Research ArticleDevelopmental Biology

Notch signaling acts before cell division to promote asymmetric cleavage and cell fate of neural precursor cells

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Science Signaling  21 Oct 2014:
Vol. 7, Issue 348, pp. ra101
DOI: 10.1126/scisignal.2005317

Abstract

Asymmetric cell divisions in the central nervous system generate neurons of diverse fates. In Drosophila melanogaster, the protein Numb localizes asymmetrically to dividing neural precursor cells such that only one daughter cell inherits Numb. Numb inhibits Notch signaling in this daughter cell, resulting in a different cell fate from the Notch-induced fate in the other—Numb-negative—daughter cell. Precursor cells undergo asymmetric cytokinesis generating daughter cells of different sizes. I found that inactivation of Notch in fly embryonic neural precursor cells disrupted the asymmetric positioning of the cleavage furrow and produced daughter cells of the same size and fate. Moreover, inactivation of Notch at different times altered the degree of asymmetric Numb localization, such that earlier inactivation of Notch caused symmetric distribution of Numb and later inactivation produced incomplete asymmetric localization of Numb. The extent of asymmetrically localized Numb positively correlated with the degree of asymmetric cytokinesis and the size disparity in daughter cells. Loss of Numb or expression of constitutively active Notch led to premature specification of the precursor cells into the fate of one of the daughter cells. Thus, in addition to its role in the specification of daughter cell fate after division, Notch controls Numb localization in the precursor cells to determine the size and fate of daughter cells. Numb also inhibits Notch signaling in precursor cells to prevent Notch-induced differentiation of the precursor cell, forming an autoregulatory loop.

INTRODUCTION

During neurogenesis, neurons of diverse fates arise from a few precursor cells. In Drosophila melanogaster, primary neural precursors called neuroblasts (NBs) divide asymmetrically to self-renew and generate an intermediate precursor cell called the ganglion mother cell (GMC). GMCs typically undergo terminal asymmetric division to generate two different neurons. However, GMCs have the potential to undergo self-renewing asymmetric divisions, at least in some lineages (1). NBs and GMCs have apical-basal polarity and divide perpendicular to the apical-basal plane. Unlike NBs, in which asymmetric division is invariably associated with asymmetric cytokinesis, only a few GMCs undergo asymmetric cytokinesis during the terminal division. One such GMC derives from NB4-2, a well-characterized NB located in the fourth row and second column of NBs. The first asymmetric division of this NB produces another NB and the GMC-1 (also known as GMC4-2a), which buds off basally and perpendicular to the plane of NB division. The GMC-1 undergoes a dramatic asymmetric cytokinesis to produce a basally located larger motoneuron (RP2) and an apically located smaller sibling (sib) cell. Similar to many other neural precursor cells, GMC-1s express the gene encoding the transcription factor Even-skipped (Eve). After division of the GMC-1, both daughter cells are Eve-positive, but as development proceeds, the sib cell becomes Eve-negative. The ultimate cell fate of the sib cell is not known, and there are no known molecular markers for this cell (110).

Notch signaling plays a central role in controlling asymmetric cell division in a variety of cell lineages, including the NB4-2-GMC-1-RP2 lineage (11). Notch is a transmembrane receptor that, when bound to ligands such as Delta, undergoes proteolytic cleavage and release of the intracellular domain of Notch (Nintra). Nintra then translocates to the nucleus and activates or modifies transcription of downstream effectors by binding to transcription factors such as RBP-J. Notch-dependent gene expression also involves transcription coactivators, including Mastermind (Mam) (1113). Genetic inhibition of Notch signaling causes GMC-1s to divide into two RP2 cells, implying that Notch activity promotes sib cell fate and represses RP2 cell fate (6, 8, 9). Notch also mediates similar binary cell fate decisions in several other lineages (1417). Numb is a phosphotyrosine binding domain (PTB)–containing protein that binds to the intracellular domain of Notch and prevents Notch cleavage, and thereby inhibits Notch signaling (11, 12, 1418). Numb invariably localizes asymmetrically to the basal end of NBs and GMCs perpendicular to the plane of cytokinesis and is inherited by the basal daughter cell. In the GMC-1 lineage, Numb promotes RP2 specification by inhibiting Notch signaling (6, 8, 9).

The adaptor protein Inscuteable (Insc) is also involved in asymmetric cell fate decisions of some, but not all, GMCs and NBs. Insc contains an Src homology 3 (SH3) binding domain, ankyrin repeats, and cytoskeletal attachment sites (19). Insc localizes asymmetrically to the apical end of NBs, and loss of Insc disrupts the localization of Numb and the orientation of the mitotic spindle in NBs (20). Numb and Insc are required for asymmetry in the GMC-1 lineage, as well as several other cell lineages (6, 21). However, there are some cell lineages, for example, the MP2 lineage, where Numb is required, but Insc is not (22). MP2 cells are similar to NBs in terms of the expression of genes that determine neural differentiation of the ectoderm but behave like GMCs by undergoing terminal cell division to generate two different neurons (16).

The bulk of the literature suggests that Notch specifies cell fates postmitotically. In contrast, I found that Notch signaling was required in the precursor cell before cell division to enable asymmetric cytokinesis and daughter cell fates. Notch signaling was required for the asymmetric localization of Numb to the basal pole of precursor cells, including GMC-1s, MP2 cells, and precursor cells of several other cell lineages. Loss of Notch signaling in GMC-1s disrupted asymmetric positioning of the cleavage furrow leading to division into two symmetrically sized RP2 cells. Likewise, flies with mutations in the genes encoding Mam or Insc also had mislocalized Numb in GMC-1s and symmetric duplication of RP2 cells. Numb was also required for the symmetric RP2 cell duplication in insc mutant embryos because fly embryos lacking both numb and insc gene products had asymmetric division of GMC-1s. Moreover, GMC-1s as well as both progeny of GMC-1 divisions in embryos lacking numb and insc, or in embryos expressing Nintra to promote excess Notch signaling, adopted characteristics of sib cell identity. Thus, in addition to the role of Notch signaling in the specification of cell fate in the progeny of asymmetrically dividing cells, Notch signaling is also required in precursor cells before cell division to restrict Numb to the smallest area within the basal pole, which likely enables the asymmetry of cytokinesis.

RESULTS

Notch signaling is required before cleavage of GMC-1 to determine daughter cell size

To better understand the temporal requirements for Notch signaling in the development of the GMC-1 lineage, I examined flies homozygous for a temperature-sensitive allele of Notch (Notchts1) that causes loss of function of Notch signaling when flies are shifted from a permissive (22°C) to a restrictive (29°C) temperature (23). As previously reported (19), in wild-type embryos, GMC-1s became positive for Eve at around 7 hours of development and divided about 30 min later to generate the large RP2 neuron and the smaller sib cell, which lost expression of Eve between 10 and 14 hours of development (Fig. 1). Thus, I shifted Notchts1 embryos to the restrictive temperature during early (6 hours), middle (6.5 hours), and late (7 hours) stages of GMC-1 development (fig. S1A). Inactivation of Notch during early GMC-1 development caused GMC-1s to divide into two equal-sized RP2 cells (Fig. 1 and fig. S1). When Notch was inactivated during mid–GMC-1 development, I observed fewer symmetrically sized duplicated RP2 cells and more asymmetrically sized duplicated RP2 cells (Fig. 1 and fig. S1). At the late stage of GMC-1 development but before division, inactivation of Notch produced fewer RP2 duplications, and the duplicated RP2 cells were typically asymmetrically sized (Fig. 1 and fig. S1). No duplication of the RP2 neurons was observed in wild-type embryos shifted at the same time as Notchts1 embryos or in wild-type or Notchts1 embryos grown at the permissive temperature (Fig. 1). Thus, Notch signaling is required before the division of GMC-1s to determine the size of the daughter cells.

Fig. 1 Notch signaling is required in GMC-1 itself for asymmetric division of GMC-1.

Immunohistochemical labeling for Eve shows GMC-1s and their progeny (RP2 motor neurons and sib cells) in wild-type or homozygous Notchts1 embryos at the indicated ages. Notchts1 embryos at 6 hours [early stage (E), soon after formation of GMC-1] or 6.5 hours [middle stage (M)] of development were shifted to 29°C for 30 min and fixed at the indicated hours (7 to 14 hours) of development. The table shows the quantification of daughter cell phenotypes in Notchts1 embryos shifted at early (6 hours), middle (6.5 hours), or late (7 hours) stages of GMC-1 development. n ≥ 144 hemisegments from at least six embryos per condition.

Notch is required in GMC-1s for asymmetric localization of Numb and positioning of the cleavage furrow

The asymmetric distribution of Numb in GMC-1s before division determines asymmetric cell fate (6, 8, 9). Therefore, I examined whether loss of Notch signaling affected Numb localization in GMC-1s. Wild-type or Notchts1 embryos aged between 5.5 and 6.5 hours of development were shifted to the restrictive temperature for 1 hour, fixed immediately, and fluorescently labeled with antibodies for Numb and Eve. Eve is abundant in the nucleus of GMC-1s during the G2 phase of the cell cycle (24). In a newly formed GMC-1s (at 6.5 hours of development), Numb was distributed around the cell cortex in a punctate pattern, Eve localization was compact in the nucleus, and the cytoplasm was evident between Eve and Numb labeling (Fig. 2A). In wild-type embryos, as GMC-1 cells proceeded toward mitosis, Numb localization became progressively restricted to the basal side, and during division, only the larger daughter cell, presumably the RP2 neuron, inherited Numb (100% of hemisegments) (Fig. 2A). In contrast, in temperature-shifted Notchts1 embryos, Numb localized evenly around the cortex throughout the transition from G2 to mitosis, and both daughter cells inherited Numb (88% of hemisegments) (Fig. 2A). In a fraction of GMC-1s in temperature-shifted Notchts1 embryos (9% of hemisegments), GMC-1s showed partial or incomplete asymmetric localization of Numb (fig. S2), which may result from the timing of Notch inactivation or a partial loss of function for Notch signaling.

Fig. 2 Notch signaling is required for the asymmetric localization of Numb and positioning of the cleavage furrow.

(A) Immunofluorescence labeling for Eve and Numb in GMC-1s of wild-type or homozygous Notchts1 (Nts1) embryos at 5.5 to 6.5 hours of development that were shifted to 29°C for 60 min and fixed at the indicated ages. Images are representative of n ≥ 59 hemisegments from at least 10 embryos per condition. (B and C) Immunofluorescence labeling for Eve and Numb (B) or Eve and Spectrin (C) in GMC-1s of wild-type or homozygous Notchts1 embryos at 7.5 hours of development. Notchts1 embryos were shifted to 29°C for 30 min at early (E; 6 hours), middle (M; 6.5 hours), or late (L; 7 hours) stages of GMC-1 development. Spectrin was used to indicate the position of the cleavage furrow (denoted by arrows). Images are representative of n ≥ 30 hemisegments from at least five embryos per condition (B) and n ≥ 20 hemisegments from at least four embryos per condition (C).

I asked whether the differences in the size of daughter cells when Notch was inactivated at different times correlated with differences in Numb localization. I shifted Notchts1 embryos to the restrictive temperature during early, middle, and late GMC-1 development and examined the localization of Numb at 7.5 hours of development. In contrast to the small basal crescent of Numb seen in wild-type embryos, when I inactivated Notch early in Notchts1 embryos, Numb was distributed all around the cortex of GMC-1s (81% of hemisegments) (Fig. 2B). In Notchts1 embryos temperature-shifted during mid–GMC-1 development, Numb was partially asymmetrically distributed in late GMC-1s, but the crescent was expanded compared to wild type and there was still Numb in the apical region (52% of hemisegments) (Fig. 2B). Moreover, similar to the differences in RP2 size, inactivation of Notch during late GMC-1 development had less effect on the localization of Numb. The crescent of Numb was either partially (32% of hemisegments) or completely (68% of hemisegments) restricted to the basal side of GMC-1s (Fig. 2B).

These data showed that Notch is required in GMC-1s before division to control the localization of Numb and the size of daughter cells. Daughter cell size is determined by the position of the cleavage furrow during cytokinesis. Thus, I asked if Notch signaling was required for the positioning of the cleavage furrow in Notchts1 embryos. I fluorescently labeled 8-hour-old embryos using antibodies targeting Eve and Spectrin, a cytoskeletal component present at the cell cortex, thus marking the cleavage furrow (25). Unlike in wild-type embryos, in Notchts1 embryos temperature-shifted during early GMC-1 development, the cleavage furrow of GMC-1s was in the middle of the cell (55% of hemisegments) (Fig. 2C). Inactivation of Notch in Notchts1 embryos during mid–GMC-1 development resulted in GMC-1s with cleavage furrows that were moderately asymmetric (43% of hemisegments), and late inactivation of Notch resulted in normal asymmetric cleavage furrows in most GMC-1s (93% of hemisegments).

Notch signals through Mam to control the localization of Numb

Notch signals through Mam to control the transcription of target genes (11, 12). Fly embryos homozygous for hypomorphic alleles of mam have RP2 duplication due to loss of asymmetric division of GMC-1 and not due to formation of additional NB4-2s (26). mamIL42 is a strong loss-of-function allele due to a premature stop codon that creates a truncated protein (26). I found that GMC-1s in mamIL42 embryos divided equally into two equal-sized RP2s in most hemisegments (Fig. 3A). mamHD10/6 is a weaker allele with a P element inserted in the first exon, which is not translated (26). Accordingly, GMC-1s in most of the hemisegments in mamHD10/6 embryos divided into two unequal-sized RP2s (Fig. 3A). I also examined the localization of Numb in mamIL42 embryos. Similar to temperature-shifted Notchts1 embryos, mamIL42 embryos failed to form a crescent of Numb on the basal side of GMC-1s. Instead, Numb localized evenly around the cortex throughout the transition from G2 to mitosis, and both daughter cells inherited Numb (87% of hemisegments) (Fig. 3B). In 11% of hemisegments, GMC-1s had partial or incomplete asymmetric localization of Numb (fig. S2). Thus, Notch signaling mediates Numb localization and asymmetric cell division through activation of Mam.

Fig. 3 The Notch-associated transcription factor Mam is required for Numb localization and asymmetric division of GMC-1s.

(A) Immunohistochemical labeling for Eve shows GMC-1s and their progeny in embryos homozygous for strong (mamIL42) or weak (mamHD10/6) loss-of-function alleles of mam. Arrows indicate symmetric RP2 duplications. Arrows with asterisks indicate asymmetric RP2 duplications. The table shows the quantification of n ≥ 240 hemisegments from at least 10 embryos per condition. (B) Immunofluorescence labeling for Eve and Numb in GMC-1s of mamIL42 embryos at the indicated ages. Images are representative of n ≥ 110 hemisegments from at least eight embryos per condition.

Insc is downstream of Notch signaling during GMC-1 asymmetric division

Insc localizes to the apical pole of GMC-1s in metaphase (6). In NBs, Insc restricts the localization of Numb to the basal end (20, 27). Loss of Insc affects the development of the GMC-1 lineage (20). Therefore, I examined Insc localization in GMC-1s of embryos with Notch or Mam loss of function. Unlike in GMC-1s of wild-type embryos, in GMC-1s of Notchts1 embryos that were temperature-shifted during early GMC-1 development (88% of hemisegments) or in GMC-1s of mamIL42 embryos (77% of hemisegments), Insc was not localized in an apical crescent but rather was widely distributed and punctate (Fig. 4A).

Fig. 4 Insc functions downstream of Notch to control Numb localization and asymmetric division of GMC-1s.

(A) Immunofluorescence labeling for DNA (propidium iodide) and Insc in wild-type GMC-1s (metaphase), Notchts1 GMC-1s (metaphase), or mamIL42 GMC-1s (prometaphase) at 7.5 hours of development. Notchts1 embryos at 6 hours of development were shifted to 29°C for 30 min. Images are representative of n ≥ 30 hemisegments from at least five embryos per condition. (B) Immunohistochemical labeling for Eve shows GMC-1s and their progeny in homozygous insc22 embryos. Arrows indicate symmetric duplicated RP2 neurons. Image is representative of n ≥ 144 hemisegments from at least six embryos per condition. (C) Immunofluorescence labeling for Eve and Numb in GMC-1s of wild-type and homozygous insc22 embryos. Images are representative of n ≥ 30 hemisegments from at least five embryos per condition. (D) Immunofluorescence labeling for Eve and Insc in GMC-1s of wild-type and homozygous numbdf embryos. Images are representative of n ≥ 20 hemisegments from at least four embryos per condition. (E and F) Immunohistochemical labeling for Eve shows GMC-1s and their progeny in wild-type embryos, homozygous numbdf embryos, or embryos homozygous for insc22 and numbdf (E) or Hsp70-Notchintra (Nintra) (F). Hsp70-Notchintra was either uninduced or induced in 6-hour-old embryos that were shifted to 29°C for 30 min. (E and F) Arrowheads show GMC-1s; arrowheads with asterisks show GMC-1s with weak Eve staining; arrows show asymmetric daughter cell division. Larger arrows show RP2 motorneurons. Smaller arrows show sib cells. Arrows with asterisks indicate asymmetric sib cell duplications. The absence of Eve or weak Eve staining suggests transformation to sib cell fate. Images are representative of n ≥ 144 hemisegments from at least six embryos per condition.

Thus, I examined whether Insc was required for Numb localization and size asymmetry during division of GMC-1s. In embryos homozygous for insc22, RP2 duplication occurred in most hemisegments (85% of hemisegments), and most of duplicated RP2s were of the same size (80% of hemisegments) (Fig. 4B). Consistent with the data on NBs (27), Numb was distributed throughout the cortex in late-stage GMC-1s of insc22 embryos (67% of hemisegments) (Fig. 4C). In contrast, the apical localization of Insc in late-stage GMC-1s was not affected by loss of Numb in embryos with a deficiency spanning over the numb gene (numbdf) (100% of hemisegments) (Fig. 4D). Thus, in GMC-1s, Insc is required for the basal localization of Numb, but the apical localization of Insc is independent of Numb.

Numb interferes with asymmetric cleavage of GMC-1s

The cleavage of GMC-1s in numb loss-of-function mutant embryos is asymmetric, producing daughter cells with different sizes (6, 8), but both daughter cells adopt the sib cell fate (6, 8, 9). This suggests that Numb is not required for asymmetric cleavage but only for cell fate specification. In embryos homozygous for a hypomorphic numb mutation, the larger daughter cell often begins to lose RP2-specific gene expression before the smaller cell, indicative of excess Notch signaling (8). Moreover, I found that in numbdf embryos, GMC-1s had reduced abundance of Eve before division (67% of hemisegments) (Fig. 4E). Because Notch signaling specifies sib fate (6, 8), this result suggested that loss of Numb enables active Notch signaling in GMC-1s, perhaps converting GMC-1s into a sib-like fate. Thus, Numb is sufficient to prevent sib specification in early GMC-1s despite its distributed and punctate localization.

Numb was mislocalized in early-stage GMC-1s in embryos with inactivated Notch, and the degree of expanded Numb localization positively correlated with the penetrance of the symmetric RP2 duplication phenotype in these embryos, suggesting that mislocalized Numb could interfere with asymmetric cleavage. Thus, to determine if mislocalized Numb interferes with asymmetric cytokinesis, I asked if the symmetric cleavage and RP2 fate specification and symmetric duplication in insc22 embryos was dependent on Numb. Similar to numbdf embryos, GMC-1s in numbdf, insc22 double-mutant embryos had reduced abundance of Eve before division (61% of hemisegments) (Fig. 4E and fig. S3, B to E). Moreover, unlike in insc22 embryos, numbdf, insc22 double-mutant embryos had asymmetrically sized sib cell pairs (65% of hemisegments) (Fig. 4E and fig. S3, D and E), indicating that the cytokinesis of the GMC-1 was asymmetric.

These results suggested that activation of Notch signaling in GMC-1s of these embryos leads to sib cell fate and asymmetric cleavage. To directly test this hypothesis, I expressed Nintra in GMC-1s before division. Numb blocks the processing and release of Nintra (16), and expression of Nintra circumvents repression by Numb and causes both the progeny of GMC-1 to adopt the sib cell fate (8, 9). I found that expression of Nintra before GMC-1 division resulted in GMC-1s losing expression of Eve before division (80% of hemisegments) (Fig. 4F) or dividing into asymmetrically sized daughter cells that were weakly positive for Eve (75% of hemisegments) (Fig. 4F and fig. S3F).

Notch signaling promotes asymmetric localization of Numb in other NBs and GMCs

To extend these findings beyond the GMC-1 lineage, I examined the localization of Numb in several other precursor cells in the absence of Notch signaling. GMC1-1a divides to generate the aCC and pCC neurons, and loss of function for Notch, Mam, or Insc causes GMC1-1a to divide into two aCC neurons (6), whereas loss of function for Numb causes GMC1-1a to divide into two pCC neurons (9). Numb localized to the basal pole of late GMC1-1a cells in wild-type embryos (100% of hemisegments), but was broadly distributed in late GMC1-1a cells in temperature-shifted Notchts1 embryos (68% of hemisegments) or mamIL42 embryos (75% of hemisegments) (fig. S4, A to C). Likewise, in MP2 cells, Numb invariably localized to the basal pole in wild-type embryos (fig. S4, D to F and N) (16), but not in most of temperature-shifted Notchts1 embryos (fig. S4, G to I and O) or mamIL42 embryos (fig. S4, P and Q). Similarly, in NB7-1 cells, which require Insc function for Numb localization (27), Numb localization was invariably asymmetric in wild-type embryos (fig. S4, J, K, and N), but not in most of the hemisegments of temperature-shifted Notchts1 embryos (fig. S4, L, M, and O) or mamIL42 embryos (fig. S4, P to R). In contrast, not all GMCs and NBs required Notch signaling for proper basal localization of Numb (fig. S5), consistent with previous results (28). For example, NB5-3, which is an S1 NB formed at the same time as MP2, had normal Numb localization in Notchts1 embryos (fig. S5). Moreover, in wild-type embryos, Numb localization in newly formed GMC-1s and GMC1-1a cells was not uniform but was distributed in a punctate pattern (fig. S6).

DISCUSSION

Here, I found that Notch signaling determined the asymmetric size and fate of daughter cells by acting on precursor cells before division. Notch signaled through Mam, presumably to activate downstream genes, which initiated the relocalization of Insc to the apical pole and Numb to the basal pole. Insc was required for localization of Numb, but Numb was not required for the localization of Insc. Thus, Notch likely acted through Insc to restrict Numb to a small area within the precursor cell, and thereby promoted asymmetric cytokinesis and fate (Fig. 5). Numb loss of function did not affect the relative sizes of the daughter cells. Therefore, this suggests that for asymmetric cleavage of GMC-1 to occur, Numb must be either restricted to a narrow region on the basal side or altogether absent. Although Notch signaling at the apical pole—where Numb inhibition is not present——may initiate cell fate specification before precursor cell division, the positioning of the cleavage furrow likely depends on progressive changes in the localization of Numb and the feedback inhibition by Numb on Notch signaling.

Fig. 5 Model of Notch, Insc, and Numb interactions in asymmetric division of GMC-1s.

Illustration of the proposed model for the role of Notch in controlling asymmetric cell division of GMC-1s. (A) In wild-type embryos, Notch signaling restricts the localization of Insc to the apical side and Numb to the basal side. This leads to inhibition of Notch by Numb on the basal side of the GMC-1 and in the basal daughter cell. Active Notch signaling in the apical daughter cell promotes sib cell fate, whereas inhibition of Notch by Numb in the basal daughter cell promotes RP2 cell fate. Restriction of Numb to the basal pole in GMC-1 allows its asymmetric cleavage. (B) Early loss of Notch signaling by inhibition of Notch or loss of Mam in the GMC-1 disrupts the localization of Insc and Numb and leads to division into two equal-sized daughter cells with RP2 cell fate. Later inactivation of Notch in GMC-1 leads to partial localization of Insc and Numb and a moderate asymmetric cleavage into two unequal-sized RP2 cells. (C) Loss of Insc in the GMC-1 causes unrestricted localization of Numb and thus inhibition of Notch signaling throughout the GMC-1, leading to division into two equal-sized daughter cells with RP2 cell fate. (D) Loss of Numb does not affect the distribution of Insc or asymmetric cleavage of the GMC-1 but leads to excess activation of Notch, thereby promoting sib cell fate.

The asymmetric localization of Numb in GMC-1s required Insc. Like GMC-1s, other neural precursor cells, including GMC1-1a and NB7-1 cells, require Insc for asymmetric division (27). However, MP2 cells do not have Insc and thus do not require Insc for asymmetric division (22). Yet, Notch loss of function disrupted basal localization of Numb in MP2 cells. One possible explanation for this observation is that Notch signaling may control the apical localization of another determinant, such as Bazooka (29), which is required for the basal localization of Numb in MP2 cells (22). A few GMCs and NBs localize Numb properly in the absence of Notch signaling (28) (fig. S5), suggesting that these precursors likely have a different pathway performing the same task as Notch signaling or that Notch signaling is fully redundant in these cells.

Newly formed GMCs and NBs inherit Numb, which is initially broadly distributed throughout the cell cortex but punctate throughout the cell cortex (Fig. 2A and fig. S6). This nonuniformity of Numb distribution may enable Notch activation at discrete foci that are sufficient to begin a cascade leading to the progressive restriction of Numb to the basal pole. The time between formation and division of GMC-1s is about 1.5 hours, suggesting a relatively fast mechanism with Notch signaling acting soon after the formation of GMC-1. I found that early, but not late, inactivation of Notch generated symmetrically sized daughter cells and that the Notch-associated transcription factor Mam was required for the localization of Numb. Thus, these results are consistent with a mechanism involving transcription of a factor (or factors) that acts on Insc, and possibly other apically localized determinants, to influence Numb localization. However, these data do not preclude the possibility of additional “noncanonical” Notch-dependent signaling pathways in this process.

Both Notch and Insc were required for the basal localization of Numb and asymmetric cytokinesis of GMC-1s, suggesting that Numb localization to the basal pole is essential for asymmetric positioning of the cleavage furrow. Furthermore, the symmetric cytokinesis of GMC-1s that occurred in insc mutant embryos was not present in insc, numb double-mutant embryos, suggesting that mislocalized Numb interferes with asymmetric cytokinesis. Insc is also required for proper spindle orientation of NB cells (30). However, because the symmetric cytokinesis in insc22 embryos was Numb-dependent, Insc-mediated spindle orientation is unlikely to mediate asymmetric cytokinesis. One model to explain asymmetric cell division posits that the lengthening of the pole arms on one side of the mitotic spindle and shortening on the other side may cause asymmetric cytokinesis (31). However, another study determined that asymmetric spindle lengthening was the result, not the cause, of asymmetric cytokinesis (32). Moreover, cells in several different mutant fly embryos, including centrosomin, spindle assembly abnormal 4 ortholog, asterless, and spindle defective 2, have identical length spindle poles but undergo normal asymmetric cell division (30, 33, 34). A second model, called the cortical expansion model, suggests that the cell cortex in one pole expands by a spindle-independent mechanism creating asymmetric forces on the spindle (32) and shifting the position of the cleavage furrow (35). Finally, a third model invokes apical-basal polarity of proteins in the cell cortex as a cue for spindle-independent positioning of the cleavage furrow in neural progenitor cells in Caenorhabditis elegans (36) and NBs in Drosophila (32). How Notch and Numb control any or all of these mechanisms remains unclear. Nevertheless, my results provide insight into the relationships between asymmetric cell fates and asymmetric cleavage during neurogenesis and implicate Notch signaling as a key factor in Numb and Insc localization in neural precursor cells before division.

MATERIALS AND METHODS

Genetics

Oregon-R wild-type flies and flies with genotypes Notchts1 (encoding Notch with a G1272D amino acid substitution), Hsp70-Nintra, insc22, insc [Df(2R)ED3791], numbdf [Df(2L)ED690 (30B3-30E4)], and a second numb deficiency [Df(2L)ED680 (30A4-30B12)] were obtained from the Bloomington Stock Center. mamIL42 and mamHD10/6 flies were obtained from B. Yedvobnick. insc22, numbdf double-mutant flies were generated by crossing insc22 and Df(2L)ED690/SM6a flies and confirmed by out-crossing to flies with an insc deficiency [Df(2R)ED3791] or flies with a different numb deficiency [Df(2L)ED680] and testing for noncomplementation and central nervous system phenotypes. All mutant flies were balanced with either green fluorescent protein (GFP)– or LacZ-containing chromosomes to facilitate the identification of homozygous mutant embryos. Mutant embryos were further identified using their mutant defects in other lineages.

Temperature shift experiments

Several different sets of temperature shift experiments were done depending on the lineage. For the analysis of the effects of Notch inactivation on the GMC-1 lineage (Figs. 1, 2, B and C, and 4A and fig. S1), Notchts1 embryos were collected for 15 min at 22°C permissive temperature, thoroughly washed, briefly dried, and then shifted to 29°C restrictive temperature at different time points during GMC-1 development: early (6 hours), middle (6.5 hours), and late (7 hours). These embryos were kept at restrictive 29°C for 30 min and shifted back to permissive 22°C and fixed after 1 to 1.5 and 4 to 5 hours (early shift), 0.5 to 1 and 3.5 to 4 hours (mid shift), and 30 min and 4 to 5 hours (late shift). To analyze the dynamics of Numb localization throughout GMC-1 development (Figs. 2A and 3B and fig. S2), wild-type, Notchts1, and mamIL42 embryos were collected for 1 hour, aged for 5.5 hours (embryos were of the ages 5.5 to 6.5 hours of development), shifted to 29°C for 1 hour, and fixed immediately. Because of the 1-hour incubation at 29°C, some embryos were equivalent to 8 hours of development. The GMC1-1a lineage and some unidentified GMCs were also analyzed from this collection of embryos (fig. S4, A to C). To analyze the dynamics of Numb localization in MP2, NB7-1, and NB5-3 (figs. S4, D to R, S5, and S6), wild-type, Notchts1, and mamIL42 embryos were collected for 2 hours, aged for another 3.5 hours (these embryos now were of the ages between 3.5 and 5.5 hours), shifted to restrictive 29°C for 1 hour, and fixed immediately. For the experiments with Hsp70-Nintra, embryos were collected for 20 min at 22°C, thoroughly washed, briefly dried, aged for 5 hours, shifted to 37°C for 20 min, and allowed to develop for another 2 to 5 hours at 22°C before fixation.

Immunohistochemistry and microscopy

For immunolabeling, embryos were washed thoroughly with running water, dechorionated with 50% bleach, rinsed with running water and then with phosphate-buffered saline containing Triton X-100 (Sigma) (0.1%), and fixed with n-heptane (Fisher Scientific) and 37% formaldehyde (Fisher Scientific) mixed in a 1:1 ratio for 2 min (immunofluorescence labeling) or for 6 min (immunohistochemical labeling). Vitelline membranes were removed by a rapid (~20 s) wash with methanol (Fisher Scientific). Embryos were processed immediately using standard protocols (1, 5). The following antibodies and concentrations were used: Eve [rabbit, 1:2000 (37)], Eve [mouse, 1:5, Developmental Studies Hybridoma Bank (DSHB), mAb2B8 and mAb3C10], Numb [rabbit, 1:100 (38)], Numb [guinea pig, 1:50 (39)], Insc [rabbit, 1:50 (6)], Spectrin (mouse, 1:200, DSHB, mAb3A9), GFP (mouse, 1:100, Abcam, mAb9F9.F9), Achaete (mouse, 1:1, DSHB, achaete), and β-galactosidase (rabbit, 1:3000, Invitrogen, A-11132, or mouse, 1:400, DSHB, 40-1a). For confocal microscopy, secondary antibodies conjugated to Cy5 (rabbit, 1:400, Invitrogen, A10523), fluorescein isothiocyanate (mouse, 1:50, Invitrogen, 62-6511), Alexa Fluor 488 (rabbit or mouse, 1:300, Invitrogen, A-11008 or A-11001), or Alexa Fluor 647 (rabbit or mouse, 1:300, Invitrogen, A-21245 or A-21236) were used. For light microscopy, secondary antibodies conjugated to alkaline phosphatase (rabbit, 1:200, Pierce, 31341) or horseradish peroxidase (HRP; rabbit, 1:200, Pierce, 31460) were used. Alkaline phosphatase was detected using 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (Promega, S3771). HRP was detected with diaminobenzidine (Sigma, D4418). For the labeling of Eve in numbdf, insc, and Hsp70-Nintra embryos, overstaining was avoided to prevent masking the sib identity. Fluorescence images were collected on a Bio-Rad Radiance 2002 confocal microscope using Lasersharp software. Bright-field images were collected on a Zeiss microscope. All images were collected and analyzed by an investigator blinded to the experimental treatment.

SUPPLEMENTARY MATERIALS

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Fig. S1. Notch signaling is required early for asymmetric division of GMC-1s.

Fig. S2. Notch signaling is required for the asymmetric localization of Numb.

Fig. S3. Inscuteable functions downstream of Notch to control Numb localization and asymmetric division of GMC-1s.

Fig. S4. Notch signaling is required for the asymmetric localization of Numb in multiple neural precursor cells.

Fig. S5. Notch signaling is not required for the asymmetric localization of Numb in some neural precursor cells.

Fig. S6. Numb is initially distributed in a punctate pattern in precursor cells.

REFERENCES AND NOTES

Acknowledgments: I would like to thank the members of the Bhat laboratory: M. A. Manavalan, Z. Zhu, and M. Krishnamurthy for assistance in staining embryos and line drawings; M. Frasch for the Eve antibody; B. Chia for the Insc antibody; and C. Desplan, C. Doe, Y. N. Jan, and J. Skeath for Numb antibodies. I would also like to thank Development Studies Hybridoma Bank at the University of Iowa for the Achaete and the spectrin antibodies, the Bloomington stock center for fly lines, and B. Yedvobnick for the mam alleles. Special thanks to C. Desplan and C. Doe for their continued support, encouragement, and discussion, and to K. Prehoda, L. Rose, G. Benian, and M. Noll for discussion on asymmetric cytokinesis. Funding: This work was funded by grants from the National Institute of General Medical Sciences (R01GM080538) and the National Institute of Neurological Disorders and Stroke (R01NS04526) to K.M.B. and the generous support from the Department of Neuroscience and Cell Biology, University of Texas Medical Branch. Competing interests: The author declares that he has no competing interests. Data and materials availability: All reagents used in this study are commercially available or available from the indicated sources.
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