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First published online 2 October 2008
doi: 10.1242/dev.024349

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Pdm and Castor close successive temporal identity windows in the NB3-1 lineage

Institute of Neuroscience, Institute of Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA.

View larger version (71K): [in this window] [in a new window]   Fig. 1. Temporal identity gene expression in the NB3-1 lineage of the Drosophila CNS. (A) NB3-1 (encircled with dashed line) sequentially expresses Hb, Kr, Pdm and Cas. A portion of one hemisegment is shown, with Engrailed marking the most-posterior neuroblast (NB) rows 6/7 (blue). Midline, left; anterior, up. Embryonic staging is from Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985): early stage 10 (E10), when NB3-1 forms; late stage 10 (L10); early stage 11 (E11); mid stage 11 (M11); late stage 11 (L11). Scale bar: 10 µm. (B) Summary of gene expression in NB3-1.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Molecular markers and cell position can be used to identify each RP motoneuron in the NB3-1 lineage. (A) RP1 and RP4 are Hb+, Kr+, Cut-, Zfh2- and are shown in insets because they are usually obstructed in the projection (n>100). RP3 is Hb-, Kr+, Cut-, Zfh2+ (n>100). RP5 is Hb-, Kr-, Cut+, Zfh2+ (n>100). A single representative hemisegment of a wild-type (wt) stage-16 CNS is shown as a maximum intensity projection. Midline, left; anterior, up. An expression summary is shown above. (B) (Top row) RP motoneurons are Islet+ HB9+ (outlined). RP1 and RP4 occupy the deepest layer; RP1 is more dorsal and expresses HB9 at higher levels than does RP4 after stage 15 (n>100). RP3 is directly ventral to RP1 and RP4 (n>100). RP5 is ventral and anterior to RP3 (n>100). White arrowheads, NB7-3-derived EW interneurons. Dashed vertical line, midline. Ventral views of two segments are shown from deep (left) to superficial (right) focal planes. (Bottom row) Each RP neuron has a non-RP neuron sibling, based the duplication of each RP neuron in sanpodo mutants. Eight Islet+ HB9+ Late Bloomer+ RP motoneurons are observed in each hemisegment (quantified in Table 1). Midline, between each pair of panels. (C) Recombination-induced activation of lacZ in NB3-1 labeled RP4, RP3, RP5 and the late-born interneurons but not RP1, showing that RP1 is the first-born neuron in the lineage. HB9, green; β-galactosidase, magenta. RP neurons and NB3-1 are outlined and labeled. One hemisegment of a stage-16 CNS is shown as a maximum intensity projection. Midline, left; anterior, up. A schematic summary of the clone is shown to the right. (D) Schematic of NB3-1 gene expression and cell lineage. The vertical dashed line represents the transition between RP neuron and subsequent interneuron specification. Nb, sibling cell fate specified by Numb; N, sibling cell fate requires Sanpodo and active Notch signaling. Scale bars: 3 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 3. Hb is necessary and sufficient to specify the first temporal identity. (A) Drosophila hb mutants lack the RP1 and RP4 neurons, but have normal RP3 and RP5 neurons (Table 1). Wild type has RP1, RP4, RP3 and RP5 neurons (see Fig. 2). (B) hb misexpression (insc-gal4 UAS-hb) generates ectopic RP1/4 neurons based on molecular markers (Table 1). (C) (Top row) Wild-type RP motoneurons express the pan-motoneuron markers phosphorylated (p) Mad (PMAD) and Late Bloomer. (Bottom row) hb misexpression (insc-gal4 UAS-hb) generates ectopic RP1/4 neurons that are double positive for pMad and Late Bloomer (100%, n=48). For all panels, a single representative hemisegment of a stage-16 CNS is shown as a maximum intensity projection. Midline, left; anterior, up. A phenotype summary is shown in the right-hand panels, with Late Bloomer expression indicated by dashed circles. Scale bars: 3 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 4. Kr is necessary and sufficient to specify the second temporal identity. (A) In Kr mutant Drosophila embryos, RP3 is missing in the majority of hemisegments examined (both rows) and RP5 is occasionally missing (second row), whereas RP1/RP4 are normal (Table 1). Wild type has RP1, RP4, RP3 and RP5 neurons (see Fig. 2). (B) Kr misexpression (insc-gal4 UAS-Kr) generates ectopic RP3 neurons, RP5 is usually absent, and RP1/RP4 are normal (Table 1). For all panels, a single representative hemisegment of a stage-16 CNS is shown as a maximum intensity projection. Midline, left; anterior, up. A phenotype summary is shown to the right. Scale bar: 3 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 5. Pdm closes the second temporal identity window in the NB3-1 lineage. (A,B) Neuroblast expression of Kr and Cas in pdm mutant and pdm misexpression embryos. One hemisegment is shown. Kr or Cas, brown; the positional marker Engrailed, blue. NB3-1 is outlined. (A) In pdm mutants, Kr expression persists through late stage 11 (it is normally switched off by mid stage 11; see Fig. 1A) and Cas expression is delayed until mid stage 12. (B) In pdm misexpression embryos (insc-gal4 UAS-pdm2), Kr expression is lost prematurely and Cas is expressed precociously (compare with Fig. 1A). (C,D) RP neuron specification in pdm mutant and pdm misexpression embryos. One hemisegment of a stage-16 CNS is shown as a maximum intensity projection. Midline, left; anterior, up. A phenotype summary is shown to the right. (C) In pdm mutant embryos there are two to three ectopic RP3 neurons; RP1, RP4 and RP5 neurons are usually normal (Table 1). (D) pdm misexpression (insc-gal4 UAS-pdm2) results in the frequent loss of the RP5 neuron (both rows) and in the occasional loss of the RP3 neuron (second row); RP1/RP4 are normal (Table 1). Scale bars: 10 µm in A,B; 3 µm in C,D.

View larger version (89K): [in this window] [in a new window]   Fig. 6. Cas closes the third temporal identity window in the NB3-1 lineage. (A) Drosophila cas mutants have persistent Pdm expression in NB3-1 until at least mid stage 12 (M12); in wild type, Pdm is gone from NB3-1 by late stage 11 (see Fig. 1A). At mid stage 16, neuroblasts in the medial column no longer express Pdm (arrowheads); one of these neuroblasts is likely to be NB3-1. (B) In cas mutants, there are up to four ectopic Cut+ RP5 neurons; RP1, RP4 and RP3 are normal (Table 1). Wild type has RP1, RP4, RP3 and RP5 neurons (see Fig. 2). (C) cas misexpression (insc-gal4 UAS-cas) results in frequent loss of RP5 and occasional loss of RP3; RP1 and RP4 are normal (Table 1). For all panels, a single representative hemisegment of a stage-16 CNS is shown as a maximum intensity projection. Midline, left; anterior, up. A phenotype summary is shown to the right. Scale bars: 10 µm in A; 3 µm in B,C.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Hb and Kr specify early temporal identity, whereas Svp, Pdm and Cas act as timer elements within the NB3-1 lineage of the Drosophila CNS. Spatial cues specify NB3-1 identity and formation, which allows the neuroblast to respond in a potentially unique way to timer genes and temporal identity genes that are expressed subsequently. Timer elements (top row) include Seven up (Svp), Pdm and Cas. These factors close successive temporal identity windows. Timer genes indirectly control cell fate through the regulation of temporal identity genes. Temporal identity genes (middle row) include Hb and Kr, which specify first and second temporal identities (TIs), respectively, in this and other lineages. Neuronal identity (bottom row) within the lineage: during the first TI, GMC-1 makes RP1/sibling neurons and GMC-2 makes RP4/sibling neurons; during the second TI, GMC-3 makes RP3/sibling neurons; during the third TI, GMC-4 makes RP5/sibling neurons; and during the fourth TI, GMC-5 makes interneurons (IN).

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