A functional analysis of inscuteable and its roles during Drosophila asymmetric cell divisions

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1 Journal of Cell Science 112, (1999) Printed in Great Britain The Company of Biologists Limited 1999 JCS A functional analysis of inscuteable and its roles during Drosophila asymmetric cell divisions Murni Tio, Michael Zavortink, Xiaohang Yang and William Chia* Institute of Molecular and Cell Biology, National University of Singapore Campus, 30 Medical Drive, Singapore *Author for correspondence ( mcbwchia@imcb.nus.edu.sg) Accepted 22 February; published on WWW 22 April 1999 SUMMARY Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localisation and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localisation and segregation of the determinant Numb during ganglion mother cell divisions ensure that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organise both neuroblast and ganglion mother cell asymmetric divisions is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localised to the apical cell cortex and is required both for the basal localisation of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. Here we define the functional domains of Inscuteable. We show that aa appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region, aa , is necessary and sufficient for apical cortical localisation and for mitotic spindle (re)orientation along the apical/basal axis. A larger region aa is necessary and sufficient for asymmetric Numb localisation and segregation; however, correct localisation of Miranda and Prospero requires additional sequences from aa The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localisation (aa ). Our data suggest that apical localisation of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Finally, we show that although inscuteable RNA is normally apically localised, RNA localisation is not required for protein localisation or any aspects of inscuteable function. Key words: Asymmetric cell division, Neuronal cell fate, Cell polarity INTRODUCTION Asymmetric cell divisions appear to be a universally employed strategy for generating diversity in cell fates (reviewed by Horvitz and Herskowitz, 1992; Guo and Kemphues, 1996; Doe, 1996; Jan and Jan, 1998). During Drosophila embryonic development the effects of both extrinsic cues and asymmetrically localised and segregated intrinsic determinants are integrated (reviewed by Campos-Ortega, 1996) to produce the distinct cell types which are organised into the stereotypic patterns seen in the central (Spana and Doe, 1996; Skeath and Doe, 1998) and peripheral nervous systems (CNS and PNS) (Guo et al., 1996; Dye et al., 1998) as well as the somatic musculature (Ruiz-Gomez and Bate, 1997; Carmena et al., 1998). Most Drosophila CNS neurons derive from stem cells called neuroblasts (NBs) (Bossing et al., 1996); NBs undergo repeated divisions, budding off a series of smaller ganglion mother cells (GMCs) from their dorsal/lateral sides; GMC s divide terminally to produce two progeny neuron/glia (Goodman and Doe, 1993). Both NB and GMC divisions are asymmetric but appear to utilise two distinct intrinsic cell fate determinants. In mitotic NBs two potential cell fate determinants, the homeo-domain protein Prospero (Doe et al., 1991; Vaessin et al., 1991; Matsuzaki et al., 1992) and Numb (Uemera et al., 1989), as well as pros RNA are localised as crescents to the basal cell cortex and segregate preferentially to the more dorsal (GMC) progeny (Rhyu et al., 1994; Hirata et al., 1995; Knoblich et al., 1995; Spana and Doe, 1995; Li et al., 1997; Broadus et al., 1998). Whereas Prospero fulfills the criteria of a cell fate determinant and acts in GMCs to promote the expression of GMC specific genes and repress the expression of NB specific genes (Doe et al., 1991; Vaessin et al., 1991), a role for Numb has not been defined in the NB cell division. However, in GMC cell divisions (and MP2, another CNS precursor undergoing terminal cell division, see Spana et al., 1995, 1996) Numb is localised as a basal crescent and acts as a cell fate determinant (Buescher et al., 1998; Skeath and Doe, 1998), possibly by directly interacting with the intracellular region of Notch (N) (Frise et al., 1996) and

2 1542 M. Tio and others thereby inhibiting N signaling, in the progeny neuron which preferentially inherits the asymmetrically localised Numb. In order for asymmetrically localised molecular components to preferentially segregate to just one progeny cell, the orientation of the mitotic spindle has to be coordinated with the site of localisation. A gene inscuteable (insc), allelic to a previously described muscle pattern mutation not enough muscles (Burchard et al., 1995), encoding an apically localised protein has been previously cloned (Kraut and Campos-Ortega, 1996). Apical cortical Insc localisation in NBs occurs prior to the formation of the mitotic spindle and the basal localisation of Numb, Pros and pros RNA (Kraut et al., 1996; Li et al., 1997); the basal localisation of these molecules as well as the orientation of the mitotic spindle along the apical/basal axis in mitotic NB requires insc; in addition, ectopic expression of Insc in ectodermal cells can cause their mitotic spindles to undergo an extra 90 degree rotation, resulting in spindles which are oriented along the apical/basal axis (Kraut et al., 1996). Moreover, unlike NBs, dividing muscle progenitors do not form Insc crescents at a fixed position on the cellular cortex. Yet despite its variable position, the Insc crescent always overlies one of the spindle poles and the Numb cortical crescent always forms on the side of the cell opposite the Insc crescent (Carmena et al., 1998). These observations suggest that insc lies at the top of a hierarchy and may act by providing positional information to coordinate these processes which together comprise the asymmetric cell division. Interestingly, apical Insc crescents are also formed in dividing GMCs and insc function is required for the asymmetric localisation and segregation of Numb; in insc mutants, Numb is distributed to both GMC progeny and (some of) the resultant sibling neurons are unable to resolve distinct fates (Buescher et al., 1998). Recently several molecules have been identified on the basis of their ability to interact with Insc or the localisation domains of Pros and Numb (reviewed by Lu et al., 1998a). These molecules appear to act downstream of insc to mediate the localisation of Pros, Numb and pros RNA and have been referred to as adapter-like molecules (Shen et al., 1998). The localisation of pros RNA requires not only Insc but also Staufen (Li et al., 1997; Broadus et al., 1998; Schuldt et al., 1998), a molecule capable of binding double stranded RNA in vitro (St Johnston et al., 1992) implicated in the transport and anchoring of oskar and bicoid RNAs in the oocyte and early egg (St Johnston et al., 1991; Ferrandon et al., 1994) which also interacts with Insc. Localisation of Pros requires Miranda (Shen et al., 1997, 1998; Ikeshima-Kataoka et al., 1997; Matsuzaki et al., 1998) which appears to interact not only with Insc but also the previously defined localisation domain of Pros (Hirata et al., 1995) as well as Staufen (Schuldt et al., 1998; Shen et al., 1998). Normal localisation of Numb in NBs requires partner of numb (pon) (Lu et al., 1998b), a protein which interacts with the previously defined localisation domain of Numb (Knoblich et al., 1997) and Miranda. The adaptors themselves are localised in an identical way to the molecules they help to localise. The localisation of Miranda and Pon in NBs requires insc; the localisation of Staufen requires not only insc but also miranda. Hence, insc is the most upstream component of asymmetric cell divisions identified to date. insc encodes a 859 residue deduced protein (Kraut and Campos-Ortega, 1996). The Insc sequence must contain information necessary and sufficient for directing: (1) its own apical cortical localisation, (2) the basal cortical localisation of Pros and Numb, (3) the orientation of mitotic spindle along the apical/basal axis, and (4) the resolution of distinct identities of sibling neurons. Here we delineate where the various insc functions (and characteristics) reside with respect to its coding sequence. We demonstrate that the various insc functions have distinct requirements. A 210aa region (aa ) is necessary and sufficient for its own apical cortical localisation and for mitotic spindle (re)orientation along the apical/basal axis. A somewhat larger region aa is necessary and sufficient for Numb localisation and segregation in NBs and cells of mitotic domain9; however, correct localisation of Miranda (and Pros) requires additional sequences from aa The requirement for the resolution of alternative sibling cell fates (RP2/RP2sib) coincides with the region necessary and sufficient for Numb localisation (aa ). aa appear to be sufficient for all aspects of insc function examined. Finally, we demonstrate that although insc RNA is normally localised to the apical cortex of interphase cells (Li et al., 1997; Knirr et al., 1997), this localisation is apparently dispensable for all aspects of insc function. MATERIALS AND METHODS Transposon constructions and germline transformation As the starting construct for making the 3 and interstitial deletions, a full length insc cdna including the 3 -UTR and in which the C terminus was tagged with a MYC epitope was constructed and introduced into pbluescriptks + (insc-myc-utr); the entire insert can be excised with SalI/NotI. Since there is a BclI site in the MYC sequence, deletions from the C terminus were made by utilizing linkers with a BclI site at the 3 -end and sequences that reconstructed the MYC-tag. So, for example, the deletion to the EcoRI site was made by klenow filling the EcoRI site and ligating to a linker 5 - CGAGCAGAAGCT-3 /3 -GCTCGTCTTCGACTAG-5 which linked the blunt ended EcoRI site to the BclI cut site in the MYC-tag of inscmyc-utr. The various C-terminal deletions were terminated at the EcoRI, DraIII, BsrBI, NheI and NarI sites at amino acids 710, 545, 368, 287 and 251, respectively. The internal deletion construct (hsbcli) deleted a fragment between aa All newly created junctions were sequenced and these constructs were subcloned as SalI/NotI fragments into the hs-casper transformation vector. To make a series of 5 deletions, a FLAG tagged full length insc was first constructed. A FLAG oligo followed by HindIII, EcoRV and NdeI sites (to provide the three reading frames) was cloned into pbluescript KS + at the XhoI and NdeI sites (pbs-flag). A N-terminal fragment of Insc was synthesised by PCR using a NdeI containing upstream primer, with a modified sequence to recreate the ATG of Insc, and a SacII containing downstream primer; this fragment was cloned into pbs-flag followed by ligation of the rest of the insc sequence (the SacII fragment cut directly from the insc cdna). N-term deletions were generated by excising fragments from one of the reading frame sites to the three corresponding restriction sites NheI, BsrBI and EcoRI, at amino acids 287, 371 and 710, respectively. The five sufficiency constructs were generated by PCR using an NdeI upstream primer and a NotI downstream primer to produce fragments consisting of aa (hsa1-a5), aa (hsa2-a4), aa (hsa2-a3), aa (hsa3) and aa (hsnls). These fragments were then inserted into pbs-flag. All the FLAG-tagged N-terminal deletion and sufficiency constructs were subcloned as XhoI (blunted)/noti fragments into HpaI/NotI cut hs-casper. Embryo injections used to produce germline transformants were performed essentially as described by Spradling (1986).

3 Inscuteable functional domains 1543 Drosophila genetics For the analyses of Miranda, Pros and Numb protein localization and rescue of the insc22 RP2 phenotype, transgenic flies carrying the different heat shock (hs) constructs which showed good levels of protein induction were introduced into the insc22 background. For second chromosome insertions: hs-transgene, insc22 recombinant chromosomes were generated and balanced over CyO Ftz-LacZ balancer. For third chromosome insertions: viable insertions were used to generate stocks of the genotype yw; insc22/cyo Ftz-LacZ; hstransgene/hs-transgene. A similar strategy was used for X- chromosomal insertions. Embryos homozygous for insc22 and the transgene can be identified by the lack of βgal staining. Heatshock, immunohistochemistry, RNA in situ protocols For the analyses of subcellular localization of Insc, Miranda, Pros, Numb and insc RNA as well as spindle orientation, up to 8 hours (h) old embryos were collected and dechorionated in 50% bleach. After several washes in PBT (PBS+0.01% Triton X-100) embryos were incubated at 37 C for 15 minutes followed by a recovery period of 1 hour in a moist chamber at 25 C prior to processing for immunocytochemistry or RNA in situ. For the analysis of RP2 rescue, embryos were collected in a 2 hour interval, aged to stage10, heatshocked and allowed to develop until stage before processing. For most analyses, embryos were fixed in 4% paraformaldehyde for 12 minutes; for tubulin staining, fixations were carried out in 37% formaldehyde for 3 minutes. After staining, embryos were mounted in either vectashield (from Vector Labs) or DNA mounting medium (Lundell and Hirsh, 1994) and analysed by laser scanning confocal microscopy (MRC 1024). Primary antibodies used were rabbit anti-insc, rabbit anti-miranda (from F. Matsuzaki), rabbit anti-numb (from Y.-N. Jan), mouse anti-eve (from M. Frasch), mouse anti-pros (from C. Q. Doe), rat anti-βtubulin, mouse anti-histones (Chemicon), mouse anti-flag (KODAK) and rabbit anti-βgal (Cappel). Secondary antibodies obtained from Jackson Laboratories were Cy3-conjugated goat anti-rabbit IgG, FITCconjugated goat anti-mouse and FITC-conjugated goat anti-rat. RNA in situ hybridisations were performed as previously described (Li et al., 1997). Analysis of asymmetric protein localization and RP2 rescue For the analysis of Miranda, Numb and Pros localization in the insc22 mutant background, the embryos were double stained with rabbit antiβgal and either rabbit anti-miranda or rabbit anti-numb and only nonβgal stained embryos were analysed. In parallel, the same collection of embryos were double stained with rabbit anti-βgal and rabbit anti- Insc/mabM5 anti-flag to ascertain that the observed non-βgal stained embryos were expressing the heat-induced transgene. As a control for RP2 rescue analysis, insc22 embryos were exposed to hs and treated in the same manner as the other insc22; hs-transgene embryos. In this case, the frequency of RP2 duplication is 49%, average determined from the twenty percent of embryos with the lowest frequency of RP2 duplication. Heat treated insc22 embryos also had a slight increase in the amount of RP2 loss and therefore the 49% is taken from the number of hemisegments with RP2 duplication/total hemisegments excluding those with RP2 loss. The percent RP2 duplications for insc22; hs-transgenes shown in Fig. 1 was calculated in the same manner (i.e. the average of the twenty percent embryos showing the best rescue or lowest percentage of RP2 duplication) and these are compared to that of the heat treated insc22 embryos. RESULTS The Inscuteable coding region The main features of the deduced Insc sequence (Kraut and Campos-Ortega, 1996), denoted in Fig. 1, comprise: a putative WW binding site (PPPPPPY) at aa (see Staub et al., 1996); a putative nuclear localisation sequence, RRGVFFNDAKIERRRYL at aa ; a putative PDZ binding site, QESFV-COOH at the C terminus, aa Fig. 1. Summary diagram of the Inscuteable functional dissection analysis. Schematic representations of the Insc protein and the various constructs and their names are shown on the left. WWBS = WW binding site, A = ankyrin-like repeats and NLS = putative nuclear localisation signal. Underlined are the first and the last amino acids; numbers in brackets correspond to amino acids for the restriction sites. hsinsc is a full length Insc and, like all 5 and 3 deletions, possesses the 3 UTR. All 3 deletion constructs contain MYC epitope (which disrupts the putative PDZ binding site) at the C terminus. All 5 deletions and the five sufficiency constructs, containing only sequences from the region of ankyrin-like repeats, have a FLAG epitope. The five sufficiency constructs do not contain 3 UTRs. Subcellular localisation of the various modified Insc proteins induced from each of the constructs was assessed in both NBs and ectodermal cells: (A represents apical cortical localisation and C represents cytoplasmic localisation). The ability of the various constructs to reorient the mitotic spindle was analysed in mitotic domain11 ( denotes spindle reorientation; // denotes no spindle reorientation. The capacity of the various constructs to correctly localise Miranda (Mir) and Numb (Nb) and resolve RP2/RP2sib fates were all assessed in the insc22 mutant background. + denotes wt localisation; denotes abnormal localisation. % hemisegments with RP2 duplication are shown (see Materials and Methods).

4 1544 M. Tio and others Many PDZ domain containing proteins localise to submembranous sites (e.g. see Ponting et al., 1997); Kraut and Campos-Ortega (1996) pointed out that a sequence motif which is somewhat related to the ankyrin repeats found in many protein is repeated five times in Insc, at residues , , , and Although the similarities are weak, we designate these regions as A1 to A5 for future reference. In all of the constructs and transformants described in the following sections, the mutant proteins can be detected by immunofluorescence with either the anti-insc serum and/or with the anti-flag monoclonal. Furthermore our western analyses using induced embryos derived from several transformant lines, including full length insc, hs5 NheI, hs5 BsrBI, hsa1-a5 and hsa2-a4 indicated the presence of protein bands of the expected size (data not shown). These results suggest that the expected mutant proteins are produced from the various constructs. Apical cortical localisation of the Inscuteable protein In wild-type (wt) embryos, Insc is expressed and apically localised in NBs and the cells of the procephalic mitotic domain9. However, we have previously shown that ectopically expressed Insc will localise to the apical cell cortex of ectodermal/epithelial cells (Kraut et al., 1996; see also Fig. 2A- C). For the purposes of assessing the regions of Insc that are necessary and sufficient for its apical localisation, we have examined both ectodermal/epithelial cells and NBs. The results are summarised in Fig. 1 with representative data shown (Fig. 2). Ectopically expressed proteins from various transgenes containing the N terminus of Insc were detected using an anti- Insc polyclonal serum; however, this serum recognises an N- terminal epitope; for detection of modified versions of Insc lacking the N terminus, an anti-flag monoclonal antibody was used (see Materials and Methods). It appears that hsa2-a3, encompassing aa , is sufficient to specify localisation to the apical cortex. All modified Insc proteins containing this minimal domain will localise apically when mis-expressed in the ectoderm. This region is also sufficient to effect apical localisation in cells of the procephalic region which normally express Insc (not shown). Conversely all N-terminal deletions which remove at least A2, e.g. hs5 BsrBI and hsa3, produce proteins which cannot localise apically when ectopically expressed; similarly, C-terminal deletions which remove A3, e.g. hs3 BsrBI, also cannot localise apically in the ectopic expression paradigm. In all cases where a truncated protein cannot localise to the apical cortex, its localisation is primarily cytoplasmic (Fig. 2D-F). Our results therefore demonstrate that aa of Insc containing A2 and A3 is necessary and sufficient for apical cortical localisation both ectopically in ectodermal/epithelial cells and in cells which normally express and localise Insc. Mitotic spindle orientations Most ectodermal cells do not express Insc and divide with the mitotic spindles oriented parallel to the apical surface of the embryo (see Fig. 3A,A showing mitotic domain11). We have previously shown that when wt Insc protein is ectopically induced in these normally non-expressing cells they will Fig. 2. Deletions into A2 and/or A3 of Insc abolish its ability to associate with the cortex in ectodermal cells, NBs and GMCs. Apical is down unless otherwise specified. Induced Insc expression (red) and DNA (green) are visualised. (A-F) hsinsc (full length Insc), hs3 RI, hs3 DraIII, hs3 BsrBI, hs3 NarI and hsbcli, respectively. Note that ectopically expressed full length Insc (A) and from the C- terminal deletions excluding (B) and including A5 (C) are still apically localised in ectodermal cells (arrowheads). Larger C- terminal deletions removing A3-A5 (D) or A1-A5 (E), and an interstitial deletion removing A1-A3 result in truncated Insc proteins which detach from the apical cortex and localise to the cytoplasm (D-F). Note that cytoplasmically localisation of these truncated Insc proteins (an example shown in E) can be detected in the ectodermal cells (arrowhead), the dividing NBs (vertical arrow) and GMCs (horizontal arrow). reorient their mitotic spindles (and divide) perpendicular to the apical surface. To define the sequences necessary and sufficient for this ectopic reorientation, we stained embryos ectopically expressing various modified versions of the Insc protein with anti-β-tubulin and assessed the effects of the expression of the various modified Insc on mitotic spindle orientation. The results are summarised in Fig. 1 with representative data shown (Fig. 3). There is an absolute correlation between constructs which exhibit apical cortical localisation and their ability to induce ectopic mitotic spindle reorientation along the apical/basal axis (Fig. 1; Fig. 3B,E). Conversely, cells

5 Inscuteable functional domains 1545 the first set of experiments we addressed the question of whether any of the mutant versions of Insc can counteract the effects of wt Insc in the process of mitotic spindle reorientation in the procephalic region (mitotic domain9) where the epithelial cells normally express Insc and normally reorient their mitotic spindles perpendicular to the apical surface. None of the mutant proteins when ectopically expressed in a wt genetic background affected the spindle reorientation normally seen in the Insc-expressing cells, indicating the lack of a dominant negative effect. In the second experiment we asked whether the minimal construct (hsa2-a3, aa ) is capable of restoring the wt spindle orientation in the cells of mitotic domain9 of insc mutant embryos. In insc22 mutant embryos cells of mitotic domain9 and most NBs fail to (re)orient their spindles perpendicular to the apical surface; when hsa2-a3 is ectopically expressed in insc22 embryos, perpendicular orientation of the mitotic spindle is restored in both mitotic domain9 (see Fig. 5R) and NBs (see Fig. 5P,Q). Our results show that the region of Insc from aa , containing A2 and A3, is necessary and sufficient for apical localisation and for mitotic spindle (re)orientation. Fig. 3. Apical cortical localisation of Insc appears to be a prerequisite for the reorientation of mitotic spindles. All panels represent surface views. (A) Control wt embryo exposed to hs and stained with anti-β-tubulin (green) and anti-insc (red), showing that cells of mitotic domain11 (consisting of several sub-domains, arrows), which do not express Insc, orient their spindles parallel to the surface (blowup shown in A ). (B) Ectopic expression of full length Insc (red) in mitotic domain11 can reorient mitotic spindles (green) along the apical/basal axis (arrowheads, one example of perpendicular spindle in the insert); note that apical Insc can be seen at this plane of focus as a red outline of the cells. Two ectopically induced truncated versions of Insc, hs3 NheI (deletion removing A2- A5, C) and hs3 NarI (removing A1-A5, D), fail to reorient mitotic spindles (insert in C shows one example of a parallel oriented mitotic spindle); note the cytoplasmic localisation of these truncated forms of Insc (red). (E) Ectopic expression of hsa2-a3 (aa ) which is apically localised (not shown), can reorient mitotic spindles along the apical/basal axis. (F) hsa3 (aa ) which is cytoplasmically localised (not shown) fails to reorient mitotic spindles. expressing constructs which produce cytoplasmically localised proteins cannot reorient their spindles and divide with their spindles parallel to the surface of the embryo (Fig. 3C,D,F). The region of Insc encompassing A2-A3 defines the minimal region which is necessary and sufficient for ectopic spindle reorientation and for apical, cortical localisation (Figs 1, 3E). We have performed two additional types of experiments. In Localisation of Miranda, Numb and Prospero proteins We next examined which regions of Insc might be involved in localising Pros and Numb. We ectopically expressed various insc constructs in insc22 mutant embryos and performed immunocytochemistry with anti-numb, anti-miranda and anti- Pros (see Materials and Methods) to determine the localisation of these proteins in mitotic NBs of the segmented CNS (and also cells of mitotic domain9 in the procephalic region). Since it has previously been shown that Miranda and Pros colocalise in NBs and that the localisation of Pros requires miranda (Shen et al., 1997, 1998; Ikeshima-Kataoka et al., 1997; Matsuzaki et al., 1998), it is likely that correct Miranda localisation reflects correct Pros localisation. We therefore used anti- Miranda to routinely monitor Pros localisation; however, we have also directly examined Pros (anti-pros) localisation for confirmation (not shown). Our results, summarised in Fig. 1 with representative data shown in Figs 4 and 5, indicate that all Insc constructs which fail to localise apically also cannot direct the basal cortical localisation of Numb (Fig. 5G-I), Miranda (Fig. 4J-L), and Pros (not shown). However, although apical localisation (of Insc) appears to be a prerequisite for the basal cortical localisation of Pros and Numb, the Insc sequences necessary and sufficient for its apical cortical localisation and for the apical/basal orientation of mitotic spindle (hsa2-a3) is not sufficient for directing the basal cortical localisation of Miranda (Pros) or Numb (Fig. 5P-R). In the cases where Miranda (Pros) and Numb localisation is not basal, these proteins are distributed either throughout the cortex or form broad cortical crescents at inappropriate (e.g. basolateral or more rarely apicolateral) positions on the cortex (Fig. 4J-L,P-R and Fig. 5G-I,P-R), similar to the phenotype seen in insc22 mutant NBs (Fig. 4D-F). Deletions from the N terminus indicate that the critical region is between aa287 and aa371; sequences up to and including aa287 (encompassing A1) are dispensable for the basal cortical localisation of Miranda and Numb in mitotic NBs and procephalic ectodermal cells of mitotic domain9; however, deletion extending to aa371 (removing A1-A2) fails to restore

6 1546 M. Tio and others Fig. 4. The ankyrin-like domains of Insc are necessary and sufficient for wt Miranda localization. Apical/ventral is down. Miranda protein (red) and DNA (green) are visualised in dividing NBs. In wt embryos (A-C), Miranda is localised to the basal cortex of NBs and eventually segregated into the more dorsal GMCs (C). In insc22 mutant embryos (D-F), Miranda is either localised throughout the NB cortex (D) or forms broad crescents along the NB cortex at inappropriate positions (E-F). Note that in D and F, the planes of division are parallel rather than perpendicular to the apical surface of the embryos, a phenotype associated with the insc22 homozygous embryos. Overexpression of a C-terminal deletion which stops short of the ankyrin-like repeats (hs3 RI) in the insc22 background is able to restore wt Miranda localisation (G-I); while the expression of a N-terminal protein (hs3 NarI missing A1-A5) can no longer do so (J-L). hsa1-a5 (aa ) is sufficient for restoring wt Miranda localisation (M-O); however, hsa2-a4 (aa ) which is sufficient for restoring wt Numb localisation (see Fig. 5M-O) cannot restore wt Miranda localisation (P-R). basal localisation of Numb and Pros when ectopically expressed in insc22 mutant NBs or domain9 cells. Deletions from the C terminus indicate that the region from aa , encompassing A5, is required for Miranda but dispensable for Numb basal cortical localisation. When ectopically expressed in insc22 mutant NBs, C-terminal deletion up to aa545 can restore basal Numb localisation but shows abnormal cortical/basolateral Miranda localisation. However, further removal of sequences to aa368 (removing A3-A5) results in the inability to localise both Miranda and Numb. Hence, at least in NBs of the segmented CNS and the procephalic cells of mitotic domain9, the requirements for Numb and Pros localisations are not equivalent. Consistent with the deletion analyses, the minimal construct hsa2-a4, encompassing aa , is sufficient to effect basal cortical Numb localisation (Fig. 5M-O) but is insufficient for correctly localising Miranda (and Pros) (Fig. 4P-R). However, the region from aa (including A1-A5) can effect correct localisation of both Numb (Fig. 5J-L) and Miranda (Pros) (Fig. 4M-O); this region lies within the previously defined Insc segment (aa ) which is sufficient for interaction with Miranda in vitro (Shen et al., 1998). Our results indicate that the regions of Insc necessary for localising the determinants Miranda (Pros) (aa ) and Numb (aa ) are similar but not equivalent; moreover they emcompass the region necessary and sufficient for directing apical cortical localisation of Insc and spindle reorientation along the apical/basal axis. Resolution of alternative sibling cell fates We have previously shown that the cell fate changes associated with insc loss of function appear to be the inability to resolve distinct sibling cell fates (Buescher et al., 1998; Carmena et al., 1998). In the CNS of insc mutants, we have shown that the great majority of RP2sib adopts the fate of its sibling, the RP2

7 Inscuteable functional domains 1547 Fig. 5. The requirements for wt Numb and Miranda localisation are similar but not identical. Apical is down and DNA is visualised in green. In dividing wt NBs (from metaphase onwards, shown in A-C), Numb (red) is localised to the basal cortex and segregates exclusively to the more basal daughters. Overexpression of a C-terminal deleted Insc (hs3 DraIII) which removes sequences including A5 can restore wt Numb localisation in insc22 mutant background (D-F). Overexpression of a C-terminal truncation (hs3 BsrBI) which removes A3-A5 no longer restore wt Numb localisation (G-I). hsa1-a5 and hsa2-a4 are both sufficient for restoring wt Numb localisation in insc22 mutants. Note that hsa2-a4 is not sufficient for restoring wt Miranda localisation (see Fig. 4P-R). The smaller hsa2-a3, which can function to reorient mitotic spindles (see Fig. 3E), is no longer sufficient for restoring wt Numb localisation (P-R); however, unlike hs3 BsrBI which cannot set up the correct plane of division (H-I), hsa2-a3 can restore wt plane of division in both NBs (P and Q) and the PNR (R). neuron, causing a duplication of the RP2 neurons at the expense of RP2sib in most hemisegments (Fig. 6B). By ectopically expressing various insc constructs in the insc22 mutant background, we can assess which regions of Insc are necessary and sufficient for the restoration of the resolution of distinct RP2 and RP2sib cell fates. Representative results are shown in Fig. 6 and summarised in Fig. 1. In the experimental paradigm we used (see Materials and Methods), the control homozygous insc22 embryos (subjected to the same treatment and analyses as experimental animals) will exhibit dupicated RP2 neurons in 49% of their hemisegments (Fig. 6C). Ectopically induced full length Insc expression shows significant rescue and reduces the frequency of RP2 duplication to 3% of the hemisegments (Fig. 6D). Similarly C-terminal deletions up to aa545 (including A5) retain the capacity to rescue, reducing RP2 duplication to 6% (for hs3 RI) and 10% (for hs3 DraIII) of the hemisegments. However, deletions to aa368 (removing A3-A5; 28% RP2 duplication), aa287 (removing A2-A5; 54% RP2 duplication) and aa251 (removing A1-A5; 39% RP2 duplication) drastically reduce the capacity to resolve distinct sibling cell fates. N- terminal deletion up to aa287 (removing A1) retains the ability to resolve distinct RP2/RP2sib cell fates, reducing RP2 duplication to 3% of the hemisegments (Fig. 6E). However, deletion further into the coding region, to aa371 (removing A1- A2; 48% RP2 duplication), compromises the capacity to resolve distinct RP2/RP2sib fates (Fig. 6F). The deletion data define the region required for the resolution of RP2/RP2sib fates to aa (encompassing A2-A4). Consistent with this, the region from aa (including A1-A5) is sufficient to rescue (to a large extent) the RP2 phenotype (reducing RP2 duplication to 17%). However,

8 1548 M. Tio and others Fig. 6. The capacity to resolve distinct sibling neuronal fates coincide with the ability to localise Numb. Ventral views of stage14/15 embryos stained with Anti-Eve. wt embryos form one RP2 neuron per hemisegment (A, arrow). insc22 homozygotes duplicate RP2 neurons in >90% of hemisegments (B, double arrows); however, a 15 minute heatshock will reduce the frequency of RP2 duplication in insc22 homozygotes such that the worse affected embryos will exhibit RP2 duplication in ~50% of hemisegments (C). Induction of full length Insc (D) and hs5 NheI (E) proteins in insc22 mutant background yield near wt RP2 patterns. An induced insc22;hs5 BsrBI embryo is shown (F) as a representative example of a construct not capable of rescuing the RP2 phenotype. Induced insc22;hsa2-a3 embryos illustrating the gross morphological defects late in embryogenesis, including high frequency of RP2 loss (arrowheads in G), occasional reduction in/loss of EL clusters (arrowheads in H), a general spatial disorder of the whole CNS (H) and defects in gut development (not shown), which are seen in all of the smaller sufficiency constructs (hsa2-a4, hsa2- A3, hsa3 and hsnls). constructs which cannot localise Numb also do not effectively rescue the RP2 duplication defect (see Fig. 1). ectopic expression of hsa2-a4, which is sufficient for directing the basal localisation of Numb in insc22 mutant NBs, causes gross morphological defects later during embryonic development making it difficult to assess the status of the RP2/RP2sib neurons (Fig. G,H). Nevertheless, there exists an absolute correlation between the ability to resolve RP2/RP2sib fates and the ability to localise Numb. All constructs which can localise Numb can rescue the RP2 phenotype; conversely, all Apical localisation of insc RNA is dispensable to its function We and others have previously reported that insc RNA is, itself, localised apically to the interphase NB cell cortex (Li et al., 1997; Knirr et al., 1997). Since Insc protein can localise apically when ectopically expressed in ectodermal/epithelial cells, we wondered whether ectopically expressed insc RNA might also similarly localise. As can be seen hs induced RNA from the construct hs3 RI (which contains the insc 3 -UTR, see Materials and Methods) localises apically in interphase ectodermal/epithelial cells (Fig. 7A-C); similar results were obtained for RNA derived from hs5 NheI, again possessing the 3 -UTR (data not shown). Since the great majority of known RNA localisation signals map to the 3 -UTR of transcripts, we looked at the localisation of RNA derived from hsa1-a5 (Fig. 7D-F) and hsa2-a4 (data not shown), both of which lack the insc 3 -UTR. Both these RNAs, when ectopically expressed, are localised throughout the cytoplasm of interphase ectodermal-epithelial cells (Fig. 7D-F). Interestingly, despite the cytoplasmic localisation of its RNA, the protein encoded by hsa1-a5 localises to the apical cortex and can restore all Fig. 7. Localisation of heatshock induced RNA expressed from insc transgenes in epithelial cells. Stage10 embryo expressing the transgene hs3 RI (A-C), which contains the intact 3 -UTR of insc. RNA induced from hs3 RI (red) is apically localised in epithelial cells during interphase (arrowhead in A,C); DNA staining is shown (green). Stage10 embryo expressing hsa1-a5 (D- F), which does not contain the insc 3 -UTR. RNA induced from the transgene (red, arrowhead in D,F) is cytoplasmic during interphase. Superimposed images of RNA signal and DNA staining are shown in C and F. White dots indicate the boundary of the epithelial cells. Apical is down.

9 Inscuteable functional domains 1549 aspects of insc function examined (Fig. 1). While these data do not unequivocably place the RNA localisation signal to the insc 3 -UTR, they do suggest that the apical localisation of insc RNA is not essential for apical localisation of Insc protein or for any aspects of its function (see Discussion). DISCUSSION Our results demonstrate that the central region of Insc (aa ), containing A1-A5, is sufficient for all aspects of insc function tested. These include the ability to localise to the apical cell cortex, orient mitotic spindles in NBs and cells of mitotic domain9 along the apical/basal axis, localise Miranda (and Pros) and Numb to the basal cell cortex during mitosis and to resolve distinct sibling cell fates (for RP2/RP2sib). However, there are differences in the requirement for each of these functions. For apical localisation (of Insc) and for mitotic spindle orientation along the apical basal axis, the region from aa (containing A2-A3) is necessary and sufficient, however, this region is incapable of directing the correct localisation of Miranda, Pros or Numb. A larger segment of Insc, from aa (containing A2-A4), is necessary and sufficent to localise Numb but not Miranda and Pros; to correctly localise Miranda and Pros, additional C-terminal sequences from aa (encompassing A5) appear to be required. For the resolution of distinct sibling cell fates, only sequences from aa (A2-A4) are required. It is interesting to note that the key functional region of Insc defined by our analyses lie within the segment (aa ) which has previously been shown to be sufficient for interaction with Miranda in vitro (Shen et al., 1998). Finally, we have shown that the apical localisation of insc RNA, is apparently not required for any aspects of its function. Apical protein localisation is a prerequisite for all aspects of inscuteable function All of the modified Insc proteins either localise correctly to the apical cortex or localise abnormally to the cytoplasm. In no case do we see cortical localisation without the formation of apical crescents, suggesting that the process of targeting Insc to the cortex and the process of forming the apical crescents may be linked. All versions of Insc which can localise apically can also direct the (re)orientation of mitotic spindles along the apical/basal axis in NBs and epithelial/ectodermal cells and all versions of Insc which show cytoplasmic localisation cannot; therefore apical localisation of Insc (within the resolution of our experiments) appears to be necessary and sufficient for spindle (re)orientation. However, the minimal sequence sufficient for apical localisation cannot effect the correct localisation of Pros and Numb, neither is it sufficient to effect the resolution of alternative sibling cell fates for RP2/RP2sib; additional sequences are necessary to facilitate these aspects of insc function. Nevertheless apical localisation appears to be a requirement for directing correct Numb and Pros localisation and resolving sibling cell fate. That its own apical cortical localisation seems to be a prerequisite for all functions mediated by insc is consistent with previously proposed ideas that Insc might act as a centrosomal cortical attachment site, and that it may be providing positional information for organising asymmetric cell divisions. Our results also demonstrate that the putative WW and PDZ binding sites are apparantly not essential for insc function, at least in our experimental paradigm. Removal (or modification) of either or both of these sites does not compromise insc function. Resolution of distinct neuronal sibling cell fates requires the ability to correctly localise Numb but not Miranda The asymmetric localisation of Numb in GMCs and the phenotype of numb loss and gain of function in the CNS all suggest that Numb acts as the intrinsic cell fate determinant for the GMC cell division and acts to effect distinct cell fates for RP2/RP2sib. Therefore it is not surprising to see an absolute correlation between the capacity to asymmetrically localise and segregate Numb and the ability to specify distinct sibling neuronal cell fates. However, it is interesting to note that the ability to localise and segregate Miranda and Pros is apparently not required for specifying distinct RP2/RP2sib cell fates; e.g. hs3 DraIII, which cannot restore normal Miranda and Pros localisation (but can effect wt Numb localisation) can effect distinct RP2/RP2sib fates. Therefore, the ability to localise Miranda and Pros is apparently not necessary for the specification of distinct sibling neuronal cell fates. Similarly in insc22 mutant embryos, the primary cell fate phenotype is a RP2sib>RP2 transformation resulting in the duplication of RP2 and not the mis-specification of GMC4-2a and loss of RP2 and RP2sib, despite the fact that Miranda is not correctly localised. These observations are somewhat surprising in view of the fact that miranda loss of function mutants exhibit a loss of RP2 phenotype because the GMC4-2a that gives rise to RP2/RP2sib is mis-specified due to the failure to asymmetrically localise and segregate Pros. So why aren t GMC4-2a mis-specified and RP2 neurons lost in strains carrying mutant/modified insc which cannot localise Miranda correctly? An obvious difference between phenotypes of miranda loss of function and insc22 mutants is that in the miranda mutants, Pros localisation is no longer cortical but rather cytoplamic; whereas with insc22 mutants Miranda and Pros remain cortical, either throughout the cortex or as broad cortical crescents which are localised to inappropriate positions with respect to the apical basal axis. It has been previously shown that pros RNA is not synthesised de novo in GMCs (Broadus et al., 1998). Therefore the specification of GMC4-2a will depend critically on the stability of the Pros it inherits from the neuroblast. Our model/explanation for the anomalus phenotype of modified insc transgenes and insc mutants is that the function of Pros in the GMC, perhaps due to its stability, depends on cortical localisation in the NB. In insc transgenes and mutants which cannot localise Miranda and Pros as a basal crescent, both proteins are nevertheless localised either throughout the cortex or as broad inappropriately positioned cortical crescents; therefore, in the great majority of cases, a sufficient amount of functional (stable) Pros segregates to GMC4-2a to correctly specify its fate despite the mislocalisation of Miranda and Pros in the NB. However, when GMC4-2a divides, Numb must be asymmetrically segregated to only one of the two postmitotic neurons to effect distinct sibling cell fates. In insc22 and modified forms of insc which cannot localise Numb in GMCs (Buescher et al., 1998), both sibling neurons inherit Numb and adopt RP2 cell fates. The situation is different for miranda loss of function because Pros cannot localise to the

10 1550 M. Tio and others NB cell cortex; consequently insufficient functional (stable) Pros is inherited by the progeny GMC4-2a resulting in its misspecification and the failure to produce the appropriate progeny neurons. Apical localisation of insc RNA is not necessary for insc function Our results are consistent with the notion that a sequence in the 3 -UTR of insc can direct the apical localisation of insc RNA. It is interesting to note that epithelial cells which normally do not express insc nevertheless have the capacity to apically localise ectopically expressed insc RNA, suggesting the presence of a similar cellular machinery for RNA localisation in interphase ectodermal/epithelial cells and NBs. Nevertheless, insc transgenes lacking 3 -UTR sequences encoding RNAs which distribute throughout the cytoplasm of interphase cells can produce proteins that will localise to the apical cortex, and cause ectopic reorientation of mitotic spindle, indicating that RNA localisation is not a prerequisite for protein localisation or spindle reorientation along the apical/basal axis; moreover, expression of transgenes (e.g. hsa1-a5) lacking 3 -UTR sequences in insc22 embryos can rescue all aspects of insc function. These results suggest that in the experimental paradigm employed in this study, apical RNA localisation is not essential for insc function. However, it has been previously shown that the basal localisation of pros RNA in mitotic NBs is apparently functionally dispensable (Li et al., 1997) except under circumstances when the level of pros function is close to being limiting (Broadus et al., 1998). 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