GMC1-1a (N; a wild type GMC1-1a is shown in M), or several other unidentified Miti/Nub-negative GMCs (O). Scale bars: 15 µm for A-I; 10 µm for J-O.

View larger version (123K): [in a new window]   Fig. 9. GMCs for the RP2/sib, U, CQ and EL neuronal lineages do not undergo additional rounds of cell division in prospero mutants. Embryos are single stained for Eve (A-D) and double stained for Eve (red) and Zfh1 (green) (E-J; only one half-segment is shown). Midline is marked by vertical lines. Embryos mutant for pros have holes in the midline and a mis-routed axon scaffold, thus the neuronal lineages are farther apart. The loss of RP2, U and CQ lineages is partially penetrant in this mutant allele (see text for details). (A) 10-hour-old wild-type embryo. The number of U neurons is usually four in wild type, CQs are hidden beneath the U cluster, but are visible in the hemisegment on the left. (B) 13-hour-old wild-type embryo. Us and CQs are out of the focal plane. ELs are also slightly out of the focal plane. (C) 10-hourold pros mutant embryo. An RP2 and a sib are shown. aCC/pCC, Us and CQs are missing in this segment. (D) 13-hour-old pros mutant embryo. An RP2 is flanked by two Us on top and three Us on the bottom. (E) 13-hour-old wild type, note that RP2 and aCC are Eve and Zfh1 positive. (F) 13-hour-old pros mutant. Both aCC and pCC are missing (marked by a star). (G) 13-hour-old wild-type embryo. Out of four Us, two are Eve and Zfh1 positive. The other two are only Eve positive. (H) 13-hour-old pros mutant embryo. There are only three U neurons, one of the two Eve- and Zfh1-positive Us is missing. (I) 13-hour-old wild-type embryo. In wild type there are three CQs, all are Eve and Zfh1 positive (one of the CQs in this panel is out of the focal plane). The number of ELs varies between eight and ten in the abdominal segments. (J) 13-hour-old pros mutant embryo. There is only one CQ in this hemisegment. The number of ELs is usually the same as in wild type (some of the ELs are out of the focal plane in this panel).