Neuron,

Vol. 9, 789403,

November,

1992, Copyright

0 1992 by Cell Press

castor Encodes a Novel Zinc Finger Protein Required for the Development of a Subset of CNS Neurons in Drosophila Dervla M. Mellerick,* Judith A. Kassis,+ Shang-Ding Zhang,* and Ward F. Odenwald* *Neurogenetics Unit Laboratory of Neurochemistry National Institute of Neurological and Stroke National Institutes of Health +Center for Biologics Evaluation Food and Drug Administration Bethesda, Maryland 20892

Disorders

and Research

Summary Using an enhancer detection screen, we have identified castor, a new gene required for embryonic CNS development in Drosophila. Embryos that lack castor expression have a diminished CNS axonal network and express engrailed aberrantly late in CNS development. castor is unique among the previously described genes involved in Drosophila neurogenesis in that its expression is restricted to a subset of delaminated CNS neuroblasts and to ventral midline glial precursor cells. The putative castor gene product contains a novel zinc-binding domain and multiple transcriptional activation domains, suggesting that it acts as a transcription factor necessary for thedevelopment of asubset of CNS neuronal precursors. Introduction Essential to understanding the development of the nervous system is the characterization of the molecular machinery involved. Neural development has been thoroughly studied at the cellular level in several organisms, but little is known about the molecular mechanisms that govern these events. The fruit fly Drosophila melanogaster is well suited for investigating the molecular basis of neurogenesis because it is amenable to genetic analysis and it has a relatively simple nervous system. In the fly, ventral cord neurogenesis begins with the segregation of the neuroblasts (NBS), neuronal precursor cells, from the ectoderm and their arrangement between the ectoderm and the underlying mesoderm (Hartenstein and Campos-Ortega, 1984; Hartenstein et al., 1987). As each NB delaminates, it acquires a unique identity and fate based on the location it takes within the developing neuroepithelium (Doe and Goodman, 1985; Pate1 et al., 1989). NB delamination occurs in three pulses (referred to as SI, SII, and SIII), giving rise to three different subpopulations of NBS. Following delamination, the ventral cord NB undergoes asymmetric divisions producing an NB and a ganglion mother cell (GMC) with each mitosis. The NB progeny, the GMCs, divide in turn, producing a pair of sibling neurons that differentiate and specialize. In contrast to those in the ventral cord, NBS in the cephalic neurogenic region can

begin division while still within the ectoderm. Moreover, cephalic NBS delaminate in less temporally defined stages than ventral cord NBS and can undergo symmetric divisions before giving rise to GMCs (for reviews see Hartenstein and Campos-Ortega, 1984; Campos-Ortegaand Hartenstein, 1985; Hartenstein et al., 1987). Mutational analysis has identified a number of genes involved in the entry of ectodermal cells into the neuronal lineage in Drosophila. The proneural and neurogenic genes mediate these early steps by programming ectodermal cells within the developing neuroepithelium to becomeeitheran NBor an epidermoblast (for references see Artavanis-Tsakonas, 1988; Jan and Jan, 1990; Campos-Ortega and Jan, 1991; Campos-Ortega, 1988, 1991). Once the neuronal fate has been established, the neuronal precursor genes are required for the proper differentiation of many or all NBS and their progeny (Gonzalez et al., 1989; Doe et al., 1991; Vaessin et al., 1991), while other genes, including the segmentation genes, establish the unique identity of individual neuronal precursor cells (Doe et al., 1988a, 1988b; Pate1 et al., 1989). Although many genes involved in the selection of NBS have been identified, our understanding of those genes that play regulatory roles at the intermediate stages of neurogenesis, prior to the formation of a functionally mature CNS, is limited. The advent of enhancer detection screens (O’Kane and Gehring, 1987; Bier et al., 1989; Bellen et al., 1989; Wilson et al., 1989) has facilitated the identification of additional genes required for neurogenesis (Uemura et al., 1989; Fasano et al., 1991; Doe et al., 1991; Vaessin et al., 1991). To learn more about the genes involved in the intermediate stages of CNS development, we employed such enhancer detection screens to search for genes required during this phase. Analysis of one of our transgenic lines, H23A, revealed two closely spaced genes, castor (cas) and pollux (p/x), located at the cytological location 83C. cas and p/x are expressed during different periods of larval CNS development and in different cells. Here we describe the gene cas, whose mRNA is expressed in a subset of CNS neuronal precursor cells but not in neurons. In embryos lacking cas expression the number of CNS axons is reduced, and the expression of the segmentation gene engrailed (en) is altered late in CNS development. This suggests that cas is required for the proper differentiation of a subset of neuronal precursor cells.

Results Molecular

Cloning of cas and p/x

Enhancer detection screening with the PHlac vector (Figure IA; Experimental Procedures) has identified a homozygous viable line, H23A, that has embryonic P-galactosidase (P-gal) expression restricted to a sub-

Neuron 790

A

-6

-10 I

-6

-4 I

0 I

-2 I

I

2 I

*5

B -Breakpoints-

* as

4 I

Deficiency

Alleles

Al,

A3

6

6

10 Kb

*6

A5

P 8

Al

Figure 1. Molecular

A2 A3 A4 A5 TM3

Organization

&

$

Al

A2 A3 A4 A5 TM3

of cas and p/x

(A) Approximately 22 kb of cloned genomic DNA from the cytological location 83C is shown. The integration site of the PHlac vector, located in the 3’ untranslated region of the p/x gene, is designated 0 on the genomic map coordinates. A restriction map indicating the positions of BamHl (B), EcoRl (R), and Xbal (X) sites is aligned with the cas and p/x transcription units. As indicated, the cas and p/x genes are transcribed from opposite strands. Hatched areas represent the longest open reading frames in the transcripts, while introns are indicated by angle bars. DNA probes, shown as bars (#l-&j, were prepared from genomic, cDNA, or P element vector fragments. The f/-/lac vector’s chimeric minimal promoter consists of the 612 bp murine Hox 1.3 region-specific enhancer (Zakany et al., 1988) fused to the first 400 bp of the Drosophila engrailed promoter (PHlac is described as construct H in Kassis et al., 1991). (6) cas deletions, created by imperfect excisions of PHlac from the P element insertion line H23A are indicated by the boxed region that is aligned with the restriction map shown in (A). The stippled area corresponds to the deleted PHlac sequences. Southern analysis of Xbal-cut genomic DNA using probes #I and #6 was employed to identify the breakpoint fragments, highlighted by triangles. Note that DNA between these two probes is deleted in Al, A3, A4, and AS, creating novel Xbal fragments that are recognized by probes #I and #6. Also note that probe#&does not identify the breakpoint fragment of A2. Cenomic DNAfrom Df(l)w67c2,yflies, the transgenic line H23A, and the TM3 balancer stock were included in the Southern analysis to identify polymorphisms. Positions of the DNA size markers (Hindlll-digested h and Haelll-cut @X174 phage) are indicated.

castor Is Required for Proper CNS Development 791

EMBRYO .: : 2 i d 4 i i

INSTAR ,*

PUPA

ADULT

2

18S--,

Figure 2. Developmental Expression

Northern

Analysis

of

t-p 49

cas and

p/x

Lanes were loaded with 4 pg of poly(A)’ RNA from each of the collections indicated. cas mRNA was detected using a mixture of #2a and #Zb (see Figure IA) as probes. After probe removal, p/x transcripts (-2.7 kb) were identified on the same blot using probe3a,andfinallyasacheckformessageintegrity, the blotwas reprobed with a ribosomal protein 49 probe (see Experimental Procedures). Notethat the most abundant cas message is approximately 3.7 kb in size. Also note the apparent reactivation of cas expression in the third instar larva.

set of cells in the developing larval CNS (data not shown). In situ hybridization to H23A polytene chromosomes indicated that a single PHlac had inserted at the chromosomal subdivision 83C (data not shown). To determine whether the PHlac B-gal expression pattern observed in the H23A embryos reflected the expression pattern of a nearby gene, DNA flanking the integration site was cloned and characterized (see Experimental Procedures). A restriction map representing approximately 22 kb of genomic DNA spanning the insertion site is shown in Figure IA. To identify transcribed sequences, cloned EcoRl fragments (Figure IA) were used as probes for in situ hybridization studies on embryos (data not shown). Those fragments that hybridized were used as probes to screen O-12 hr embryonic cDNA libraries, and two cDNA populations were isolated that did not cross-hybridize. Sequence analysis of these cDNAs and the corresponding genomic regions revealed two genes that are transcribed from opposite strands and separated by 99 bp (Figure IA). Based on their close apposition, we have named these genes cas and p/x after the Greek mythological brothers. Southern analysis of the genomic and cDNA clones, using DNA fragments flanking the insertion site as probes (see Experimental Procedures), demonstrated that PHlac had integrated into the 3’untranslated sequence of theplx gene (data not shown).

Developmental Northern blots were performed to determine whether these closely linked genes are unique in their temporal expression patterns (Figure 2). Results from this analysis showed that cas and p/x differ both in message size and in temporal expression, the latter suggesting that they may employ separate cis regulatory elements to control their expression during development. The highest levels of the -3.7 kb cas message coincide with embryonic CNS development and with the PHlac IacZ expression pattern observed in the H23A line. cas expression is also detected in third instar larvae, and longer autoradiogram exposures of the cas Northern blot reveal that message can be detected in adults, albeit at a lower steady-state level (data not shown). In contrast, levels of p/x mRNA (-2.7 kb) are constant up to and including the first instar larval stage, while lower levels are detected at other stages of development. As the description of both the cas and plx genes is beyond the scope of this text, the remainder of this paper will focus on the characterization of cas. A detailed analysis of p/x will be described elsewhere (S.-D. Z., 1. A. K., D. M. M., and W. F. O., unpublished data). cas Encodes a Putative Transcription Factor with Novel Zinc Fingers Sequencing of overlapping cDNA and genomic clones has generated the primary structure of the -3.7 kb cas transcript shown in Figure 3A. A single long open reading frame encoding a 799 amino acid protein with a calculated molecular mass of 88 kd was found. The open reading frame begins with seven clustered ATGs between nucleotides 688 and 756. Analysis of the sequence flanking these ATGs indicates that the first methionine codon is in a favorable context for the initiation of translation (Cavener, 1987). Preceding this open reading frame, there are multiple termination codons in all three reading frames. The gene has three introns (depicted as arrowheads in Figure 3A), which are 554 bp, 70 bp, and 60 bp long, respectively. In the 3’ untranslated region, there are four consensus polyadenylation signals (AATAAA; Proudfoot and Brownlee, 1976) and three mRNA instability motifs (ATTTA; Shaw and Kamen, 1986). ldentity between the cDNA and genomic sequence ends at the 5’ end of the poly(A)’ cDNA tail. The most prominent feature of the putative cas protein is its four consecutive repeats that share partial homologywith thezinc-binding motifs of known transcription factors (Vallee et al., 1991). Optimal alignment of these repeats (Figure 38) indicates that each repeat has a novel C2-HZ&-H2zinc finger motif, which is separated by a 19 amino acid linker. Part of the motif, the second &Hz, closely resembles the zinc fingers found in the Xenopus TFIIIA transcription factor (Miller et al., 1985; Figure 3B). In comparison with other described Drosophila zinc finger proteins (e.g., see Boulay et al., 1987; Fasano et al., 1991), the second C2-H2 domain of cas is unusual in that 4 amino acids separate the 2 cysteines. This spacing and the acidic

Neuron 792

A

449 561 673 1

~~~A~~~~~~G~A~G~AAC~~TG~~GTAATTTGCCGTAATA~C~~~GCAAA~~GCC~GCGA~~~~GGA~AAAA~~CC~C~GC~GA~C~G~GC~C~~C~T~CA~~~GC~G~CC c~~~c~AA~cAcG~~GGccATGcGGGATTCCTTCACAAA~ACACGC~cC~c~~~cGcA~~~~~~~~~AcCGAAAAACc~C~C~~~~C~AGA~AAA~~~ACA~~ACCGC~GG ~GTTTTATTGGT~GAGAAAAG~GAG~AAAAAACGGAAACCAGCCGAAAACAAGCTGGGTTTTCCCAGAGCATTTTCCTGCGAAGGGTCGGTTAAAAAACTAGGGGATGAAGG ~cGAAccAccAcCAccAAcGGAcTTCTCATTTTATTCGTT~~~~AAGAGAGCGAGAGcGGCGAGA~CGG~~~~~~AAGAGAACCGG~GGCGAGAGGG~~GCAC~~GGGCA~G GA~~~GCGCAA~TTTTGCTATATTAGCCGGAGGCCGCGGAAGAG~~GAAGCAG~~~GAGCC~CGCAGCCGAAC~~~GAGGA~CGC~GAGACGAGACGCCG~GCAAGAAAG~C GAT~~AAAAT~CGAAATTCGAAATCCGTGCTGTGATCTGTGCTGAATATAGTGAAGAAATCTTAAAGAATTTTCAAAAAGAAATCCCAGTGCTGTGCTGAATATATCCGCGA AAGTGTGCCACAAAAATGTCC AAC CAA ATG GAG TTT ATT ATG CAA CTC TAC ATG ATG AAC TTG ATG AAG CAG CAG CAG CAA ATG CAG CTC CAG Met ser asn gln Met glu phe ile Met gin leu tyr Met Met asn leu Met lys Q,:: @%~#a Met gln leu gln

765 27

CTT CCG CAG CAG CAA CAG CAG CAG CAG CTG GCT GGA TAC ACT TAC ACA CAG AAC GAG OAT ATC TCT AGC AGC ACA XC GTC GAG CAG CAA :‘@$ leu ala gly tyr thr tyr thr gln asn glu asp ile ssr ser ser thr ser val glu & @t$ leu pro #& @oAt:B

655 57

CAG CAG CAG CAG CAG GAG CAA CTG CAG CAG CCA CAG CCA OAT CTC CGC AAG ACA AGA ACG CAC AAG GCC ATT AGC TCG CAA AAC ACC AAC n leu gln gln pro gln pro asp leu arg lys thr arg thr his lys ala ile ser ser gln ssn thr asn

945 67

AGT AGC CGT AGT TGT AGT CCC AAT AGT AAT TTG ATT GCT TTT CAA CCA CAA CAA TAC CCA GGC GCT ACA GGC GCA ACT CCT TCC cys ser ser ser arg ser cys ser pro asn ser asn leu ile ala phe gln pro gln gln tyr pro gly ala thr gly ala thr pro ser

1035 117

ACT CCC AAC AGC CAC CCC AGC AAC CTG OTC AAC CAG ATG CTG CTC CAA AGC CTG CC0 CCG CTG ACG CAG CTC ATG TTG CAG thr pro asn ser his pro sBr asn leu val asn gln met leu leu gln ser ieu pro pro leu thr gln leu met leu #I&)

1 113 225

337

1125 147

CAG CAG J& gm CAG CAA CAT TTG CTG ACC ACC TCC AAC CTT CTA CTA ACA CCC ACC CAC ACC CCC AGT TCG CTG GGC AAG CAG GAT CCA CTG CAG CAT CCG $$&.$#t his leu leu thr thr ser ssn leu leu leu thr pro thr his thr pro ser ser Ieu gly lys gln asp pro leu gln his pro

1215 177

TTG TTG CTG GGC CAG TTT GCC GGC KC GAA CAG ATG GCC ACA AAT AAC TTC CTT CAA TCA TCC ACA GTG ACC TCC ACG CCC ATC GAG AGA leu leu leu gly gln phe ala gly ser glu gln met ala thr asn asn phe leu gln ser ser thr val thr ser thr pro ile glu arg

1395 237

GAG AAG GCA GCC ACA CCA GCA CCC TCC GCC GGA GCA ACT GCC CGC AAC CTT TCG GCG GCC CAG OTC AAG TTT GAG CAG GAG TCC GCC GAC glu lys ala ala thr pro ala pro ser ala gly ala thr ala arg asn leu ser ala ala gln val lys phe glu gln glu ser ala asp GAC OAT GAG GAC GAT GAT AAG CCG CTG TCC AGC CTC ACC AGC TGC AGC KC TCG GGC CAC ACC AAC GCC AGC TCC GAG AAG CTG CTG TTG asp asp glu asp asp asp lys pro leu ser ser leu thr ser cys ser ser ser gly his thr asn ala ser ser glu lys leu leu leu

1485 267

TCG GGC OTC CAT CC0 CTG GAG TCA ACC ACC GAC AGC CTA GAC TCA CCC AGC ArG-Ar ACG CCC GTC AAG CAG CCG GCG GAC TCA TCG TAC ser gly val his pro leu glu ser thr thr asp ser leu asp ser pro ser met tyr thr pro val lys gln pro ala asp ser ser tyr

1575 297 1665 327

GGA CTC ATC ACA CCC OTC GAC AGT GAT CTG ACC CCC AAC ACA CCC CTC CAA CC0 ACC CAA ACA ATA TCC CTG CTG ACG CCG CCG TCG AGT gly leu ile thr pro val asp ser asp Ieu thr pro asn thr pro leu gln pro thr gln thr ile ser leu leu thr pro pro ser ser -- --- --GAG CAG AGC AAG AGC CTG GTG AGC CTC TCG GCG GCC AGC GGC CTG OAT GCT CTG CTC CAG AAC GAG GAG GTA CTG AAG AAC CTG COG AAA glu gln ser lys ser leu val ser Ieu ser ala ala ser gly leu asp ala leu leu gln asn glu glu val leu lys esn leu arg lys

1755 357

GTG KC XC TAC CTG GAG TGC GAG AAC AGC CTG TGC AGO CAG GAG AAC CTG COG GAG CAC TTC CAT TGC CAC GAG GAG CCC TGC CAG GGC val ser ser tyr leu glu cys glu asn ser leu cys arg gln glu esn leu arg glu his phe his cys his glu glu pro cys gln gly

1845 367

AAG ATC CTG AGC AAG AAG GAC GAC AK ATC COG CAC CTG AAG TOG CAC AAG AAG CGC AAG GAA AGC CTC AAG CT0 GGC TTT GCC CGC TTC lys ile leu ser lys lya asp asp ile ile erg his leu lys trp his lys lys arg lys glu ser leu lys leu gly phe ala erg phe

1935 417

TCC TCC KG GAC GAC TOT GCA CCA GCC TAC GGA GAG GOT TGC GCC TAC AAC TOG AAA CAG ACC CAC TAC CAC TGC OTC TAC GAG CAC TGT ser ser ser esp asp; cys ala pm ala tyr gly glu gly cys ala tyr esn trp lys gln thr his tyr his cys val tyr glu his cys

2025 447

CCC AAG GTG TAT GTG AGC ACC AGT GAC OTC CAG ATG CAT GCC AAT TTC CAC CGC AAG GAC XC GAG ATC GTG AAC GAG GGC TTC COG CGC pro lys val tyr val eer thr ser asp val gln met his ala asn phe his erg lys asp ser glu ile val asn glu gly phe arg arg

2115 477

TTC COG GCG CAC GAG ACC TGC CGC ATC GAG OAT TGC CCC TTT TTC GGC AAG AAG AX TCC CAC TAC CAC TGC TOT CGC GAG GGA TGC ACC phe arg ala his glu thr (oys arg lie glu esp cys pro phe phe gly lys lys ile ser his tyr his cys cys erg glu gty cys thr

2205 507

CAC ACC TTC AAG AAC AAG GCC GAT ATGbAC AAG CAC AAA ACT TAC CAT CTG AAG GAC CAC CAG CTG AAG ATG GAC GGC T7C AAG AAG ATC his thr phe lys asn lys ala asp met asp lys his lys thr tyr his leu lys asp his gln leu lys met asp gly phe lys lys ile

2295 537

CTG AAG ACC GAG GTG TOT CCC TTC GAC GCC TGC AAG TTC TCC ACC GTC TGC AAC CAC ATC CAC TGC OTC CGC GAG GGC TGC GAC TAC ATC leu lys thr glu val ; cys pm phe asp ala cys lys phe ser thr val cys asn his ile his cys val arg glu gly cys asp tyr ile

2365 567

CTG CAC TCC AGC AGC CAG ATG ATC AGC CAC AAG AGA AAG CAC GAT CGT CAG GAC GGA GAG CAG GCG TAC CAG CAG TTT AAG ATC AAG CAG leu his ser ser ser gln met ile ser his lya arg lys his asp arg gln asp gly glu gln ala tyr gin gln phe lys L lys gln

2475 597

GAC GTG GAG GAG TCC TCC CT0 GAT GCC ATG CCC CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CCC ACC AGC CTT AGT CAA TCC CAG AGC pm thr ser leu ser gln ser gln W sap val glu glu] ser eer leu asp ala me4 pro #;3

2565 627 2855 657

AGC AGC TCT GTA TGC GGC GGC ACG AAC ACC XC ACT CCA CTC TCC TCT TTG TCC &“#, ,@ val cys gly gly thr asn -_thr -_ ser ---thr pro --lsu -----ser wr leu --ser CCC AAG AAA AT&AA CTG CCA GCC GAC GCT CAG CAG TCG GAA GCC AAG CGC CTG pro lys lys ile gln leu pro ala asp ala gln gln ser glu ala lys arg leu

2745 667

CTG CCC CAG W C CAG CCA GCA GCC GCT GTG CAT CC0 CTG ACA AGT GGA CTC T’IT CCC GOT CTC CTG CCC CGT GCC GCC GCT CCT GGA GTG leu pro gln ser gln pro ala ala ala val his pro leu thr ser gly leu phe pro gly leu leu pro arg ala ala ala pro gly val

2635 717

OAT CCC ACA GCC CCT AAC TX CGA CTC ACC CAC CTG ATG GCC CTC TTC CAG TTG CAG AAT CCC CTC TTC TAC CAG AAC CTC TAT CCG GGA asp pro thr ala pro asn phe arg leu thr his leu met ala leu phe gln leu gln asn pro leu phe tyr gln asn leu tyr pro gly

2325 747

ATG ACC CAG AAT TCC KC ATG CTC GGC AAC CTG GCA GCA CTT AGC GCC GCA TCG GCA GCA GCG GCG GCG GCA GCG GCG GCA AAT GGA GCC met thr dn asn ser ser met leu gly asn leu ala ala leu ser ata ala ser &.?#f&.@& $j& &N ;*#‘q& I#& :Q esn gly ala

3015 777

GGA GTC CAG CAG CCG AAC GAG AGT TTA GCT TTA AGC CAG AGT TTA AGG AGT AGO AAG OAT GCG TOT CCT TAGGGTATTTTGCCAGCTACTTAGTT gly val gin gln pro esn glu ser leu ala leu ser gln ser leu arg ser srg lys ssp ala cys pro AM

iii

-- -- --

GCC GAG CAC TTC CTG GCT CGG AAA CGC GGC AGG CCA ala glu his phe leu ala arg lys arg gly arg pro AAA GTC GAA GAC GAG TCC TCT AAT CCC GCC ATG CTC lys val glu asp glu ser ser asn pro ala met leu

3110 TGACCGACffiACGTTGGACATTGTACATACGCGCTT~AAGCAGG~~~AGG~GGG~~~~TT~G~G~~~CAGGCAGA~CCAGGACAAGGCA~CC~~CCC~CCACAAAAACCAA 3222 CTCAGCCACACTTTGAGAACCTAGAAGTCCCTATACAAAGTGTAGCTGAATTGGCTTTTGCCAAACATTTTCCCCTTTAGCTTAGATTCTCTCAGTGCCACATTAATTTTTA 3334 AAATTTTCAGCCATTTTCAGGAAGAACTTTCTGAA~AGTTTGACTAAACTGTGATGTTTTCATTAGAGTAGCCTCTTTTAATTGATAGAGCGAGTGCAGGCT 3446 ATGCAGGATATGCAGGACTTAGGAGACTTGGATTGTCAATAACA~AGCG~C~AAGCA~~ACA~~G~A~G~GA~ATAC~AA~GGC~AA~~G~T~~~GC~~~A~A~ 3656 ~AGAGAATAATATTATAATTCTAATTATAAACTTTAGTAAAATCAGCATTCTTCTA~AAGTCAACATTACCT~~~~ATTATGCCTCAGAGCTTA~~ 3670 UAAAAAAAAAAAAAAAAAAA

castor Is Required 793

for Proper CNS Development

B 363 CEN...S~CRD.ENLREHFHCHE~PCQGK~LSKKDD~IRHLKWHKKRK.ESLKLGFARFSSSDD I : :I : ::I:11 I:/: ::I:: I: I:: /:/ : 1 :: 422 CAPAYGEGCAY*NWKD~HYHCVYEHCPKVYVSTS.DVDMHANFHRKDS,EIVNEGFRRFRAHET I /:I:: ::I :iI// I I:::: ::: I:: / ::/ /I :: 483 CRI...EDCPFFGKKISHYHCCREGCTHTFKNKA.DMDKHKTYH~KDH.~~KMDGFKK~~KTEV / : : ::(I : :(:I1 IIll:: ::::: :I :/I: / ::: : 542 CPF*..DACKF*STVCNHIHCVREGCDYltHSSS.~MlSHKRKHDR~DGEaAYaDFKlKaDVEE

421 //

11:

:: 482

I:

: I I : : :

541 : : I I

: : 600

CONSENSUS

cas

C..,,,E.C.~,.-K..HYHCVR~GC---~-SKS~DMI-HK--H-KD-~E----GFKRF---E-

TFIIIA

TGEKPYVC--DGCDKRFTKK-.-LKRH---H

Figure 3. Primary

Structure

of the cas Transcript

and Its Predicted

Protein

(A) The nucleotide sequence of most of the cas transcript is shown, together with the amino acids encoded by its longest open reading frame. The 5’ end of the gene has not been identified. Homopolymeric runs of 4 or more amino acids are stippled. The acidic domain is underlined, and two regions that are rich in putative PEST residues are highlighted by a dashed underline. The boxed amino acids represent the four contiguous repeats in the metal-binding domain. The positions of the three introns are indicated by arrowheads. In the 3’ untranslated region, polyadenylation signals are boxed, and RNA instability motifs are underlined. (B) Optimal alignment of the metal-binding repeats. The four repeats are shown in the single-letter amino acid code. To maximize homology, gaps have been introduced that are indicated by asterisks. Vertical bars between the repeats represent identical residues, and colons indicate aminoacids that are similar (according to the similarity criteria of Lipman and Pearson, 1985 and Jacobs and Micheals, 1990). Invariant amino acids are depicted in bold lettering. Amino acids that appear two or more times at any one position in the aligned The 30 amino acid TFIIIA consensus was reported by Miller et al. (1985). repeats are shown in the cas consensus.

residue at the third position between the cysteines in thecasfingersarealsofound in theTFlllAzincfingers (see Figure 36). A search of the Zinc Finger Gene Data Base (Jacobs and Micheals, 1990) reveals that the additional putative zinc ligands, the first &-Hz in each of the repeats, are not homologous to other known zinc-binding domains. The putative cas protein contains additional domains found in regulatory proteins important for development. Multiple runs of four or more homopolymeric amino acids, including five polyglutamine repeats (opa repeats; Wharton et al., 1985), a polyalanine stretch, and a polyserine stretch, comprise 6% of the protein. In addition, the protein has multiple homotriplet amino acid stretches of alanine, leucine, and serine. An acidic domain, composed primarily of aspartic acid residues, spans residues 236-242. Adjacent to the acidic domain, there is a region (from residues 270-330) that is rich in serine and proline residues (19% serine; 17% proline). Multiple potential PEST sequences, which are thought to contribute to rapid protein turnover (Rogers et al., 1986), are present throughout the protein. Expression

of cas during

Embyrogenesis

The spatial distribution of cas transcripts was investigated using whole-mount embryo in situ hybridization. cas expression is first detected in the cellular blastoderm in aventral patch of cells located between 12% and 35% of the blastoderm length (0% at the posterior pole; data not shown). These cells cover a region that corresponds to portions of the presumptive mesoderm and theabdominal ventral neurogenic anlage (see blastoderm fate map; Hartenstein et al., 1985). While cas expression is detected in the cellular blastoderm, this phase of expression appears to be

transient, due to our inability to detect cas message at the beginning of gastrulation. cas message is next detected in cells that border the anterior midgut primordium in early stage 8 embryos (Figure 4A). Additionally, two groups of bilaterally symmetrical cells (containing l-3 cells per group) located in the lateral and dorsal-lateral ectoderm of the procephalic neurogenic region also contain cas transcripts (Figure 5A). Shortly afterward the complexity of cas expression in the procephalic lobes increases (Figures 5B and 5C). These large cas-positive ectodermal cells may be NBS (Hartenstein et al., 1987). Coincident with the onset of cas expression in the procephalic regions, cas mRNA is detected in segmental clusters of mesoectodermal cells in the ventral midline (Figure 4A). Simultaneous in situ hybridization with cas- and en-specific probes positions the caspositive cells in the anterior portion of each parasegment (Figure4B), the site occupied by the midlineglial precursor cells (KIHmbt et al., 1991). The number of cas-positive cells and the intensity of midline expression are segment dependent, with a gradient of expression from anterior to posterior (Figure 4A). In slightly older embryos (Figure 56), at least three pairs of midline cells that are cas positive are present in the gnathal segments, whereas only 3-4 cas-expressing cells are found in the thoracic segments. Cross-sectional analysis shows that the lateral ventral cord NBS are already delaminating from the ectoderm when cas expression is detected only in the ventral midline mesoectodermal cells (Figure 4C). During stage 9, the highest level of cas expression is detected in the cephalic neurogenic regions. Three clusters of cas-expressing cells are seen at the margins of each procephalic lobe (Figure 5D). In contrast to

Neuron 794

castor Is Required 795

for Proper CNS Development

their initial surface location, serial cross sections of stage 9 embryos show that some cas-positive cells within the cephalic lobes are now positioned between theectoderm and the underlying mesoderm, the location of delaminated NBS. In the ventral-lateral neurogenie regions, cas expression is first detected in fully delaminated NBS during stage 9. In each segment, with the exception of a8 and a9, a symmetrical pair of cells that flank the midline express cas (Figure 4D; Figure 5D). In situ hybridization with both cas and en probes indicates that these oblong cells are located at the posterior edge of each segment, with their long axis perpendicular to the midline (data not shown). The numberof cas-expressing cells increases during late stage IO and stage 11. As Figure 4G demonstrates, the cas-positive cells in both the ventral cord and the cephalic lobes are now predominantly delaminated NBS, with the exception of a minority of enlarged casexpressing ectodermal cells in the cephalic neurogenie region. Thetas-positive cells in the presumptive head outline the developing supraesophageal ganglia and express high levels of the transcript (Figure 5F). The cas-expressing cells in the ventral neurogenic region are also arranged in characteristic segment-specific patterns. Views of whole-mount and serial cross sections show that there are 7-10 large, rounded NBS that express cas per hemisegment (Figures 4E and 4F). In situ hybridization with both cas and en probes (data not shown) indicates that most of the cas-expressing cells in stage 11 embryos are located close to the seg-

Figure 4. Expression

of the cas Gene in the Developing

Ventral

ment boundaries. In the ventral-lateral neurogenic region, many of the cas-expressing cells reside close to the anterior border of each segment (Figure 4E). Jacobs et al. (1989), using a /acZ line (F263) that marks the longitudinal glial lineage, have shown that the longitudinal glioblasts initially reside within this same region, attheanterior margin of each segment boundary. To determine whether one of the cas-expressing cells in this region is a longitudinal glioblast, we carried out in situ hybridization with a cas-specific probe followed by anti-B-gal immunocytochemistry on F263 embryos. No overlap in cas mRNA expression and immunoreactivity was observed, indicating that the cas-expressing cells are not longitudinal glioblasts, but most likely neighboring NBS (data not shown). cas expression reaches its maximum during stages 11 and 12. In the cephalic neurogenic region the casexpressing cells are arranged similarly to those in stage 9 embryos, with the number of cas-positive cells increasing (Figure 5E). In a segment-dependent manner, there are now IO-17 lateral NBS per ventral cord hemisegment that expresscas (Figure4H). One prominent midline cell per segment also expresses the cas transcript. In stage 11 embryos, en is expressed in the midline NB (Pate1 et al., 1989). When simultaneous in situ hybridization analysis was done on stage 11 embryos with the en and cas probes, only a single midline cell was stained coincident with the ectodermal enstripe, indicatingthat themidline NBexpresses both en and cas (data not shown). Serial cross sections

Cord

The distribution of cas message during ventral cord development is shown. Whole-mount in situ hybridizations using digoxigeninlabeled probes (blue) and immunostaining of mature neurons (brown) with anti-HRP antibodies u and K) were carried out according to Experimental Procedures. With the exception of the 10 pm thick sections (C, F, G, and I), whole-mount views are either ventral or ventral-lateral. (A) Following gastrulation, cas mRNA is initially detected at stage 8 in segmental clusters of ventral midline mesoectodermal cells (large arrow) and in cells that line the anterior midgut primordium (small arrow). Note the anterior to posterior gradient of cas-expressing midline cells. (B) Simultaneous in situ hybridization with cas and en probes on stage 9 embryos revealed that the cas-positive midline cells are in the anterior of each segment. (C) During stage 9, the lateral delaminating NBS (arrows) do not express detectable levels of cas. The transcript is detected only in midline mesoectodermal cells. (D) The first lateral cells that express cas message are symmetrical pairs of lateral NBS that flank the midline at the posterior edge of the gnathal, thoracic, and abdominal al-7 segments. These cas-positive cells are not detected in segments a8 and a9 (see dorsal view showing a&9 in Figure 5D). (E) By stage 11, the number of cas-expressing cells in the thoracic segments has increased to approximately9-IO cells per hemrsegment, with 7-8 cells expressing detectable levels of cas in the first three abdominal hemisegments segments. (F) Transverse sections of late stage 11 embryos show that most, but not all, lateral NBS are expressing cas (arrow indicates a negative blast cell). (C) Sagittal sections reveal that ventral cord NBS express cas mRNA after they have delaminated from the ectoderm. Note that in the cephalic lobes, cas expression is detected in some NBS while still within the ectoderm (arrow, also see Figure 5). (H) From late stage 11 to the middle of stage 12, the number of ventral cord cells expressing cas is at its peak. (I) Transverse sections through stage 12 embryos show that the cas-positive cells, which include most but not all NBS, are smaller than those in stage 11 embryos (see [F]). The arrow indicates a blast cell with no detectable cas message. (J) In situ detection of cas message followed by anti-HRP immunostaining of neurons in stage 14 and older embryos suggests that most neurons do not express detectable levels of cas message. Note that the cas-expressing cells now reside predominantly on the ventral and ventral-lateral surfaces of the developing neuromeres. (K) From stage 15 onward there is a progressive reduction in the number of cas-positive cells in the contracting ventral cord. This reduction in cas expression is more pronounced in the abdominal neuromeres. (L) By stage 16, cas expression in the abdominal neuromeres is limited to only a few cells while cas is still expressed in cells lining the ventral surface of the thoracic neuromeres. Note the sharp expression boundary between the third thoracic and first abdominal neuromeres.

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Figure 5. cas Expression

in the Procephalic

Neurogenic

Regions

Shown are whole-mount lateral (A, B, C, and I), dorsal (F and C), laterodorsal (D), or transverse section (E and H) views of cas in situ hybridization (blue) and neuron immunostaining (brown) with anti-HRP antibodies (H). (A) Stage 9, in the procephalic lobes (anterior, up; dorsal, right), cas mRNA is first detected in a pair of adjacent ectodermal cells on the anterior border of the neurogenic region and in a single ectodermal cell on the posterior border of the neurogenic region. The ventral view of an embryo of similar age is shown in Figure 4A. (B) By stage 10, the number of cas-expressing cells in the dorsoposterior neurogenic region has increased to 6 per lobe (embryo oriented as in [A]). (C) In early stage 11 embryos, cas expression is detected in ectodermal cells that line the posterior and ventral borders of the zone of reduced NB density (orientation same as in [A]). There are two additional groups of cas-expressing expressing cells, which are out of focus, in the dorsal and lateral anterior regions of the cephalic lobes (see [D]). (D) During stage 11, the clusters of cas-expressing cells continue to grow in size. Note that NBS in the optic lobe anlage are expressing cas (arrows). (E) Cephalic lobe transverse sections of stage 11 embryos show that most NBS contain cas transcripts (arrow indicates a cas-negative NB). Note that many cephalic lobe NBS have not segregated from the ectoderm. (F) The number of cas-expressing cells per cluster continues to increase until it reaches a maximum in early stage 12 embryos. (C) By stage 13 the clusters of cas-positive NBS have condensed and are now predominantly found on the surface of the growing supraesophageal hemispheres. Note that the average diameter of the cas-positive cell has decreased from earlier stages.

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reveal that many ventral cord NBS express detectable levels of cas message at these stages (Figure 41). As the ventral cord contracts, the number and average diameter of cas-expressing cells per developing neuromere decrease. A reduction in the diameter of the cas-positive cells lining the supraesophageal ganglia is also evident (Figure 5G). These cells are most likely the same cells that expressed cas earlier, as the diameter of NBS decreases with each division (Hartenstein et al., 1987). As the neuropil develops, the cas-positive cells in the ventral cord become arranged in a pattern of triangular clusters per hemisegment (Figures 4J and 4K), an arrangement characteristic of older NBS (Hartenstein and Campos-Ortega, 1984). lmmunostaining with anti-horseradish peroxidase (HRP) antibodies (Jan and Jan, 1982), on embryos where cas-expressing cells have been identified by in situ hybridization, indicates that the cas-positive cells reside predominantly on the ventral and ventral-lateral surfaces of the developing neuromeres (Figures 4) and 4K; Figure 5H). In Figure 5H, there is little or no overlap between the cas-positive cells (blue) and the neurons of the ventral cord and esophageal ganglia (brown), suggesting that most mature neurons do not express the cas transcript. Moreover, cas-expressing cells are not detected on the dorsal surface of the connectives and commissures, indicating that mature glia also do not express cas. During stages 16 and 17, the continued reduction in the levels of cas message throughout the CNS is most evident in the abdominal neuromeres, with a sharp boundary of expression forming between the third thoracic and first abdominal neuromeres (Figure 4L). At this time, cas expression is primarily restricted to cells on theventral surfaceof the thoracic neuromeres and to cells outlining the esophageal ganglia (Figure 4L; Figure 51). cas Expression May Be Necessary for the Development of a Subset of Neuronal Precursors To generate flies with mutations in cas and p/x, the PHlac vector was mobilized by crossing the H23A line to P[ry+;d2-31 (99B) flies, which constitutively produce transposase (Robertson et al., 1988). Imprecise excisions occur at relatively high frequency and often generate small deletions in sequences flanking the P element insertion site (Searles et al., 1982; Voelker et al., 1984). Complementation tests among the resulting third chromosome recessive lethal mutants identified two allelic groups. Cenomic Southern analysis of these lines revealed that imperfect excision events created deletions in PHlac sequences and in sequences flanking the insertion site. One allelic group contained deletions that spanned the cas transcrip-

tion unit (Figure IB), while the other set of alleles contained lesions in the other flank (data not shown). The fact that these two allelic groups complement each other demonstrates that the deletions created in one of these genes do not affect the function of its neighbor. Crosses between flies that were heterozygous for cas deficiencies revealed that homozygous cas mutant embryos failed to exit their egg chambers and showed no signs of movement but they appeared fully developed with normal cuticles and wild-type denticle belt patterns (data not shown). To facilitate the identification of homozygous mutant embryos, alleles were maintained over P[ftz/lacZ] TM3 (Nambu et al., 1990), where embryos hetero- or homozygous for the balancer chromosome display P-gal expression in pair-rule ectodermal stripes and in the CNS due to the fushi-tarazu (ftz) promoter driving /acZ gene expression (Hiromi et al., 1985). Anti-p-gal immunostaining, on embryos where cas or p/x expression was monitored by in situ hybridization, verified that in the homozygous cas mutants no cas transcripts were detected, while p/x expression was indistinguishable from that observed in parental H23A embryos (data not shown). When the embryonic ventral cord axon scaffold of cas null (case) embryos was examined with BP102, a monoclonal antibody (MAb) that recognizes all CNS axons, the level of staining was less than that observed in embryos carrying a functional cas gene. This defect was observed only late in neurogenesis, near the time of dorsal closure. Prior to this stage, when the anterior and posterior commissures are forming, the staining is relatively normal in cas-embryos. The ventral cords from wild-typeand mutant stage14embryos immunostained with the BP102 MAb are shown in Figures 6A and 6B. Staged embryoswere selected for comparison based on morphological criteria and comparable levels of CNS condensation (the axon scaffolds in Figures 6A and 68 are superimposable). As judged by BP102 immunostaining, the overall number of axons within the ventral cord of mutant embryos is reduced, although the overall architecture of the CNS appears normal. lmmunostaining with antibodies that bind to the surface of neuronal cell bodies (anti-HRP and BP104) did not reveal any observable differences between neuron densities and numbers in case and wild-type CNSs at any stage of embryonic development (data not shown). The reduction of axonal density is most striking in the posterior commissures, in the connectives, and in the number of axons projecting laterally from the connectives. To address the possibility that early axon guidance and fasiculation may be affected in embryos lacking cas expression, which may have caused altered axonal outgrowth later in develop-

(H) During head involution there is a progressive reduction in the number of cas-expressing ceils within the cephalic lobes. At this stage and later, most cas-positive cells are found on the ventral surface of the supraesophageal ganglion (plane of section is at the level of the posterior commissure of the second thoracic segment). (I) By stage 17, the number and size of the cas-expressing cells have reduced significantly (lateral view of a supraesophageal hemisphere).

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Figure 6. Loss of cas Function Results in a Reduction in the Axonal Density and in Aberrant en Expression Late in CNS Development Dorsal views of ventral cords, dissected from stage 14 (A and B) and stage 16 (C and D) embryos, following immunolocalization, are presented. Anterior is up in all photographs. (A)Theventral cord axonal network in wild-type embryos stained with the BP102 MAb, which recognizes all CNS axons, reveals the hexagonal array of longitudinal connectives and the anterior (a) and posterior (p) commissures, as well as axons projecting laterally from the connectives. (B) Although the commissures and connectives are relatively intact and have separated normally in cas- embryos, the axonal density detected with the BP102 MAb is significantly reduced, particularly in the connectives and the posterior commissures. (C) en expression in stage 16 wild-type ventral cords is detected in two distinct bilateral clusters of mediolateral and lateral neurons (outer large open arrows), in the ventrally located midline cells (small arrowhead) in the midline NB progeny (arrowhead), in two pairs of leaf-shaped support cells flanking the midline (larger arrow), and in two pairs of interstripe neurons (small arrows) located anterior to the lateral en-positive clusters. There are two sets of leaf-shaped en-positive support ceils per segment

ment, we examined a subset of early growth cones and axons usingthe22ClOMAbthat selectively recognizes the MPI, dMP2, vMP2, aCC, VUM, and SPI neurons (Fugita et al., 1982; Grenningloh et al., 1991; same as SOXII MAb of Goodman et al., 1984). During early fasiculation, 22ClO immunostaining was normal in cas- embryos (data not shown). Moreover, staining with the anti-fasciclin III MAb 2D5 (Pate1 et al., 1987) indicated that RPI growth cone pathfinding was also normal in cas- embryos (data not shown). To determine whether the diminished axonal network correlated with the aberrant expression of other genes known to play a role in CNS development, we examined the expression of ftz, even-skipped, and en (Doe et al., 1988a, 1988b; Pate1 et al., 1989). Prior to stage 13, the immunostaining patterns of all of these proteins in cas-embryos were indistinguishable from their wild-type expression patterns, during both segmentation and early CNS development (data not shown). Starting at early stage 13, only the CNSspecific expression of en was altered (data not shown), suggesting that the differentiation of some of the enexpressing neuronal precursors may be affected in cas-embryos. With increasing contraction of the ventral cord, the alteration in en expression became more dramatic in mutant embryos (Figures 6C and 6D). Normally in stage 16embryos, en is expressed in the contracted ventral cord in the posterior end of each neuromere in two distinct bilateral clusters of neurons, referred to as the mediolateral and lateral neurons and composed of approximately 5 and 6 neurons, respectively (Doe et al., 1991; N. Patel, personal communication). These neurons are derived from NBS in rows 6 and 7, which express en (Pate1 et al., 1989). Anterior to these en-positive neurons, two pairs of interstripe neuronsalsoexpress en (Figure6C). In contrast to the other en-expressing neurons, these neurons originate from non-en-expressing NBS, probably in row 4. For the more lateral pair, their GMC is born at the onset of germ band shortening and clearly expresses en before giving rise to the pair of interstripe lateral neurons shown in Figure 6C. The GMC of the more medial pair arises somewhat later, and it also expresses en before dividing into its progeny neurons. At the midline, en is expressed in six to eight midline NB progeny and in a row of 4ventrally located cells, the origin of which is unknown (Pate1 et al., 1989; Doe et al., 1991; N. Patel, personal communication). en is also expressed in two pairs of leaf-shaped support cells bordering the ventral midline, posterior to the en-positive midline NB progeny (Figure 6C). In stage 16cassembryos, en expression in the inter-

(larger arrow). Note that one of the pairs is out of the focal plane. (D) In case stage 16ventral cords, en expression in the mediolateral neurons is normal. However, the number of lateral neurons expressing en has increased approximately2-fold. en expression is not detected in the interstripe neuronsand in the support cells flanking the midline. A reduction of en expression in the midline NB progeny is also detected.

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Development

stripe neurons is not detected (Figure 6D). en expression in their GMC precursors was also not detected in younger embryos (data not shown).The leaf-shaped en-positivesupportcellsattheventral midlinearealso not detected in stage 16 (Figure 6D) and in younger embryos (data not shown), and the number of midline NB progeny expressing en is also reduced. Moreover, the expression of en in the lateral neurons, coincident with the en stripe, is altered. Whereas in wild-type stage 16 embryos, two clusters of strongly stained enpositive lateral neurons are detected per hemisegment, en is expressed in a more continuous row of cells in the hemisegments of cas-embryos. The number of lateral cells expressing en is approximately 2-fold more than in embryos having a functional cas gene, but the intensity of staining detected in the lateral cluster is lower than that observed in wild-type embryos. In stage 13 mutant embryos the number of lateral cluster GMCs is also elevated (data not shown). Identical patterns of diminished axonal density and altered en expression were observed in A3, the casallele containing the smallest deletion of genomic DNA, and in the other cas- allele examined, Al (see Figure IB).

the four repeats in cas. The presence of two additional sets of potential zinc ligands, the first G--Hz in each of the repeats, distinguishes the putative cas metalbinding domain from other zinc-binding proteins and allows for the potential binding of two zinc atoms per motif, creating the possibility for multiple zinc-ligand tetrahedral coordination complexes. The conserved spacing between the cas motifs (19 amino acid long linkers), as well as the invariant phenylalanine residue in the linker, suggests that this region is also important for function. In addition to the putative DNA-binding domain, cas contains regions that may be involved in the activation of transcription. Functional analysis of known transcription factors has demonstrated that acidic domains (Ma and Ptashne, 1987; Gill and Ptashne, 1987), glutamine-rich regions (Courey and Tjian, 1988; Courey et al., 1989), and proline-rich domains (Mermod et al., 1989) are involved in transcriptional activation. All three of these potential activating domains are part of the predicted cas protein. Similar to cas, multiple runs of homopolymeric amino acids are part of the mastermind(Smoller et al., 1990) and prosper0 (Vaessin et al., 1991) proteins. Both genes function as regulators in Drosophila nervous system development.

Discussion W e have identified a gene, cas, that encodes a putative transcription factor, whose expression appears necessary for the normal development of a subset of neuronal precursors. cas was identified by the analysis of genes flanking a P element integration at chromosomal subdivision 83C. The PHlac vector inserted into the 3’end of its close neighbor p/x without disturbing its function. However, the expression pattern of the reporter gene mimics theexpression of cas. The selective use of the cas cis regulatory elements over those controlling the expression of p/x indicates that our chimeric minimal promoter is not completely permissive to flanking enhancers. To our knowledge, the integration of an enhancer detection vector into a gene and its reporter construct reflecting the expression pattern of an adjacent gene is a rare event. CJS Has Many Features Common to Transcription Factors A number of features found in the primary structure of the cas protein suggest that it may be involved in the regulation of transcription. cas contains a putative metal-binding domain that shares homology with other zinc finger DNA-binding proteins (Klug and Rhodes, 1987; Vallee et al., 1991). Four consecutive repeats, spanning 229 amino acids each, contain a novel C2-H2C2-H2 motif, with the second CT-HZ part of each motif being similar to the DNA-binding TFIIIA zinc fingers (Miller et al., 1985). Like TFIIIA, the Cl-HZ zinc ligands are separated by 12 or 13 residues. Moreover, the aromatic residues (phenylalanines/tyrosines) considered important for the secondary structure of the finger region of TFIIIA are found in two of

cas Function Is Required for the Normal Development of a Subset of CNS Neurons and for Normal CNS-Specific Expression of en The developmental profile of cas expression suggests some interesting functions for its gene product. Based on the fact that the majority of cells that express cas mRNA are located between the ectoderm and the mesoderm, combined with the enlarged and rounded appearance of these cells, we conclude that cas is expressed primarily in a subset of NBS during neurogenesis. No lateral-ventral cord NB-specific cas expression was detected when the initial SI and SII pulses of NB delamination occur. A significant number of ventral cord NBS expressed cas only after the final Sill phase of delamination (in stage 11 or older embryos; see Hartenstein and Campos-Ortega, 1984; Hartenstein et al., 1987). The generally enlarged appearance of those cas-positive NBS suggests that many arose during the Sill phase of delamination, although a minority of the cas-positive cells are smaller and may be NBS from the earlier phases of delamination that have gone through a number of divisions (see Hartenstein et al., 1987). The developmental time point when cas expression is initially detected in NBS stronglysuggests that its gene product is not involved in their primary selection, like the proneural and neurogenic genes, but functions at a secondary stage in neurogenesis. W e observe two CNS-specific defects, namely, a reduction in the axonal scaffold and an alteration in the pattern of expression of the homeodomaincontaining protein en. Both phenotypes are observed relatively late in neurogenesis. As the expression of en (as well as four other segmentation genes; data not shown) in

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ectodermal stripes is normal in mutant embryos and their larval denticle belts are unaltered, this suggests that late embryonic lethality is most likely due to the absence of cas expression in neuronal precursors during CNS development. Klambt et al. (1991) have shown that the separation of the commissures is dependent on the migration of the midline glia. Thus, although cas is expressed in the midline glioblasts, the maturation of the midline glia does not seem to be affected by the absence of cas expression in their precursors, as the commissures separate normally in cas- embryos. Moreover, functional compensation by other proteins may explain the relatively weak phenotype that we observe in casembryos. For instance the expression domain of the zinc finger gene snail in lateral NBS partially overlaps that of cas (Alberga et al., 1991) and may compensate for the lack of cas function. Such functional redundancy is not uncommon with proteins involved in neurogenesis (e.g., Elkins et al., 1990). Aswedetected noalteration duringearlyaxonogenesis in cas- embryos, one possible explanation is that many cas-expressing NBS give rise to neuronal progeny that differentiate and undergo axonogenesis later, after the primary hexagonal scaffold has formed. Indeed, large numbers of cas-expressing cells are detected only by the middle of stage 11. According to Hartenstein et al. (1987), SI NBS should have gone through three to four mitoses, SII NBS, two to three, and Sill NBS, perhaps one mitosis, suggesting that a significant number of neurons develop from the progeny of thoseearlydivisions devoid of the requirement for cas function. The fact that the nervous system-specific expression of en in a subset of neuroectodermal cells is disturbed late in embryogenesis, with the lateral interstripe neurons and the leaf-shaped midline cells that normally express the gene failing to do so, suggests that some neuronal precursors have differentiated incorrectly (see Figure 6). The aberrant expression of en in the lateral cluster and in the midline NB progeny of cas- mutants is noteworthy because from simultaneous in situ hybridization analysis with cas and en probes, we know that many cas-expressing NBS are located in rows 6 and 7 from stage 11 onward, the position of those NBS that give rise to these enpositive neurons (Pate1 et al., 1989). Moreover, at the height of cas expression, some NBS in each of the seven NB rows per segment express the cas gene. Thus, our finding that the progeny of NBS in row 4, i.e., the interstripe en-expressing neurons (N. Patel, personal communication), are affected in cas- embryos is not unexpected. cas May Be a Neuronal Precursor Gene The restricted expression of cas to a subset of NBS, the altered pattern of en expression in the CNS, combined with the abnormal appearance of the axonal network in cas- mutants suggest that cas may be essential for the differentiation of a subset of neuronal

precursors. It should be noted, however, that theanalysis of CNS lineages is limited by the availability of well-established lineage markers, making it difficult to distinguish whether cas expression is necessary for the identity of individual NBS or their differentiation. That having been said, however, cas shares many properties in common with genes that have been classified as neuronal precursor genes (Vaessin et al., 1991). Like prospero, cas expression is not detected in NBS until delamination is completed, and mature neurons do not express the cas transcript. Moreover, both genes are transiently expressed in the blastoderm and also in midline glioblasts, but not in their progeny. Like the neuronal precursor genes asense (T8) and prosper0 (Gonzalez et al., 1989; Vaessin et al., 1991), cas encodes a protein with a putative DNAbinding motif. However, in contrast to the neuronal precursor genes thus far described, which are expressed in both the developing CNS and PNS (Gonzalez et al., 1989; Doe et al., 1991; Vaessin et al., 1991), cas expression is restricted to a subset of CNS precursor cells. Within the developing CNS, cas expression is also considerably more restricted than that of deadpan,asense(T8),andprospero,in thatonlysomeofthe total complement of NBS express the cas transcript. Hence, it is perhaps not surprising that the mutant phenotype of cas is more subtle than that of neuronal precursor genes with broader expression domains. For example, absence of prosper0 expression, which is normally expressed in most, if not all, neuronal precursors, results in aberrant expression of the segmentation genes en, ftz, and evenskipped, as well as misrouting of motor axons in the PNS, the absence of longitudinal axons, and the presence of only one commissure per segment in the CNS (Doe et al., 1991; Vaessin et al., 1991). Vaessin et al. (1991) have shown that prosper0 regulates the expression of the other neuronal precursor genes deadpan and asense (78), suggesting that there is a hierarchy of expression among neuronal precursor genes. In the case of cas, the expression pattern of deadpan and prosper0 is wild type in cas- mutants (data not shown). Thus cas may be downstream of these genes in the neuronal precursor regulatory gene hierarchy. This hypothesis can be tested in these neuronal precursor gene mutant backgrounds and may provide additional clues to the pathways for neuronal precursor differentiation. Experimental

Procedures

Drosophila Transformations and Imperfect Excision Mutants Df(7Jw67c2,y (Lindsley and Zimm, 1987) embryos were injected with 300 n&I of PHlac (construct H; Kassis et al., 1991) and 150 nglpl of the helper plasmid px25.7wc (Karess and Rubin, 1984) using standard techniques. Transformed flies were selected based on their white+ phenotype. g-Gal histochemistry was performed on embryos according to Hiromi et al. (1985). Of the 12 lines generated, H23A was 1 of 10 that exhibited temporally restricted /ad staining in the embryonic nervous system. Cytogenetic locations of the P element vector insertions were determined by salivary gland polytene chromosome in situ hybridization using vector-specific probes accordingto Kassis et al. (1991).

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801

Homozygous lethal cas and p/x alleles were generated by transposase-mediated mobilization of the PHlac vector in H23A flies using the immobilized transposase insertion line P[ry+;dZ3](99B) (Robertson et al., 1988) as described in Kassis et al. (1991). Briefly, Fl males with mosaic eye color were backcrossed to Df/7)w67c2,y virgin females, and their sons, with eye color that differed from H23A, were mated to virgin females from the third chromosome balancer stock yw;TM3,y+ripPsepbx34ee”Ser/Sb. Sibling crosses were then carried out to identify third chromosome homozygous lethal lines. Allelic groups were identified by complementation crosses between the homozygous lethal lines. For the lethality analysis, heterozygous flies were backcrossed to Df(l)w67c2,y. Phenotypic analysis of homozygous mutant embryos was carried out on lines balanced with TM3 P[ftz//acZ] (Hiromi et al., 1985; Nambu et al., 1990). Southern analysis of genomic DNA, employing standard techniques, was performed oneachofthecasallelesusingprobesl-6(Figure1).DNAprobes were labeled with [P*P]dCTP by the random primer method of Feinbergand Vogelstein (1983) usinggel-purified DNAfragments as templates. Molecular Cloning and DNA Sequence Analysis Agarose gel electrophoresis, restriction enzyme digestions, Northern and Southern transfers, and hybridization conditions were according to standard procedures. Amplification of genomic DNA flanking the 5’end of the H23A insertion (Figure 1) was carried out by the inverse polymerase chain reaction (PCR) protocol of Ochman et al. (1988) with the following modifications. Cenomic DNA (600 ng) was cut with either EcoRl or Xbal restriction enzymes. Following phenol-chloroform extractions and ethanol precipitations, the DNA was ligated with T4 DNA ligase (12.5 U, New England Biolabs) in a total volume of 400 ul for 20 hr at 15OC. After phenol-chloroform extractions and ethanol precipitations, the DNA was used as templates for PCR amplifications. Standard PCR reagents (Perkin-Elmer Cetus) and conditions were employed (38 cycles of 94OC for 1.5 min, 55OC for 1.5 min, and 72°C for 3 min, followed by 72°C for 7 min). The sense inverse PCR primer S-GCAAGCATACGTTAAGTGGATC-3’ corresponded to P element sequences adjacent to the 31 bp inverted terminal repeat (O’Hare and Rubin, 1983), while the antisense primer S-CATTAACCCTTAGCATCTCCGTGG-3’corresponded to the polycloning site (containing the EcoRl and Xbal sites) located at the 5’ end of the P element vector (Figure 1). Agarose gel-purified inverse PCR products from EcoRI- and Xbal-cut DNA were used as probes to screen duplicate lifts of an Oregon R genomic phage library (Promega). Recombinant clones that hybridized to both probes were plaque purified and characterized. Adjacent 6.0 kb and 3.5 kb genomic EcoRl fragments were then used as probes (#2 and #3, Figure 1) to isolate cas and p/x cDNAs from embryo cDNA libraries constructed in lgtl0 (Poole et al., 1985) and 5zap (Stratagene) from mRNA from the Oregon R strain. Both strands of genomic DNA and overlapping cDNAs in the Bluescript KS+ vector (Stratagene) were sequenced using the T3 and T7 primers or custom primers by the chain termination sequencing method (Sanger et al., 1977J modified for Sequenase (v.2.0, U. S. Biochemicals). Data base searches and sequence comparisons wereconducted using the FASTA package (Pearson and Lipman, 1988) with the Zinc Finger Gene Data Base Service (Jacobs and Micheals, 1990). Analysis of RNA Expression PolyfA)’ RNA from different developmental stages was size fractionated in a formaldehyde-agarose gel and blotted onto Nytran membrane (Schleicher & Schuell) using standard procedures. Hybridization with DNA probes and washes were carried out according to the manufacturer’s specifications (last wash, 0.1 x SSPE, 0.1% SDS at 65OC for 30 min). DNA probes were prepared by the random primer method from gel-purified cDNA fragments that were generated by PCR using cDNA templates and custom primers. The cas probes 2a and 2b correspond to se(Figure 2). The quences 1769-2365 and 2538-2958, respectively

p/x probe (3a) corresponds to a 765 bp p/x cDNA fragment that was amplified using the PCR primers S’CTACAGACTCAGTAATGCC3’and 5’-CGGAGGAAGCATATCAGCC-3’. The ribosomal protein 49 probe used was the HRO-6 subclone (see O’Connell and Rosbash, 1984). Whole-mount in situ hybridization to embryoswas performed according to Tautz and Pfeifle (1989) using digoxigenin-labeled DNA probes that were generated by the random primer method with the Genius Kit (Boehringer Mannheim). The probes used included the EcoRl genomic fragments (Figure 1) and the PCRgenerated cas cDNA fragments described above. For the detection of en transcripts a 1 kb fragment containing the first exon was used as template (Poole et al., 1985). lmmunohistochemistry The procedure used for double antibody staining was adapted from a protocol provided by Nipam Pate1 (Carnegie Institute, Baltimore, MD). Briefly, embryos that had been dechorionated in Chlorox for 3-4 min were rinsed in water and then fixed for 10 min by shaking in a I:1 mix of heptane and PEM-FA (PEM is 0.1 M PIPES, 2 m M EGTA, 1 m M MgS04 adjusted to pH 6.95 with concentrated HCI. PEM-FA is 9 parts PEM mixed just before use with 1 part 37% formaldehyde [Fisher]). Vitelline membranes were removed by replacing the aqueous phase with an equal volume of methanol followed by shaking for 30-60 s. Embryos were stored in methanol at -2OOC. Following rehydration in phosphate-buffered saline containing 0.1% Tween 20 (PBT), embryos were incubated in PBTfor 30-60 min at room temperature and then were preincubated in PBT containing 4% horse serum (Difco, PBT-HS) for a minimum of 1 hr at room temperature. Then they were incubated with the primary antibody diluted in PBT-HS overnight at 4OC or for l-2 hr at room temperature, after which they were washed seven to ten times with PBT over a 90 min period. The incubation with the secondary antibody conjugated to biotin in PBT-HS (Vector Labs) was for l-2 hr at room temperature. After washing as above, embryos were incubated with streptavidin-HRP (BRL) for 15-20 min and washed seven to ten times with PBT and then with 50 m M citric acid, 50 m M ammonium acetate (pH 5) (HRP buffer; Kania et al., IVVO), prior to incubation with 0.1 ml of HRP buffer containing 0.006% HzOz, 0.02% NiClz and CoCl?, and 2.7 mglml diamnobenzidine, according to Kania et al. (1990). Color development was monitored microscopically, and at the appropriate time the reaction was stopped by washing three to four times with PBT. Embryos were then incubated in PBT-HS overnight at 4°C and then with rabbit anti-B-gal antibody for l-2 hr at room temperature. The antibody incubationstepsandwashingstepsused wereasoutlined above. Prior to color development embryos were washed with 0.1 M Tris (pH 7.2) in place of HRP buffer. Diaminobenzidine (0.5 mgi ml) (BRL) and H20, (0.006%) were used as substrates for color development, which was stopped by washing with PBT. In some cases the HRP was developed according to Kania et al. (1990) with nickel and cobalt enhancement (see above). For immunolocalization after mRNA detection, embryos were briefly dehydrated through a graded ethanol series following in situ hybridization. Then theywere rehydrated and rinsed in PBT. The antibody incubation, washing, and color development steps were as described above. Stained embryos were mounted in 70% glycerol for observation, and in somecases the ventral cord was dissected using fine needles (Hamilton, #90033), as described by Pate1 et al. (1989). The antibodies and dilutions used were rabbit anti-P-gal (Promega 1: 250), rabbit anti-HRP (Cappel 1:500), anti-en MAb (l:l), MAb BP102 (1:5), and MAb 2D5 (1:5). All secondary antibodies were preabsorbed with whole embryos. Sectioning of Plastic-Embedded Embryos Following whole-mount in situ hybridization and immunohistochemistry, embryos were postfixed in 2% glutaraldehyde in phosphate-buffered saline and then individually encased in albumin plugs to facilitate handling (Christian, 1991). Standard procedures for the dehydration and Epon embedding of tissues were employed. An LKB ultramicrotome, equipped with glass

NWKNl 802

knives, was used to cut 10 ~rrn thick sections.

serial transverse

or sagittal

Acknowledgments We are indebted to Dr. Nipam Pate1 for communicating unpublished results, for his advice on the antibody staining and the mutant analysis, as well as for giving us the anti-engrailed, BP102, BP104, and 2D5 MAbs. We are also grateful to Drs. Seymour Benzer, Yuh Nung Jan, and the members of their laboratories for antibodies used during the course of these studies (22C10, Benzer lab; anti-deadpan and -prospero, Jan lab). Drs. Corey Goodman and Vanessa Auld kindly provided the F263 lad line. We would like to thank Dr. Peter Vos for his help in preparing the figures, Dr. George Micheals of the Zinc Finger Data Base for help with the similarity searches, and the National Institute for Neurological Disorders and Stroke electron microscopy facility for assistance. Sincere thanks to Drs. Jim Battey, Gregory Dressler, Colin Hodgkinson, Jim Kennison, Mary Whiteley, and Andreas Zimmer for providing helpful comments on the manuscript. We are also indebted to Dr. Harold Gainer for his advice and support throughout the duration of this project and for suggesting the Dioscuri as names. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

June 2, 1992; revised

August

17, 1992.

Synergistic transcription

activation by the glutamine-rich factor Spl. Cell 59, 827-836.

domains

of human

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CenBank

Accession

The accession

Number

number

for the castor

DNA sequence

is L04487.