Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1

Developmental Biology 290 (2006) 139 – 151 www.elsevier.com/locate/ydbio

Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1 Anne Lanjuin a,1, Julia Claggett b, Mayumi Shibuya a, Craig P. Hunter b, Piali Sengupta a,* b

a Department of Biology, Brandeis University, Waltham, MA 02454, USA Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA

Received for publication 5 August 2005, revised 8 November 2005, accepted 14 November 2005 Available online 22 December 2005

Abstract Members of the HES subfamily of bHLH proteins play crucial roles in neural patterning via repression of neurogenesis. In C. elegans, loss-offunction mutations in ref-1, a distant nematode-specific member of this subfamily, were previously shown to cause ectopic neurogenesis from postembryonic lineages. However, while the vast majority of the nervous system in C. elegans is generated embryonically, the role of REF-1 in regulating these neural lineage decisions is unknown. Here, we show that mutations in ref-1 result in the generation of multiple ectopic neuron types derived from an embryonic neuroblast. In wild-type animals, neurons derived from this sublineage are present in a left/right symmetrical manner. However, in ref-1 mutants, while the ectopically generated neurons exhibit gene expression profiles characteristic of neurons on the left, they are present only on the right side. REF-1 functions in a Notch-independent manner to regulate this ectopic lineage decision. We also demonstrate that loss of REF-1 function results in defective differentiation of an embryonically generated serotonergic neuron type. These results indicate that REF-1 functions in both Notch-dependent and independent pathways to regulate multiple developmental decisions in different neuronal sublineages. D 2005 Elsevier Inc. All rights reserved. Keywords: bHLH; ref-1; Hes; C. elegans; Asymmetry

Introduction Basic helix– loop – helix (bHLH) proteins act in a lineageand cell type-specific manner to regulate neurogenesis and neuronal cell fate specification (reviewed in Bertrand et al., 2002; Dambly-Chaudiere and Vervoort, 1998; Hatakeyama and Kageyama, 2004; Lee, 1997; Ross et al., 2003). Proneural bHLH proteins such as members of the Achaete-Scute and Atonal subfamilies promote the generation of neuronal fates, whereas members of the HES subfamily of bHLH proteins inhibit neurogenesis (Fisher and Caudy, 1998; Garcia-Bellido, 1979; Goulding et al., 2000; Guillemot et al., 1993; Jarman et al., 1993; Knust et al., 1987; Orenic et al., 1993; Skeath and Carroll, 1991; Zhao and Emmons, 1995). The roles of different

* Corresponding author. E-mail address: [email protected] (P. Sengupta). 1 Present address: Department of Molecular and Cellular Biology, Harvard, University, Cambridge, MA 02138, USA. 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.11.018

members of this large family of proteins in different lineages and cell types are not fully understood. Hes bHLH genes are upregulated upon Notch signaling between initially developmentally equipotential cells (Bailey and Posakony, 1995; Jarriault et al., 1995; Lecourtois and Schweisguth, 1995). HES proteins antagonize proneural bHLH protein function by either directly repressing proneural gene expression, and/or via physical interaction with proneural proteins, thereby promoting adoption of a non-neuronal fate (Alifragis et al., 1997; Chen et al., 1997; Fisher and Caudy, 1998; Giagtzoglou et al., 2003; Ishibashi et al., 1995; Orenic et al., 1993; Paroush et al., 1994; Skeath and Carroll, 1991; Van Doren et al., 1994). Thus, loss-of-function mutations in Hes genes result in the generation of ectopic neuronal cells (Ishibashi et al., 1995; Nakamura et al., 2000; Wrischnik and Kenyon, 1997), indicating that precise regulation of Hes gene expression and function is critical for patterning the nervous system. The C. elegans genome is predicted to encode 39 bHLH domain proteins (Ledent et al., 2002). Analyses of the

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functions of a subset of these genes indicate that similar to observations in other organisms, individual bHLH proteins are required for correct neurogenesis and neuronal specification in specific cell types (Frank et al., 2003; Hallam et al., 2000; Karp and Greenwald, 2004; Krause et al., 1997; Portman and Emmons, 2000; Wrischnik and Kenyon, 1997; Zhao and Emmons, 1995). For example, mutations in the lin-32 Atonal homolog result in loss of subsets of sensory neuron types (Portman and Emmons, 2000; Zhao and Emmons, 1995), whereas mutations in the lin-22 Hes homolog result in the generation of ectopic neuroblasts (Wrischnik and Kenyon, 1997). However, it is unclear whether these genes function in only the lineages examined, or whether they also play roles in the development of additional neuron types. In addition, the functions of a majority of bHLH proteins in C. elegans are unknown. Six Notch interactions have been shown to be critical in patterning the C. elegans embryo. Two of these interactions occur in the E blastomere lineage and are required to create a left/right (L/R) asymmetric twist in the intestine (Hermann et al., 2000). The remaining four interactions involve daughters of the AB blastomere which gives rise to the majority of neuron types in C. elegans (Hutter and Schnabel, 1994; Hutter and Schnabel, 1995; Mango et al., 1994; Mello et al., 1994; Moskowitz and Rothman, 1996; Moskowitz et al., 1994; Priess et al., 1987; Sulston et al., 1983). These embryonic interactions are essential to create L/R asymmetry in the AB lineage, such that L/R pairs of initially equivalent blastomeres adopt distinct lineage patterns and cell fates. Later in development, L/R symmetry is re-imposed through extensive cell rearrangement (Schnabel et al., 1997; Sulston, 1983; Sulston et al., 1983). As a consequence, many bilaterally symmetric neurons arise from AB-derived neuroblasts that are not initially L/R symmetric. A subfamily of HES-related bHLH proteins have been recently shown to be targets of Notch signaling in the embryo (Neves and Priess, 2005). This nematode-specific subfamily consists of six genes, each of which is predicted to encode proteins containing two HES-related bHLH domains (Alper and Kenyon, 2001; Neves and Priess, 2005; Ross et al., 2005). ref-1, a member of this family, was initially identified on the basis of defects in posterior hypodermal cell fusions, and the presence of ectopic neuroblasts generated from fate conversion of the V6 epidermal cell (Alper and Kenyon, 2001). REF-1 has also been shown to antagonize the proneural LIN-32 bHLH protein in the formation of male sensory rays (Ross et al., 2005; Zhao and Emmons, 1995). Although REF-1 function in these postembryonic lineages may be Notch-independent, expression of members of this gene family was shown to be upregulated in response to Notch signaling in both the AB and E lineages in the embryo (Neves and Priess, 2005). In the E lineage, REF-1 functions in a Notch-dependent manner to generate the L/R asymmetrical intestinal twist (Neves and Priess, 2005), and two AB-derived hypodermal cells were shown to be absent or mispositioned in ref-1 mutant embryos (Alper and Kenyon, 2001; Neves and Priess, 2005). The role of REF-1 in the generation and specification of embryonically generated neurons has not been explored.

Here, we describe the role of ref-1 in the generation and differentiation of neurons derived from the AB blastomere during embryogenesis. We show that multiple neuron types are ectopically generated in ref-1 mutants consistent with the ectopic generation of an AB-derived neuroblast. Intriguingly, ectopic cells are observed only on the right side of animals, and these cells exhibit gene expression patterns characteristic of their counterparts on the left. In addition, we observe developmental defects in a serotonergic neuron type. Taken together, these results implicate ref-1 in regulating distinct developmental decisions in different neuronal sublineages. Materials and methods Nematode strains Worms were grown using standard protocols (Brenner, 1974). Integrated marker strains used in this work were the following: kyIs104 (str-10gfp), oyIs44 (odr-10rfp), oyIs51 (srh-1420rfp), yzIs71 (tph-10gfp), ynIs3 (flp80gfp), oyIs18 (gcy-80gfp), otIs3 (gcy-70gfp), otIs131 (gcy-70rfp), ntIs1 (gcy-50gfp), oyIs14 (sra-60gfp), kyIs140 (str-20gfp), ynIs37 (flp-130gfp), otIs38 (ser-20gfp) (Chang et al., 2003; Kim and Li, 2004; L’Etoile and Bargmann, 2000; Li et al., 1999; Peckol et al., 1999; Sze et al., 2000; Troemel et al., 1995, 1997, 1999; Tsalik et al., 2003; Yu et al., 1997).

Isolation of ref-1(oy40) A strain carrying stably integrated str-10gfp transgenes was mutagenized using EMS. The progeny of ¨8000 mutagenized F1 animals were examined using a dissection microscope equipped with epifluorescence, and oy40 was identified on the basis of ectopic str-10gfp expression. oy40 was mapped with respect to genetic markers and deficiencies, and rescue of the mutant phenotype was obtained with the cosmid R05H5 containing the predicted ref-1 gene (T01E8.2). The mu220 and ok288 alleles were obtained from the Caenorhabditis Genetics Stock Center. Strains were outcrossed at least two times prior to further analyses. The presence of ref-1 alleles in marked strains was confirmed by the size or sequence of ref-1 amplicons generated from genomic DNA.

Molecular biology methods The ref-1 transcript was obtained by amplification from cDNA generated from oligo-d(T)-primed wild-type mRNA. The transcript includes 365 bp of ref-1 3V UTR sequences and contains a single PCR-generated T to A transition resulting in a F93Y conservative substitution. The F93 residue lies in a poorly conserved linker region between the two bHLH domains of REF-1, and the F93Y missense mutation does not appear to affect examined REF-1 functions. The ref-1 minigene was constructed by fusing 2.0 kb ref-1 promoter sequences amplified from genomic DNA to the ref-1 transcript in a C. elegans expression vector (gift from A. Fire). The ref-1 [RAGGCAA] minigene was constructed by replacing the ref-1 promoter in the wild-type ref-1 minigene construct with a ref-1 promoter in which eight LAG-1/Su(H) binding sites were mutated (Neves and Priess, 2005). The functional gfp-tagged ref-1 fusion gene was constructed by fusing ref-1 genomic sequences, including 2.0 kb upstream sequences, in frame to gfp coding sequences prior to the STOP codon.

RNAi Bacterial strains carrying the die-1(RNAi) or the control vector clones were grown overnight, diluted, and expression was induced by treatment with IPTG for 5 h at 37-C. Cultures were plated in duplicate onto worm growth plates and allowed to dry overnight. L4 larvae of wild-type or a ref-1(ok288) strain carrying stably integrated gcy-50gfp transgenes were placed on these plates and grown overnight at 20-C. Adult animals were removed 24h later and placed onto fresh plates containing the die-1(RNAi) expressing or the control bacterial

A. Lanjuin et al. / Developmental Biology 290 (2006) 139 – 151 strains and allowed to lay eggs. Adult animals were examined after 3 days at 20-C.

Temperature-shift experiments Animals were grown at either 20-C or 25-C for one to two generations. Synchronized L1 larval populations were obtained by treating gravid hermaphrodites with sodium hypochlorite and allowing the embryos to hatch overnight at the same temperature in the absence of food. Larvae were then placed on food and moved to the converse temperature or maintained at the same temperature until adulthood. Adult animals were examined for the pattern of str-10gfp expression.

Embryonic expression pattern analysis 4-cell embryos from a strain carrying integrated copies of the functional gfp-tagged ref-1 fusion gene were collected from cut mothers using a mouth pipet. The embryos were mounted on agarose pads and viewed using an Olympus IX70 microscope. Images were acquired using DeltaVision Spectris software. Nomarski optical sections along the full z-axis (every 0.9 – 1.0 Am) were gathered at every minute to determine the identities and invariant positions of all cells up to the 28-cell stage, and of the cells in the ABara(a/p) lineages through the next two cell divisions. Fluorescent z-series were likewise gathered after each AB-lineage cell-division. 80 min and 110 min embryos (grown at 21 – 22-C) were used for the final images in Fig. 6. Fluorescent optical sections along the full z-axis were gathered at exposure times of 0.5 – 0.7 s. Fig. 6D was generated using Quick Projection, which displays the maximum intensity voxels for all sections.

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allele from the C. elegans Gene Knockout Consortium. Genomic sequences from the second to the last predicted exons are deleted in ok288, including sequences encoding part of the first and the entire second bHLH domains (Fig. 1B). Thus, ok288 is likely a null allele. We further characterized the str-10gfp expression defect in all three ref-1 mutants. We observed ectopic str-10gfp expression in all three ref-1 mutant strains with the penetrance of the defect being highest in ok288 and lowest in mu220 animals (Table 1). ref-1(oy40) is temperaturesensitive for this defect (Table 1). Ectopic expression of str10gfp was first observed at approximately the same developmental stage as wild-type expression. Ectopic expression of this marker likely corresponds to the presence of additional AWB-like neurons, since we also observed ectopic expression of another AWB marker, odr-10rfp (L’Etoile and Bargmann, 2000) (Table 1), and the sensory endings of the extra neurons exhibited forked structures characteristic of AWB neurons (data not shown). Intriguingly, we noted that although in wild-type animals, the AWB neuron subtype consists of a L/R symmetrical pair of neurons, ectopic cells were present only on the right side of the animal in all three ref-1 alleles. This phenotype was rescued by a genomic fragment containing ref-1 coding sequences and 2 kb of 5V promoter sequences, as well as a ref-1 minigene (Table 1).

Results Ectopic AWB olfactory neurons are generated in ref-1 mutants

Markers for an entire neuronal sublineage are ectopically expressed on the right side of ref-1 mutants

oy40 animals were isolated in a screen for mutants exhibiting altered expression of the AWB olfactory neuronspecific marker str-10gfp (Fig. 1A) (Troemel et al., 1997). Mapping, complementation, and sequence analyses showed that oy40 was allelic to the previously described gene ref-1. The previously described ref-1(mu220) allele is a point mutation resulting in an R to Q substitution in the first basic domain (Fig. 1B) (Alper and Kenyon, 2001). ref-1(oy40) is also a point mutation resulting in a G82R substitution after the first bHLH domain (Fig. 1B). We obtained the ref-1(ok288)

To further characterize the defects in ref-1 mutants, we analyzed the expression of additional cell-specific markers. The AWB olfactory and ADF chemosensory neuron pairs arise from a terminal cell division of their shared ABpraaappa precursor on the right, and the ABalpppppa precursor on the left (Fig. 2A) (Sulston et al., 1983). Markers for the ADF neurons such as srh-1420rfp (Peckol et al., 1999) and tph10gfp (Sze et al., 2000) were also ectopically expressed in ref-1 mutants (Fig. 2B and Table 2). Similar to str-10gfp expression, ectopic expression of these markers was observed

Fig. 1. str-10gfp is expressed ectopically in ref-1 mutants. (A) Expression of str-10gfp on the right side of wild-type and ref-1(oy40) animals. Arrows point to cell bodies of expressing neurons. Lateral view; anterior at left. Scale bar = 10 Am. (B) Molecular nature of ref-1 mutations. Sequences encoding bHLH domains are shaded. Dashed bracket indicates the extent of the deletion in ok288.

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Table 1 AWB neurons are generated ectopically on the right side in ref-1 mutants Straina

-C

% of animals expressing in AWB neurons L

str-1?gfp str-1?gfp ref-1(oy40); str-1?gfp ref-1(oy40); str-1?gfp ref-1(mu220); str-1?gfp ref-1(ok288); str-1?gfp ref-1(ok288); str-1?gfp ref-1(oy40); odr-1?rfp b ref-1(ok288); str-1?gfp; Ex[ref-1 genomic]c ref-1(ok288); str-1?gfp; Ex[gfp-tagged ref-1]d ref-1(ok288); str-1?gfp; Ex[ref-1 minigene]e ref-1(ok288); str-1?gfp; Ex[ref-1 [RAGGCAA] minigene]f lag-1(q385); str-1?gfp g

R

L

R

. .

. . .

20 25 20 25 25 20 25 25 20

100 100 100 71 91 23 30 75 100

0 0 0 29 9 77 70 25 0

25

98

2

25

99

1

20

98

2

20

94

6

n = 40 – 100 for each. Adult animals grown at the indicated temperatures were examined, except as indicated. a The expression of stably integrated str-10gfp and odr-10rfp transgenes was examined. For strains carrying extrachromosomal arrays, transgenic animals from at least two independent lines were examined. pRF4 was used as the coinjection marker. b odr-10rfp drives expression in both the AWC and AWB neurons (L’Etoile and Bargmann, 2000). Neurons ectopically expressing odr-10rfp also frequently filled with DiI, a characteristic of AWB neurons. However, a subset of odr-10rfp-expressing ectopic neurons may represent AWC neurons. c ref-1 genomic refers to ref-1 coding sequences including 2 kb of upstream and 352 bp of downstream non-coding sequences. d gfp-tagged ref-1 refers to a ref-1 genomic fragment which includes gfp coding sequences inserted in frame into the last exon prior to the STOP codon. See Materials and methods for details. e ref-1 minigene refers to 2 kb of ref-1 promoter sequences driving expression of a ref-1 cDNA. f ref-1 [RAGGCAA] minigene refers to a ref-1 minigene in which eight predicted LAG-1 binding sites in the promoter have been mutated (Neves and Priess, 2005). g lag-1(q385) homozygotes arrest as L1 larvae (Lambie and Kimble, 1991). Dead or dying L1 progeny expressing str-10gfp from lag-1(q385)/+; str10gfp/+ mothers were examined. lag-1(q385) homozygotes were further confirmed by the absence of a rectum/anus and presence of a protrusion at the anal area (Lambie and Kimble, 1991).

only on the right side. To determine whether the generation of ectopic AWB and ADF cells was correlated, we examined transgenic ref-1(oy40) animals expressing both str-10gfp and srh-1420rfp. The ref-1(oy40) allele was selected for this analysis as the ectopic gene expression phenotype is incompletely penetrant in this mutant background. 30% of ref-1(oy40) animals express str-10gfp or srh-1420rfp ectopically, thus, if the cell fate transformations are independent, we would expect that only ¨30% of animals expressing one marker ectopically would coexpress the second marker ectopically. However, we found that 97% of ref-1(oy40) mutants exhibiting ectopic srh-1420rfp expression also expressed str-10gfp ectopically on the right side (Fig. 2B

and Table 3). These results indicate that the generation of ectopic AWB and ADF neurons is correlated, and likely arises due to defects at or prior to their shared precursor stage in the lineage. To identify the stage at which REF-1 acts to mediate lineage decisions, we analyzed additional markers for cells closely related by lineage to the AWB/ADF neurons. The ABpraaap and ABalpppp precursors are generated at approximately 120V after fertilization. Following a stereotyped pattern of cell divisions, these precursors give rise to the AUA, ASJ, ASE, ADL and OLL neurons in addition to the AWB/ADF neurons (Fig. 2A). We found that markers for all these cell types were ectopically expressed on the right side to a similar extent as str-10gfp and srh-1420rfp in ref-1(oy40) and ref-1(ok288) animals (Tables 2 –4). Ectopic cells were located at approximately the same positions as their wild-type counterparts. For example, the cell bodies of OLL neurons are located anteriorly, and cell bodies of most chemosensory neurons are located posteriorly to the nerve ring, which is the primary site for signal integration (White et al., 1986). OLLspecific marker expression was always observed in a cell located anteriorly to the nerve ring, whereas chemosensory neuron-specific markers were always expressed in more posteriorly located cells (data not shown). The ADL, ASJ, and AWB neurons are three of six neuron types that fill with lipophilic dyes such as DiI or DiO (Perkins et al., 1986). Consistent with the generation of multiple ectopic neuron types, we observed >6 dye-filled neurons only on the right side of ref-1 mutant animals (data not shown). We correlated ectopic marker expression in different neuron types with ectopic expression of srh-1420rfp in the ADF neurons on the right side. In all cases examined, we observed a strong correlation between the cell types (Table 3), suggesting that the lineage defect occurs at or prior to the ABpraaap/ ABalpppp precursor stage. We further traced the lineage defect by examining markers for neuron types which are more distantly related to the AWB/ADF lineage. The AFD thermosensory neurons are generated from the ABpraaaa (ABalpppa) sister of the ABpraaap (ABalpppp) precursor (Fig. 2A). We also observed ectopic expression of the AFD-specific marker gcy-80gfp on the right side, although at a lower penetrance (Table 2). It is possible that the ectopic AFD neurons do not differentiate fully, such that a subset fails to express the gcy-80gfp marker. Alternatively, only a subset of affected lineages may exhibit defects at the earlier ABpraaa/ABalppp precursor stage, while the remaining lineages are affected at the next ABpraaap/ABalpppp precursor stage, thereby accounting for a lower frequency of ectopic AFD neurons. We found that 100% and ¨91% of ref-1(oy40) and ref-1(ok288) mutants with ectopic AFD marker expression, respectively, also ectopically expressed srh-1420rfp in the ADF neurons (Table 3; based on the penetrance of ectopic expression of each marker alone in this experiment, if ectopic marker expression in each neuron type is determined independently, only 49% of ref-1(ok288) and 15% of ref-1(oy40) animals ectopically expressing gcy-80gfp would be expected to ectopically

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Fig. 2. Markers for multiple ABpraaa or ABalppp-derived neuron types are ectopically expressed on the right side in ref-1 mutants. (A) Shown are the bilaterally symmetrical lineages arising from the non-symmetric ABpraaa and ABalppp neuroblasts on the right and left sides, respectively. Neurons for which marker expression was examined are indicated in red. The identities of additional postmitotic cell types derived from these lineages are shown, along with cells that undergo programmed cell death (indicated by X). Circles indicate precursors discussed in the text. Precursors that may be affected in ref-1 mutants are colored yellow. (B) Ectopic expression of str-10gfp (AWB—green) and srh-1420rfp (ADF—red) on the right side of wild-type and ref-1(oy40) animals is correlated. Lateral view; anterior at left. Scale bar = 10 Am.

express srh-1420rfp). This result suggests that ectopic expression in these neuron types was correlated, and that the lineage defect in a subset of ref-1 mutants can be further traced back to the ABpraaa/ABalppp precursor stage during embryogenesis (Fig. 2A). REF-1 functions prior to the first larval stage to regulate neuronal lineage decisions To confirm that REF-1 functions embryonically, we carried out temperature-shift experiments using the temperaturesensitive ref-1(oy40) allele. Animals were grown at the permissive (20-C) or restrictive (25-C) temperature, and growth-synchronized L1 larvae were shifted to the restrictive or permissive temperature, respectively. str-10gfp expression was then examined in adult animals. As shown in Fig. 3, loss of REF-1 function prior to the L1 larval, but not at later stages, results in ectopic str-10gfp expression. These results are consistent with the hypothesis that REF-1 acts embryonically to regulate lineage decisions. Notch signaling may not be required for REF-1-dependent suppression of the ectopic neuroblast lineage Upon activation of Notch signaling, the intracellular domain of Notch is cleaved and translocates to the nucleus where it directs LAG-1/CBF1/Suppressor of Hairless [Su(h)]-mediated transactivation of Hes gene expression by direct binding to

promoter sequences (Artavanis-Tsakonas et al., 1999; Mumm and Kopan, 2000; Yoon and Gaiano, 2005). The promoter of ref-1 contains multiple predicted LAG-1 binding sites (Neves and Priess, 2005), and mutating these sites was shown to abrogate Notch-mediated upregulation of ref-1 gene expression in the embryo (Neves and Priess, 2005). To explore whether REF-1 functions in a Notch-mediated pathway to regulate the ectopic lineage decision, we replaced promoter sequences in the rescuing ref-1 minigene with sequences in which all predicted LAG-1 binding sites were mutated (Neves and Priess, 2005). This minigene fully rescued the ectopic str-10gfp expression phenotype (Table 1). Moreover, 94% of lag1(q385) mutants also exhibited the wild-type pattern of str10gfp expression (Table 1), suggesting that REF-1 may function independently of Notch to mediate this lineage decision. The ectopic ASER neuron exhibits ASEL-like characteristics Although cells derived from the ABpraaa and ABalppp precursors are bilaterally symmetric overall, the left and right ASE neurons exhibit asymmetric features in that they express distinct sets of signaling molecules and respond to different chemical stimuli (Hobert et al., 1999; PierceShimomura et al., 2001; Yu et al., 1997). ASER expresses the gcy-5 guanylyl cyclase gene whereas ASEL expresses the gcy-7 and the lim-6 LIM-homeobox gene. To investigate whether the ectopic ASE neuron observed on the right side

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Table 2 Markers for neurons generated from the ABpraaa/ABalppp neuroblast are ectopically expressed on the right side in ref-1 mutants Straina

Marker for

% of animals expressing in L

srh-1420rfp ref-1(ok288); srh-1420rfp ref-1(oy40); srh-1420rfp c tph-10gfp ref-1(ok288); tph-10gfp ser-20gfp ref-1(ok288); ser-20gfp ref-1(oy40); ser-20gfp sre-10gfp ref-1(ok288); sre-10gfp flp-80gfp ref-1(ok288); flp-80gfp gpa-90gfp d ref-1(oy40); gpa-90gfp d gcy-80gfp ref-1(ok288); gcy-80gfp ref-1(oy40); gcy-80gfp

ADF ADF ADF ADF ADF OLL OLL OLL ADL ADL AUA AUA ASJ ASJ AFD AFD AFD

R

L

R

. .

. . .

100 32 70 100 38 100 38 81 98 30 100 35 100 82 100 84 100

0 68 30 0 62 0 60 19 2 67 0 65 0 17 0 15 0

Otherb

0 0 0 0 0 0 2 0 0 3 0 0 0 1 0 1 0

n = 30 – 100 for each. Adult animals were examined except as indicated. ref1(ok288) animals were grown at 20-C; ref-1(oy40) animals were grown at 25-C. There were no differences in the expression of examined transgenes in wild-type animals grown at either temperature. a All transgenes were stably integrated into the genome. ser-20gfp, sre10gfp, flp-80gfp, tph-10gfp, and gpa-90gfp are also expressed in additional cell types. b Rarely, we observed loss of expression in one cell, or two expressing cells on a side with none on the other. c This strain also contained stably integrated str-10gfp transgenes. Expression of str-10gfp and srh-1420rfp in the corresponding control wild-type strain was similar to those of strains expressing each transgene individually, and is not shown for brevity. d This strain also contained stably integrated srh-1420rfp transgenes. Expression of gpa-90gfp and srh-1420rfp in the corresponding control wild-type strain was similar to those of strains expressing each transgene individually, and is not shown.

of ref-1 mutants exhibit ASER or ASEL-like properties, we examined gcy-50gfp and gcy-70gfp expression in ref-1 mutants. Surprisingly, expression of the ASER marker gcy50gfp was unaffected in these animals. Instead, we observed ectopic expression of the ASEL markers gcy70gfp and lim-60gfp on the right side of ref-1 mutant animals (Table 4). To confirm that the ectopic ASE cell generated on the right was expressing ASEL-specific genes, we examined ref-1 mutants coexpressing gcy-50gfp and gcy-70rfp. In wild-type animals, GFP expression was only observed in a single cell on the right side and RFP expression in a single cell on the left. However, in ref-1 mutants, while we still observed a single cell expressing gcy-50gfp on the right, we observed two cells expressing RFP in a L/R symmetrical manner (Fig. 4 and Table 4). These results indicate that the ectopic ASE neuron expresses ASEL-like features in ref-1 mutants. A complex regulatory cascade involving transcription factors and miRNAs has been shown to regulate the left/

right decision in the ASE neurons (Chang et al., 2003, 2004; Johnston and Hobert, 2003). In particular, the DIE-1 zinc finger transcription factor acts in the ASEL neuron to promote an ASEL fate such that, in die-1 mutants, the ASEL neuron inappropriately expresses ASER characteristics (Chang et al., 2004). To determine whether the ectopic ASEL-like neuron on the right side of ref-1 mutants is subject to the same L/R regulatory mechanisms as the wildtype ASEL neuron, we used RNAi to knockdown ref-1 function in die-1(ot26) mutants (Table 4). In both cases, we observed not only the expected misexpression of the ASER marker gcy-50gfp on the left side, but we also observed an additional cell expressing gcy-50gfp on the right. These results indicate that the ectopic ASEL-like neuron on the right side of ref-1 mutants is subject to DIE-1-mediated regulation of L/R asymmetry. Daughters of an additional AB-derived blastomere also exhibit defects in ref-1 mutants The ectopic lineage observed in ref-1 mutants could arise from the generation of an ectopic neuroblast, or cell fate transformation of a lineally related or unrelated precursor(s). In the latter case, we would expect to observe loss of expression of markers for terminally differentiated cells arising from the affected precursor. Moreover, this loss should correlate with the observed ectopic expression of cell markers. To address this issue, and to explore the role of REF-1 in the generation of additional neuron types, we examined markers for cells generated from multiple AB daughter-cell blastomeres (Fig. 5). The ectopic neuroblast likely does not arise solely from complete cell fate transformation of the ABalpp, ABplp(a/p), ABpla(a/p), ABprp(a/p), or ABpra(a/p) blastomeres, since terminal differentiation markers for subsets of cells derived embryonically from these precursors are expressed in the wild-type manner in ref-1 mutants (Tables 1, 2, and 5). It has previously been shown that postembryonic lineages derived from the ABarp(a/p), ABpla(a/p), and ABprap blastomeres are affected in ref-1 mutants (Alper and Kenyon, 2001; Neves and Priess, 2005). For example, the ABarpp-derived V6(L/R) epidermal cells were shown to infrequently adopt a V5 epidermal cell-like fate in ref1(mu220) mutants resulting in the presence of ectopic V5derived postdeirid structures (Alper and Kenyon, 2001). We explored the possibility that ABarpp may be undergoing cell fate transformation, followed by regulatory compensation by additional epidermal cells (Sulston and White, 1980). However, no correlation was observed between the presence of ectopic postdeirids and ectopic AWB cells in ref-1(ok288) animals (data not shown). Development of additional cell types derived from these precursors was also unaffected by ref-1 mutations (Table 5), suggesting that examined precursors are not undergoing complete cell fate transformation, and that REF-1 functions may be differentially required in different sublineages. Since additional members of the ref-1 family exhibit partly overlapping gene expression patterns

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Table 3 Ectopic expression of markers for multiple neurons are correlated Strain

Marker 1 (neuron)

Marker 2 (neuron)

% of animals with ectopic Marker 1 expression that also exhibit ectopic expression of Marker 2a (n)

ref-1(oy40) ref-1(oy40) ref-1(oy40) ref-1(oy40) ref-1(ok288) ref-1(oy40) ref-1(oy40)

srh-1420rfp (ADF) srh-1420rfp (ADF) srh-1420rfp (ADF) gcy-80gfp (AFD) gcy-80gfp (AFD) srh-1420rfp (ADF) srh-1420rfp (ADF)

str-10gfp (AWB) gpa-90gfp (ASJ) sre-10gfp (ADL) srh-1420rfp (ADF) srh-1420rfp (ADF) gcy-70gfp (ASEL) gcy-50gfp (ASER)

97 91 87 100 91 88 0

(36) (22) (23) (4)b (11)b (16) (12)

ref-1(oy40) animals were grown at 25-C; ref-1(ok288) animals were grown at 20-C. Adult animals were examined. a Ectopic cells were always on the right side. b Few ref-1 mutant animals expressing gcy-80gfp ectopically were examined due to the low penetrance of the gcy-80gfp ectopic expression phenotype.

with ref-1 (Neves and Priess, 2005), we also determined whether animals mutant in these genes exhibit gross neuronal developmental defects. Loss-of-function mutations are available for hlh-28 and hlh-29. However, hlh-28(tm458) and hlh-29(tm284) mutant animals exhibited wild-type patterns of dye-filling in both head and tail neurons, and exhibited wild-type expression of srh-1420rfp (data not shown), suggesting that development and differentiation of nine AB-derived neuronal subtypes are unaffected upon loss of these ref-1 family members. Interestingly, we observed defects in the expression of a marker for the serotonergic NSM(L/R) neurons which are generated from the ABaraa blastomere. We observed loss of expression of the tryptophan hydroxylase fusion gene tph10gfp in ¨74% of ref-1(ok288) mutants in either the left or the right NSM neurons, and rarely in both (Table 5). Since tph10gfp is also expressed in the ADF neurons, we correlated loss

of expression in the NSM neurons with ectopic expression in ADFR. However, expression in the NSM(L/R) neurons was lost at a similar percentage in ref-1 mutants with either one or two ADFR neurons (80% and 70% of ref-1 mutants with one or two ADFR neurons, respectively, showed loss of tph-10gfp expression in either NSML/R) (Table 5). Thus, loss of tph10gfp expression in the NSM cells and generation of the ectopic neuroblast lineage are likely independent events, indicating that the ABaraa blastomere is not undergoing complete cell fate transformation in ref-1 mutants. We are unable to rule out the possibility of a partial cell fate transformation. In addition, since we could not examine expression of markers for every AB-derived postmitotic cell type, it remains possible that the ectopic cells are generated from fate transformation of later-born precursor(s). The observed effects of ref-1 mutations on cells generated from multiple AB daughters are summarized in Fig. 5.

Table 4 Ectopic ASE neurons on the right side express ASEL-specific markers Straina

Marker for

lim-60gfp b ref-1(oy40); lim-60gfp b gcy-70gfp ref-1(ok288); gcy-70gfp ref-1(oy40); gcy-70gfp gcy-50gfp ref-1(ok288); gcy-50gfp ref-1(oy40); gcy-50gfp gcy-50gfp; gcy-70rfp

ASEL ASEL ASEL ASEL ASEL ASER ASER ASER ASELc ASERd ASELc ASERd ASER ASER ASER ASER

ref-1(oy40); gcy-50gfp; gcy-70rfp gcy-50gfp e ref-1(RNAi); gcy-50gfp die-1(ot26); gcy-50gfp e ref-1(RNAi) die-1(ot26); gcy-50gfp f

% of animals expressing in number of neurons on a side (L/R) None

1(L)

1(R)

1(L)/1(R)

1(L)/2(R)

0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0

100 86 100 38 79 0 0 0 100c 0 84c 0 0 0 0 0

0 0 0 0 0 100 100 100 0 100d 0 100d 100 100 0 0

0 14 0 62 21 0 0 0 0 0 14c 0 0 0 100 80

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20

n = 50 – 100 for each. ref-1(ok288) animals were grown at 20-C; ref-1(oy40) animals were grown at 25-C. There were no differences in the expression of examined transgenes in wild-type animals grown at either temperature. a Transgenes were stably integrated in the genome. b lim-60gfp is expressed in additional cell types. c Expression of gcy-70rfp was examined. d Expression of gcy-50gfp was examined. e Strains were grown on bacteria containing vector alone (L4440). f Numbers shown are the average derived from two independent experiments. See Materials and methods for details.

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Fig. 3. REF-1 functions during embryonic stages to regulate lineage decisions. Animals of the indicated genotypes were raised at either 20-C or 25-C for at least one generation, shifted to the converse temperature as L1s, and allowed to reach adulthood. As controls, animals were maintained at the same temperature. The percentage of animals exhibiting ectopic str-10gfp expression as adults is shown. Numbers shown are from three independent experiments using 30 – 60 animals each. All strains carry stably integrated str-10gfp transgenes.

ref-1 is expressed dynamically in the embryo To examine the spatiotemporal expression pattern of ref-1, we generated a GFP-tagged ref-1 fusion gene carrying 2 kb of upstream promoter sequences. This fusion gene rescued the ectopic marker expression defects of ref-1 mutants (Table 1). We observed broad and dynamic GFP expression in the embryo (Fig. 6). GFP was localized to the nuclei of all expressing cells as expected for a predicted transcription factor. We focused our attention primarily on the AB lineage. We observed GFP expression in the nuclei of the ABplp(a/p), ABalp(a/p), ABprp(a/p), ABara(a/p), and weakly in the ABarp(a/p) blastomeres at approximately 100V after fertilization (Figs. 6A –C). We did not observe expression in the ABpraa mother of the ABpraaa precursor which generates the sublineage affected in ref-1 mutants on the right side. However, expression was observed in the ABalpp neuroblast which gives rise to the bilaterally symmetrical set of neurons on the left side (Fig. 6A). GFP expression was maintained in the progeny of the ABara(a/p) blastomere through the next three cell divisions but was lost in additional blastomeres (Fig. 6D). In the subsequent cell divisions, GFP expression was maintained in the daughters of the ABaraap precursors which give rise to the NSM(L/R) neurons, but was downregulated in additional ABara(a/p) daughter and grand-daughter cells (data not shown). GFP expression was weaker in subsequent cell divisions and was not followed further.

regulated balance between the functions of neurogenic and proneural genes which promote neural fates, and antineural genes which antagonize neural fates is essential for neuronal patterning. Members of the bHLH protein family have been implicated in both proneural and antineural functions, and the functions of individual members of these families are wellconserved across species (Bertrand et al., 2002; Hatakeyama and Kageyama, 2004). Moreover, in both vertebrates and invertebrates, the functions of these proteins have been shown to be regulated by intercellular signaling primarily involving the Notch receptor, as well as cell-intrinsic mechanisms (Bertrand et al., 2002; Ross et al., 2003). In C. elegans, the unicellular zygote divides to give rise to the AB and P1 blastomeres. The AB blastomere generates the majority of the nervous system, whereas the P1 blastomere generates other tissue types including the germline (Sulston et al., 1983). Cell – cell inductive events involving Notchmediated signaling direct the lineage patterns and cell fates of a subset of AB daughters. In particular, L/R asymmetry in the AB progeny blastomere cell fates is directed by Notch signaling events (Hutter and Schnabel, 1994, 1995; Mango et al., 1994; Mello et al., 1994; Moskowitz et al., 1994; Priess et al., 1987). However, additional AB daughters appear to acquire their cell fates in the absence of intercellular signaling. Thus, similar to other organisms, a combination of extrinsic and intrinsic mechanisms are required to regulate the cell fates, and hence the numbers and types of neurons, generated from individual AB progeny. Somewhat surprisingly, genes that act to mediate these early patterning events are largely unknown. In particular, despite the large number of bHLH proteins predicted by the C. elegans genome, their roles in patterning the embryonic nervous system have remained obscure. Previous work implicated the LIN-22 and REF-1 HES protein family members in antineural roles similar to their counterparts in other organisms. In lin-22 mutants, the V1 – V4 epidermal cells inappropriately acquired the V5 fate resulting in the generation of ectopic postdeirid neurons (Wrischnik and Kenyon, 1997). Similarly, in ref-1 mutants,

Discussion Correct patterning of the nervous system requires that the appropriate numbers and types of neurons be generated from precursor cells in precisely delineated locations. However, equally critical for patterning is the suppression of inappropriate adoption of neuronal fate by other cell types. Thus, a

Fig. 4. Ectopic ASE neurons on the right express ASEL-specific markers in ref1 mutants. Shown is the expression of the ASEL-specific marker gcy-70rfp (red), and the ASER-specific marker gcy-50gfp (green) in wild-type and ref1(oy40) animals. Top down view; anterior at left. Scale bar = 20 Am.

A. Lanjuin et al. / Developmental Biology 290 (2006) 139 – 151

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Fig. 5. Summary of effects of ref-1 mutations on AB-derived neuronal and non-neuronal cell types. Cell types for which marker expression was examined in ref-1 mutants are shown, along with the AB-derived precursors from which they are generated. ABalpp-derived neuron types are present ectopically on the right side in ref-1 mutants and are shown in bold. Italics indicate additional cell types affected in ref-1 mutants. Effects on cell types marked with asterisks were shown previously (Alper and Kenyon, 2001; Neves and Priess, 2005). Lack of effect on cells indicated in parentheses were inferred indirectly by dye-filling. AB-derived blastomeres shown to express ref-10gfp in this study are underlined. Double underlines indicate blastomeres in which ref-10gfp expression is maintained through subsequent cell divisions; dashed lines indicate blastomeres that express ref-10gfp weakly.

the V6 cells infrequently adopted V5 neuroblast fate at the expense of V6 epidermal fate (Alper and Kenyon, 2001). Recently, REF-1 has also been shown to antagonize the LIN32 proneural protein in the generation of male sensory rays (Ross et al., 2005). The functions of REF-1 in these postembryonic lineages appear to be Notch-independent. In the embryo, REF-1 was recently shown to be upregulated in

response to Notch signaling in the E lineage which gives rise to intestinal cells, and in this lineage, REF-1 acts in a Notchdependent manner (Neves and Priess, 2005). REF-1 was also upregulated in response to Notch signaling in the AB lineage, and may act in a Notch-dependent manner to regulate the development of two ABplaaa-derived epidermal cells (Moskowitz and Rothman, 1996; Neves and Priess, 2005). We have

Table 5 Effect of ref-1 mutations on expression of markers for additional AB-derived cell types Straina

Marker for

Derived fromb

% showing wild-type pattern

tph-10gfp ref-1(ok288); tph-10gfp tph-10gfp ref-1(ok288); tph-10gfp sra-60gfp ref-1(ok288); sra-60gfp str-20gfp d str-20gfp; ref-1(ok288) d Wild-type (DiI) ref-1(ok288) (DiI) Wild-type (a-ODR-7 ab)f ref-1(ok288) (a-ODR-7 ab)f flp-130gfp ref-1(ok288); flp-130gfp

NSM L/R NSM L/R HSNL/HSNR HSNL/HSNR ASHL/ASHR ASIL/ASIR ASHL/ASHR ASIL/ASIR AWCL/AWCR AWCL/AWCR PHAL/PHAR PHBL/PHBR PHAL/PHAR PHBL/PHBR AWAL/AWAR AWAL/AWAR DD1, DD3, DD5/DD2, DD4, (DD6)g DD1, DD3, DD5/DD2, DD4, (DD6)g

ABaraa ABaraa ABplap/ABprap ABplap/ABprap ABplpa/ABprpa ABplaa/ABpraa ABplpa/ABprpa ABplaa/ABpraa ABplpa/ABprpa ABplpa/ABprpa ABplpp/ABprpp ABplap/ABprap ABplpp/ABprpp ABplap/ABprap ABplaa/ABpraa ABplaa/ABpraa ABplpp/ABprpp ABplpp/ABprpp

100 26c 100 100 97 91 100 84e 96 87 86 90 100 100

n = 30 – 100 for each. a The expression of stably integrated transgenes was examined. Animals were grown at 25-C and adults were examined. b See Fig. 4 for summary. c Since tph-10gfp is also expressed in ADF (Table 2), we correlated loss of expression in NSML or R with ectopic expression in ADFR. 33/53 animals exhibited ectopic expression in ADFR. Of these, 15 lacked expression in NSML and 8 in NSMR. 20/53 animals exhibited wild-type expression in one ADFL and one ADFR. Of these, 8 lacked expression in NSML, 7 in NSMR, and 1 in both NSML/R. d str-20gfp is expressed stochastically in either AWCL or AWCR (Troemel et al., 1999). e 11% did not exhibit any expression. f Anti-ODR-7 antibody staining was carried out as previously described (Sarafi-Reinach and Sengupta, 2000). g Expression in DD6 was variable and frequently difficult to observe. Numbers shown reflect flp-130gfp expression in D1 – D5.

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Fig. 6. Embryonic expression pattern of ref-10gfp. (A – C) Expression of a functional GFP-tagged REF-1 protein in the indicated nuclei of a 28-cell embryo. The weakly expressing ABarp(a/p) nuclei are not shown. The differences between the observed expression pattern and previously reported results (Neves and Priess, 2005) may be due to differences in the fusion genes examined in the studies. (D) GFP-tagged REF-1 expression is maintained in the progeny of the ABar(a/p) blastomere through the subsequent three cell divisions. Numbers correspond to the nuclei of AB-derived cells as follows: 1/2-ABaraap(a/p); 3/4-ABaraaa(a/p); 5/6ABarapa(a/p); 7/8-ABarapp(a/p).

shown that mutations in ref-1 result in the generation of an entire ectopic neuronal sublineage derived from the AB blastomere embryonically, as well as defects in differentiation of the AB-derived serotonergic NSM neuron type. Interestingly, although the LAG-1 binding sites upstream of ref-1 were shown to be required for upregulation of ref-1 expression in the AB lineage (Neves and Priess, 2005), we found that these sites were dispensable for regulation of the ectopic lineage decision and that moreover, ectopic cells were not observed in lag-1 mutants, suggesting that the functions of REF-1 in this decision are Notch-independent. Thus, similar to other HES genes, REF-1 may also exhibit Notch-dependent and independent roles in different lineage decisions (Fisher and Caudy, 1998; Ohsako et al., 1994; Van Doren et al., 1994). Our analysis of REF-1 functions suggests that REF-1 may act at multiple steps in different lineages. REF-1 is likely to act early to suppress the generation of ectopic neurons from an early neuronal or non-neuronal precursor(s). REF-1 retains strong expression in the descendants of the ABara(a/p) blastomeres, and may play a later role in the subtype specification of the NSM neuron type which derives from ABaraa. Similar early and late roles for correct nervous system development have been proposed for other bHLH domain proteins (Hallam et al., 2000; Jarman et al., 1993; Jarman et al., 1995; Portman and Emmons, 2000; White and Jarman, 2000). However, it is possible that the phenotypes of ref-1 mutants are not indicative of the temporal requirements for REF-1 function, but instead reflect the partly redundant roles of each of six REF-1 family members. Members of this subfamily are expressed in overlapping spatiotemporal domains (Neves and Priess, 2005), and are likely to act combinatorially and redundantly in a subset of cell types, whereas a single member may act in other cell types. The origin of the ectopic neuronal subtypes is unclear. Although we attempted to perform mosaic analyses to determine the site(s) of action of REF-1, overexpression of ref-1 together with markers commonly used for such experiments resulted in strong embryonic lethality (our unpublished data) precluding this analysis. The constellation of ectopic neurons generated is similar to that generated by ABalppp, suggesting transformation of an entire sublineage.

The complete cell fate transformation of several neuroblasts at the ABalppp developmental stage may be ruled out since we showed that there is no correlated loss of neurons generated from these neuroblasts. However, the ectopic sublineage may arise from partial cell fate conversion of an early precursor, or fate conversion of a later precursor. Alternatively, loss of REF-1 function may lead to the duplication of a precursor. Interestingly, mutations in ref-1 result in the ectopic generation of all descendants of the ABpraaa/ABalppp neuroblasts. This observation suggests that the lineage program of the affected precursor may be intrinsically determined, such that this neuroblast once specified, is Fpreprogrammed_ to go through a defined set and pattern of cell divisions giving rise to a defined subset of neuronal subtypes. Studies in vertebrates and Drosophila have indicated that proneural bHLH proteins direct specific developmental programs in a context-dependent manner (e.g. Chien et al., 1996; Goridis and Brunet, 1999; Gowan et al., 2001; Jarman and Ahmed, 1998; Jarman et al., 1993, 1994; Mizuguchi et al., 2001; Perez et al., 1999). The lineage program followed by the affected neuroblast may be directed by such a proneural protein(s). It is also formally possible that the ectopic cells arise from the coordinated transformation or generation of many individual cell fates from unrelated lineages. Detailed lineaging experiments may resolve these issues in the future. Unexpectedly, we observed a L/R bias in the functions of REF-1 in the regulation of the ectopic lineage decision. It is possible that the early Notch-mediated signaling events induce a REF-1-dependent lineage intrinsic memory that is then translated into L/R differences. Redundancy in ref-1 functions may account for the relatively restricted L/R defects observed in ref-1 mutants. We noted that although ectopic cells were on the right side, the ectopic ASE neurons (the only affected neuron type for which L/R markers are available) exhibit characteristics specific for ASEL. However, these neurons retain the ability to adopt ASER fate in a die-1 mutant background. Recently, it has been shown that the ASE(L/R) neurons express hybrid characteristics of both neuron types prior to adopting terminal L or R fates presumably in response to one or more spatially restricted transient signaling events (Johnston

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et al., 2005). These events may not be available to the ectopic lineage due to either spatial or temporal constraints, resulting in the adoption of a Fdefault_ ASEL-like terminal fate. Alternatively, the ectopic neurons may arise on the left and migrate over to the right. It is also possible that the presence of the wild-type lineage inhibits adoption of ASERlike characteristics by the ectopic ASE neurons on the right. These results suggest that a global L/R positioning mechanism is likely not necessary for maintenance of ASE(L/R) asymmetry. In summary, we have described a role for a nematodespecific member of the HES subfamily of bHLH proteins in neuronal development and differentiation. Our results together with those of others indicate that members of the REF-1 subfamily function in both Notch-dependent and independent pathways to execute neuronal lineage decisions. Analyses of the functions of additional family members singly, and in combination will likely provide additional insights into the roles of these genes in patterning and L/R asymmetry decisions in multiple tissue types. Acknowledgments We thank Maura Berkley and Caron Gauthier for technical assistance, the Caenorhabditis Genetics Center for strains, the C. elegans Gene Knockout Consortium for the ok288 allele, Andy Fire for the C. elegans expression vectors, Jim Priess for the ref-1 2.0kb[RAGGCAA] promoter, Gary Ruvkun for the die1(RNAi) clone, Shohei Mitani for the hlh-28(tm458) and hlh29(tm284) alleles, and Oliver Hobert for comments on the manuscript. This work was supported by the NSF (IBN 0129370-P.S.) and NIH (GM 069891-C. P. H.). References Alifragis, P., Poortinga, G., Parkhurst, S.M., Delidakis, C., 1997. A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system. Proc. Natl. Acad. Sci. U. S. A. 94, 13099 – 13104. Alper, S., Kenyon, C., 2001. REF-1, a protein with two bHLH domains, alters the pattern of cell fusion in C. elegans by regulating Hox protein activity. Development 128, 1793 – 1804. Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal integration in development. Science 284, 770 – 776. Bailey, A.M., Posakony, J.W., 1995. Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev. 9, 2609 – 2622. Bertrand, N., Castro, D.S., Guillemot, F., 2002. Proneural genes and the specification of neural cell types. Nat. Rev., Neurosci. 3, 517 – 530. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71 – 94. Chang, S., Johnston, R.J., Hobert, O., 2003. A transcriptional regulatory cascade that controls left/right asymmetry in chemosensory neurons of C. elegans. Genes Dev. 17, 2123 – 2137. Chang, S., Johnston Jr., R.J., Frokjaer-Jensen, C., Lockery, S., Hobert, O., 2004. MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode. Nature 430, 785 – 789. Chen, H., Thiagalingam, A., Chopra, H., Borges, M.W., Feder, J.N., Nelkin, B.D., Baylin, S.B., Ball, D.W., 1997. Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein

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