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1 Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA
2 Genome Biology Lab, Center for Genetic Resource Information, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan
3 Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
*Author for correspondence (e-mail: or38{at}columbia.edu)
Accepted February 27, 2001
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Homeobox, C. elegans, Neuronal differentiation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A prominent class of transcription factors that acts to determine terminal differentiation characteristics of neurons is the LIM homeobox class of regulatory factors (Hobert and Westphal, 2000). In the mouse, Isl1 is required for early aspects of motoneuron differentiation (Pfaff et al., 1996), while later acting Lhx genes, such as Lhx3 or Lhx4 define the identity of specific subtypes of motoneurons in the spinal cord (Sharma et al., 1998; Tsuchida et al., 1994). Lim1 and Lmx1b coordinately direct axonal outgrowth patterns in vertebrate limbs (Kania et al., 2000). In Drosophila, genetic and neuroanatomical studies on apterous, islet and lim3 have revealed their involvement in several aspects of neuronal differentiation, including axon fasciculation, pathfinding and neurotransmitter choice (Lundgren et al., 1995; Thor et al., 1999; Thor and Thomas, 1997). As in vertebrates, combinatorial expression of Lhx genes in Drosophila determines motoneuron differentiation and axonal targeting choices in the ventral nerve cord (Thor et al., 1999).
As revealed by the genome sequencing project, C. elegans contains seven Lhx genes, which are expressed in largely non-overlapping sets of neurons (Hobert and Westphal, 2000). mec-3, one of the founding members of the Lhx gene family, represents the end-point in a cascade of transcriptional regulatory factors that determines the fate of touch sensory neurons (Mitani et al., 1993; Way and Chalfie, 1988). mec-3 directly regulates the transcription of genes required for touch sensory neuron function such as the mec-7 ß-tubulin gene and the mec-4 ion channel gene (Duggan et al., 1998). The lin-11, lim-6 and ceh-14 genes act to control as yet undefined subtype characteristics of specific neurons and other tissue types (Cassata et al., 2000; Freyd et al., 1990; Hobert et al., 1998; Hobert et al., 1999), while the lim-4 gene functions as a binary cell-type switch in a class of odorsensory neurons (Sagasti et al., 1999).
There are three central aspects of Lhx gene function that remain poorly understood in any system. First, the precise nature of cellular defects evoked by loss of Lhx protein function is often unclear owing to the paucity of specific cell fate and neuroanatomical markers. Second, with a few exceptions (Benveniste et al., 1998; Duggan et al., 1998), target genes of Lhx proteins in the nervous system have remained largely elusive. Finally, the nature of upstream regulators of Lhx genes and hence the integration of Lhx gene function into larger transcriptional regulatory cascades or networks is little understood.
We address these key questions for the C. elegans Lhx gene ttx-3. We have previously shown that ttx-3 mutant animals display defects in thermoregulatory behavior that mimic the defects seen upon ablation of a class of interneurons in the thermoregulatory neuronal circuit, the AIY interneuron class (Hobert et al., 1997). The expression of ttx-3 in the AIY interneurons of wild-type animals hence suggested that ttx-3 is required for the AIY neurons to function appropriately (Hobert et al., 1997). We now show that within the AIY interneurons ttx-3 is part of a transcriptional regulatory cascade, which involves two other homeobox genes, ceh-10 and ceh-23. We demonstrate that ttx-3 is not required for the adoption of pan-neuronal features of the AIY interneurons but is rather required for the subtype specification of this interneuron class. We describe several genes that participate in defining AIY cell fate and that represent either direct or indirect targets of the ttx-3 transcription factor. Neuroanatomical defects caused by loss of ttx-3 function resemble those seen in axon pathfinding mutants, suggesting that ttx-3 is also involved in determining patterns of axonal outgrowth. Furthermore, we show that a transcription factor regulated by ttx-3, the ceh-23 homeobox gene, participates in the regulation of a presumptive ttx-3 downstream target gene.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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otEx44 – otEx49, otEx97 – otEx101=Ex[ttx-3full::gfp; pRF4]
otEx57=Ex[ttx-3full::gfp; pBX]
otEx65=Ex[unc-119::ttx-3; pRF4]
otEx75=Ex[unc-33::gfp; unc-4(+)]
otEx93=Ex[pflp-1::gfp; pRF4]
otEx95, otEx96=Ex[unc-119::ttx-3; unc-122::gfp]; unc-122::gfp is a new injection marker (see below)
utEx54=Ex[C36B7.7::gfp; pRF4] (courtesy of T. Ishihara and I. Katsura).
Chromosomally integrated arrays
evIs111=integrated Ex[F25B3.3::gfp;dpy-20(+)] (kindly provided by Joe Culotti)
kyIs5IV=integrated Ex[ceh-23::gfp; lin-15(+)] (Forrester et al., 1998)
mgIs18IV and mgIs32III=independently integrated Ex[ttx-3prom::gfp; plin-15]
otIs7=integrated Ex[zig-2::gfp; pRF4] (O. Aurelio and O. H., unpublished)
otIs14=integrated Ex[zig-3::gfp; pRF4] (O. Aurelio and O. H., unpublished)
otIs24=integrated Ex[sre-1::gfp; dpy-20(+)]
otIs33IV=integrated Ex[kal-1::gfp; pBX] (H. Buelow and O. H., unpublished)
otIs45=integrated Ex[unc-119::gfp]
otIs62, otIs123=integrated Ex[psra-11-3::gfp; pRF4] and integrated Ex[psra-11-3::gfp; unc-4(+)], respectively
otIs97IV, otIs98, otIs99=integrated otEx65
otIs107=integrated adEx1450
otIs117=integrated otEx75.
The injection marker pBX was used in a pha-1(e2123ts) background; unc-4(+), lin-15(+) and dpy-20(+) were used in unc-4(e120), lin-15(n765ts) and dpy-20(e1362) backgrounds, respectively; and pRF4 and unc-122::gfp in a wild-type background. (The protocol for integrating extrachromosomal arrays can be found at http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/hobert/protocols.htm)
Behavioral assays
Thermotaxis assays were performed as described (Hedgecock and Russell, 1975). In our specific set-up, an aluminum slab bridged two water baths, one at 61°C, the other at 5°C; the ambient temperature was 25°C. Animals were grown at 20°C and assayed on a gridded 10 cm square petri dish that had equilibrated on the aluminum plate to establish a stable gradient from 22°C to 16.5°C (measured with a surface temperature probe). Animals were placed into the center of the gradient. After an assay time of 40-60 minutes, the animals were anaesthetized. For scoring, the plate was divided into six regions of 1.3 cm width (see Fig. 3A) and animals were counted in each region. Animals that had not left the 1.7 cm2 quadrant in which they had been initially placed were not scored.
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Isolation and analysis of ceh-23 null mutant animals
A ceh-23 null allele was isolated using a transposon-tagging strategy (Zwaal et al., 1993). First, a Tc1 insertion in the ceh-23 gene was isolated by PCR screening of a Tc1 library made from the MT3126 strain, using Tc1- and ceh-23-specific primers. The isolated strain YK16[mut-2(r459); ceh-23(ms16::Tc1); dpy-19(n1347)III] contained the Tc1 insertion in the last exon in the middle of the homeodomain at position 12957 (numbering of cosmid sequence ZK652). YK16 was then PCR screened for Tc1 excision events using ceh-23-specific primers. An imprecise Tc1 excision was identified in strain YK23[mut-2(r459); ceh-23(ms23); dpy-19(n1347)III] at position 11098-12961. The junction sequence is: 5'...TTGAGCTTT-G[deletion]AGTCGGAGCA...3'. YK23 was outcrossed several times with Bristol N2. As YK23 has no obvious phenotype, the genotype was scored using a triplex PCR, in which the three primers U2, D2 and D3 gave a 1 kb product on homozygous ceh-23(ms23) animals, a 1 kb/730 bp doublet on heterozygous animals and a 730 bp product on wild-type animals. The primer sequences are: U2, 5'-TACCTGCACAACACACTGAC-3'; D2, 5'-TGGCAAAATCATGG-CGGAAC-3'; and D3, 5'-TTGGATCCAGCAATTGTTTGAA-GAACGT-3'.
ceh-23::gfp expression in several neuron classes other than AIY, including ADF, ADL, AWC, AFD, PHA, PHB and CAN (Forrester et al., 1998), prompted us to assay differentiation of some of these neurons in ceh-23 null mutants. We found that the AFD and ASE cell fate markers gcy-8::gfp and gcy-5::gfp, respectively, the ADF, ADL, PHA, PHB cell fate marker srb-6::gfp, the AWC cell fate marker str-2::gfp and the CAN cell fate marker kal-1::gfp were all correctly expressed in ceh-23(ms23) animals (data not shown). Moreover, ADL and PHA/PHB show normal dye filling behavior. In those cases where the neuron could be visualized in relative isolation (AFD, ASE, AWC, CAN, PHA/B), the anatomy of the neuron appeared wild type.
Genetic screen for new ttx-3 alleles
To screen for new ttx-3 alleles, we made use of two previously described consequences of loss of ttx-3 function, the partial suppression of the dauer constitutive phenotype of a daf-7 loss-of-function allele and the loss of positive autoregulation of the ttx-3 gene (Hobert et al., 1997). A daf-7(e1372)III; mgIs18IV strain, that contains an integrated ttx-3prom::gfp array, was mutagenized with EMS at the L4 larval stage. The F1 offspring were allowed to grow to the adult stage at 15°C. Eggs were prepared from these animals using standard bleaching protocols. Approximately 100-300 eggs of this F2 generation were placed on single plates and cultivated at 25°C. Only plates that contained dauer bypass mutants quickly grew to starvation. Those plates were chunked and again grown to starvation at 25°C twice, in order to enrich for homozygous animals with the bypasser phenotype. Animals were isolated from plates that showed downregulated ttx-3prom::gfp expression, and after testing for X-linkage of the defect the ttx-3 genomic locus was sequenced. Out of >100,000 haploid genomes screened, we retrieved three ttx-3 alleles, mg158, ot22 and ot23. In addition, we obtained another ttx-3 allele, nj14, that was isolated by Ikue Mori in an independent screen for mutants that affect ttx-3prom::gfp expression.
Pan-neuronal marker genes
We reasoned that the unc-33 gene, which appears to affect axon pathfinding throughout the nervous system (Hedgecock et al., 1985; McIntire et al., 1992), may be pan-neuronally expressed. We fused a 2.7kb genomic fragment that is immediately upstream to the start site of the previously described unc-33 mRNA transcript (Li et al., 1992) to gfp and found this reporter gene to be expressed throughout the whole nervous system (see Fig. 6A). Like unc-119::gfp (Maduro and Pilgrim, 1995), the unc-33::gfp reporter is expressed in dividing neuroblasts at pre-comma embryonic stages (data not shown). unc-33::gfp expression can be observed outside the nervous system in two amphid socket cells and weakly in non-neuronal pharyngeal cells. We also generated a fusion of the gfp gene to the promoter of the F25B3.3 gene, the C. elegans ortholog of the Ca2+-regulated ras nucleotide exchange factor CalDAG-GEFII/RasGRP, which is ubiquitously expressed in the vertebrate nervous system (Ebinu et al., 1998; Kawasaki et al., 1998). F25B3.3 and vertebrate CalDAG-GEFI/RasGRP both contain a unique combination of domains, a nucleotide exchange factor domain, two EF hands and a Cys-rich diacylglycerol binding motif; the respective C. elegans and rat proteins show 35% identity and 57% similarity throughout the whole length of the protein. The F25B3.3::gfp construct contains 3.5 kb genomic sequences upstream of the predicted ATG start site and the first six amino acids of the predicted protein and is exclusively expressed throughout the nervous system in C. elegans (see Fig. 6A). In contrast to unc-119::gfp and unc-33::gfp, which are already expressed in neuroblasts, F25B3.3::gfp is a postmitotic pan-neuronal marker, i.e. its onset of expression is observed after the terminal division of neurons (around 450 minutes of embryonic development; see Fig. 6B).
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We examined ectopic expression of AIY fate markers other than ttx-3prom::gfp by using the extrachromosomal line otEx65, which we crossed with otIs33 (kal-1::gfp), kyIs5 (ceh-23::gfp), otIs107 (ser-2::gfp) and otIs123 (sra-11::gfp). As kal-1::gfp and ser-2::gfp are already expressed in a substantial number of neurons in the head ganglia it was difficult to determine ectopic expression outside their normal expression domains in the head ganglia; it was clear, however, that pan-neuronal expression of ttx-3 did not cause a significant broadening of the expression of either of the markers in the nervous system.
UNC-17 antibody staining
We used a modification of the freeze-cracking method kindly provided to us by J. Duerr. Briefly, mixed stage animals that were grown at 20°C were transferred onto poly-L-lysine coated slides and covered with a coverslip. Slides were then frozen in liquid nitrogen and animals were cracked by flipping coverslips off. Animals were quickly fixed in methanol and acetone, incubated in blocking serum and co-stained with mouse monoclonal anti-UNC-17 (kindly provided by J. Duerr and J. Rand) and polyclonal anti-GFP (Molecular Probes) antibodies.
Other DNA constructs
The ttx-3 expression constructs ttx-3resc and ttx-3prom::gfp shown in Fig. 1B, have been described already (Hobert et al., 1997); ttx-3full::gfp represents a GFP PCR fusion product in which the genomic region contained within ttx-3resc has been PCR-fused to gfp::unc-54-3'UTR from the pPD95.75 vector. (Protocol for PCR fusion can be found at: http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/hobert/protocols.htm). The sra-11::gfp construct is termed psra-11-3::gfp and contains a 3.9 kb XbaI genomic fragment, which encompasses most of the coding region of sra-11 and 2.8 kb of sequence upstream of the predicted start codon. This construct is different from the one previously published (Troemel et al., 1995) and is more stably expressed in similar neuronal subtypes, namely AVB and AIY, but is also weakly expressed in AIA (see Fig. 5). The flp-1::gfp construct contains a genomic fragment from the 5' region of the flp-1 gene (Nelson et al., 1998), which extends from position -513 to -9 relative to the flp-1 start codon. This construct is strongly and exclusively expressed in the AVK interneuron pair. unc-122::gfp is a reporter gene construct that is strongly and exclusively expressed in coloemocytes and used as an injection marker (P. Loria and O. H., unpublished). Genomic fragments were amplified by PCR and subcloned into the standard GFP vectors pPD95.75 or pPD95.77. kal-1::gfp, zig-2::gfp, zig-3::gfp, ser-2::gfp and C36B7.7::gfp will be described elsewhere.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown schematically in Fig. 1C, two other homeobox genes are also expressed in AIY, ceh-10 and ceh-23. ceh-10 was identified by sequence homology to homeobox genes (Hawkins and McGhee, 1990) and independently in a screen for mutants affecting neuronal migration (Forrester et al., 1998); it contains a paired-type homeobox and represents the C. elegans ortholog of the vertebrate Chx10/Chx10-1 genes, which are expressed in a restricted set of interneurons in the retina and spinal cord (Chen and Cepko, 2000). ceh-23 was identified due to its proximity to the C. elegans Hox cluster (Wang et al., 1993) and represents a diverged homeobox gene with no clear ortholog in other species and no conserved motif outside the homeodomain (Fig. 2A).
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ceh-23 mutant allele
We isolated a ceh-23 deletion allele, ms23, through PCR-based screening of a Tc1 transposon library (see Materials and Methods). ceh-23(ms23) contains a 1864 bp deletion that extends from 301 bp 5' of the ATG start codon into the last exon of the gene and deletes more than 75% of the coding sequence, including half of the homeobox (Fig. 2B). It is thus likely that this allele represents a complete loss-of-function allele. Despite expression of ceh-23 in the CAN neurons, a neuron class that is essential for survival (Forrester and Garriga, 1997), ceh-23(ms23) mutant animals are viable and display no obvious neuroanatomical, morphological or locomotory abnormalities (see Materials and Methods).
ttx-3 mutant alleles
Prior to this study, only one ttx-3 allele, ks5, was available. ks5 mutant animals show thermotaxis defects that are similar to those seen in animals in which the AIY interneurons have been ablated; additionally, when placed over a chromosomal deficiency, ttx-3(ks5) behaves as a complete loss-of-function allele (Hobert et al., 1997). However, the molecular nature of ks5, a splice site mutation, left open the possibility that small amounts of truncated and partially active TTX-3 protein could be generated through incorrect splicing. To undertake a detailed analysis of AIY interneuron fate in ttx-3 mutants, we thus sought to identify new mutant alleles of the ttx-3 gene, whose molecular nature may more unambiguously imply a complete loss of ttx-3 function. Through screening for mutants with characteristic ttx-3-like defects, we identified a total of three new ttx-3 alleles, termed mg158, ot22 and ot23 (see Materials and Methods). All three alleles result in characteristic thermotaxis defects (Fig. 3A), which mimic the thermotaxis defects seen upon laser ablation of the AIY interneurons (Mori and Ohshima, 1995). Each allele was defective in autoregulation of a ttx-3prom::gfp reporter gene (Fig. 3B,C) and suppressed the dauer-constitutive phenotype of daf-7(e1372) at 25°C (Fig. 3D). While the behavioral defects of the new alleles were comparable with those seen in the previously available ks5 allele, the autoregulatory defects of the new alleles were stronger than in those observed with the ks5 allele (Fig. 3B,C). Sequencing of the new ttx-3 alleles confirmed the notion that at least two of them (mg158, ot22) are likely to be null for ttx-3 function (Fig. 2C).
ceh-10, ttx-3 and ceh-23 constitute a regulatory cascade of transcription factors in the AIY neurons
The availability of mutant alleles in all three homeobox genes has allowed us to address whether these transcriptional regulators act either in a linear pathway or, alternatively, in independent pathways within the AIY interneuron class. In a presumptive linear pathway, ceh-10 would likely be the most upstream acting gene, as ceh-10 expression in AIY is only observed during embryogenesis and fades after hatching (Svendsen and McGhee, 1995), while expression of both ttx-3 and ceh-23 is initiated in embryogenesis and maintained throughout adulthood (data not shown). Thus, we first tested whether ceh-10 is involved in initiation of ttx-3prom::gfp and ceh-23::gfp expression. We found that in the ceh-10 null allele gm58, ttx-3prom::gfp expression is abolished in the AIY interneurons (Fig. 4A). Conversely, a ceh-10::lacZ reporter gene construct is still expressed in ttx-3 mutants (data not shown). Forrester et al. have previously shown that ceh-10 is required for ceh-23::gfp expression in AIY (Forrester et al., 1998). These findings demonstrate that ceh-10 either directly or indirectly regulates both ttx-3 and ceh-23 expression.
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ttx-3ceh-23 regulatory cascade couples to AIY interneuron development and function. Cell type diversification in the nervous system is a consequence of differential gene expression within a given neuronal subtype. Hence, individual neuronal subtypes are defined by the expression of a specific and unique combination of molecular markers. The AIY interneuron class can be described by such a unique combinatorial gene expression profile: in a genome-sequence based survey of expression patterns of putative neuronal cell surface molecules, we identified a FnIII-domain-containing cell surface protein with homology to the human Kallmann’s syndrome gene, termed KAL-1, which is expressed in AIY and several other head neurons (H. Buelow, Z. A.-G. and O. H., unpublished). As the AIY neurons may constitute an integration point of temperature and food sensory inputs (Hobert et al., 1997; Mori and Ohshima, 1995) and since serotonin (5-HT) is involved in food signaling (Sze et al., 2000), we reasoned that 5-HT receptors may be expressed in AIY; we indeed found that a seven-transmembrane receptor with similarity to 5-HT and octopamine receptors, termed SER-2 (identified by T. Niacaris and L. Avery), is expressed in AIY. Moreover, an orphan serpentine receptor of unknown function, SRA-11 (Troemel et al., 1995), an acetylcholine transporter protein, UNC-17 (Alfonso et al., 1993; J. Duerr and J. Rand, personal communication) and a novel secreted protein of unknown function, C36B7.7 (T. Ishihara and I. Katsura, pers.comm.) had been previously identified as being expressed in AIY and several other neurons. While the expression of none of these eight markers (ceh-10, ttx-3, ceh-23, kal-1, ser-2, sra-11, unc-17 and C36B7.7) is entirely restricted to AIY, the expression of all eight markers exclusively overlaps in the AIY interneuron class. AIY thus has a unique ‘address’, which defines its identity and allows it to be distinguished from other neurons. This ‘address’ has enabled us to investigate if and how the three homeobox genes ceh-10, ttx-3 and ceh-23 influence AIY interneuron differentiation.
ceh-23 is required to maintain one feature of the AIY differentiation program
We examined the function of ceh-23, the transcription factor that is most downstream in the ceh-10ttx-3ceh-23 cascade, by analyzing the expression of AIY marker genes in a ceh-23 null mutant background. We found that ceh-23 has no impact on the regulation of kal-1, C36B7.7, ser-2 and unc-17 expression, yet it affects expression of the orphan seven transmembrane receptor sra-11. While sra-11::gfp expression in ceh-23(ms23) L1 larvae appears normal, expression decreases significantly in adult animals (Fig. 5 and Table 1).
We also tested whether ceh-23 is required for the known functions of the AIY interneurons, thermotaxis and dauer arrest. Both behaviors were normal in ceh-23(ms23) mutant animals (Fig. 3A,D). We also observed that AIY interneuron structure, i.e. cell body position and axon morphology appears wild-type in ceh-23 null mutants (Fig. 4C). In summary, ceh-23 has no role in the regulation of known aspects of AIY function, but it is required for the maintenance of one AIY differentiation characteristic, the expression of the sra-11 gene. Additionally, ceh-23 may affect other as yet unknown parameters of AIY structure and function.
ttx-3 is required for the expression of all known subtype-specific aspects of AIY interneuron differentiation
The finding that loss of ceh-23 function has neither an obvious effect on axonal morphology of the AIY interneurons nor on the known behaviors mediated by the AIY interneurons demonstrates that the function of ttx-3, the upstream regulator of ceh-23, must go beyond regulation of the ceh-23 gene, as ttx-3 animals display severe axonal and functional defects of the AIY interneurons (Fig. 3 and see Fig. 7). We examined parameters of AIY differentiation in ttx-3(mg158) mutant animals using the AIY cell fate markers described above. We found that the expression of every known AIY subtype marker depends on ttx-3 function (Fig. 5 and Table 1). Expression of ceh-23, kal-1, C36B7.7, ser-2, unc-17 and sra-11 is either significantly reduced or absent in ttx-3(mg158) and ttx-3(ks5) mutant animals (Fig. 5 and Table 1). As shown in Fig. 5, the effect of loss of ttx-3 function on expression of the sra-11 gene is stronger (loss in larvae and adults) when compared with the loss of ceh-23 function (loss only in adults), suggesting that the effect of ttx-3 on sra-11 gene expression cannot be solely explained by loss of ceh-23 function. Rather, both ttx-3 and ceh-23 seem to be contributing individually to the regulation of sra-11 expression. The finding that ttx-3 is required for expression of all known AIY subtype markers is in accordance with the complete loss of AIY function in thermotaxis and dauer arrest as shown in Fig. 3 and suggests that ttx-3 is a central regulator of AIY interneuron differentiation. At this point it can not be assessed how aberrant expression of these marker genes relates to the defects of AIY structure and function in ttx-3 mutants, as mutations in these marker genes have – with the exception of unc-17 – not been described so far. unc-17 null mutants die as embryos (Alfonso et al., 1993) and can thus not be readily tested for AIY structure and function. unc-17 hypomorphs show normal AIY morphology (see Table 3).
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As ceh-10 acts upstream of ttx-3, it can be assumed that AIY development is at least as severely affected in ceh-10 mutants as it is in ttx-3 mutants. Consistent with this notion we found that several AIY subtype markers failed to be expressed in ceh-10-null mutants (Fig. 4 and data not shown). To address whether AIY lost pan-neuronal characteristics in ceh-10-null mutants, we examined expression of the F25B3.3::gfp pan-neuronal marker in ceh-10-null mutants and found it to be unaffected (Fig. 6C). The integrity of AIY generation and positioning in ceh-10(gm58) had also been noted previously by DIC microscopy (Forrester et al., 1998). Thus, ceh-10 and ttx-3 both act downstream or in parallel to the determination of pan-neuronal cell fate but upstream of the determination of the subtype-specific differentiation program.
Ectopic ttx-3 expression reveals ceh-10-dependent constraints on ttx-3 function
The results shown above reveal that ttx-3 is an essential regulator of subtype-specific aspects of the AIY differentiation program. To address whether ttx-3 is also sufficient to induce AIY-like features in other neurons, we ectopically expressed ttx-3 throughout the nervous system, using the pan-neuronal unc-119 promoter (see Materials and Methods). Several independently obtained transgenic ttx-3(ks5) mutant animals that express unc-119::ttx-3 show normal expression of ttx-3prom::gfp in the AIY interneurons and are thus rescued for the autoregulatory defect of ttx-3 expression (Fig. 7). All transgenic lines are viable and show no obvious morphological abnormalities (see Materials and Methods). A pan-neuronally expressed gfp reporter construct that visualizes several structural aspects of the nervous system (including presence and localization of axon fascicles; fasciculation within nerve bundles; presence and localization of ganglia) reveals no obvious abnormalities in animals that express ttx-3 pan-neuronally (data not shown). Intriguingly, unc-119::ttx-3 transgenic lines behave indistinguishably from wild type in thermotaxis assays, either in the presence or absence of endogenous ttx-3 gene activity (data not shown); thus, unc-119::ttx-3 can substitute for loss of endogenous ttx-3 function but is not sufficient to functionally disrupt other neurons in the thermotaxis circuit.
We tested whether other neurons adopted AIY-like characteristics by examining the expression of five AIY cell fate markers, ttx-3prom::gfp, ceh-23::gfp, sra-11::gfp, kal-1::gfp and ser-2::gfp in transgenic animals that express unc-119::ttx-3 (see Materials and Methods). Pan-neuronal expression of ttx-3 did not lead to a widespread expansion of the expression domains of any of the five markers. However, ectopic expression of the ttx-3prom::gfp fate marker could be unambiguously observed in two classes of neurons, the RID motoneuron and the CAN neurons (Fig. 7A). Ectopic expression of the ceh-23::gfp marker as well as the sra-11::gfp marker could also be detected in RID (Fig. 7B). Ectopic expression of ser-2::gfp and kal-1::gfp in RID could not be tested, as both markers are already expressed in RID in wild-type animals. Similarly, ceh-23::gfp, ser-2::gfp and kal-1::gfp are already expressed in CAN in wild-type animals. Hence, RID and CAN already share several characteristics with AIY in wild-type animals, yet ttx-3 is able to confer even more AIY-like characteristics to these cells upon ectopic expression (Fig. 7D). However, the axonal morphology of RID and CAN is not obviously affected by ectopic ttx-3 expression (Fig. 7A and data not shown); thus, ttx-3 is capable of imposing only a subset of the AIY differentiation program on the RID and CAN neurons. We conclude that the activity of ttx-3 is highly constrained by the presence of other regulatory factors, and that only in certain contexts does ttx-3 have the capacity to partially re-program neural differentiation.
Notably, the only two neuron classes in which AIY-like characteristics could be induced by pan-neuronal ttx-3 expression, RID and CAN, normally express ceh-10 (Fig. 1), suggesting that it is the presence of ceh-10 that constrains ectopic ttx-3 activity to these neurons. We tested this hypothesis by examining ectopic ttx-3prom::gfp expression in RID and CAN in animals that carry an integrated unc-119::ttx-3 expressing array, but lack ceh-10 function. We found that in these animals ttx-3prom::gfp expression is downregulated or completely turned off in RID, CAN and AIY (Fig. 7C). This observation points to a requirement of ceh-10 for ttx-3-dependent regulatory events. The mere co-existence of ceh-10 and ttx-3 is however not sufficient to confer AIY-like characteristics to a neuron, as pan-neuronal misexpression of ttx-3 does not appear to induce the expression of AIY-like characteristics in other ceh-10-expressing cells (such as RMED, AIN etc.; data not shown). We conclude that ttx-3 is able to regulate the expression from its own promoter and of presumptive target gene promoters only in particular cellular contexts, which are directly or indirectly dependent on the presence of the ceh-10 homeobox gene.
ttx-3 is also implicated in axon outgrowth and pathfinding
Our identification of genes whose expression depends on ttx-3 function represents one route to assess the impact of ttx-3 on AIY differentiation. Another approach is to find genes whose mutant phenotype is similar to the ttx-3 mutant phenotype and whose function may hence be related to ttx-3 function. We have previously shown that the AIY interneurons display a characteristic set of neuroanatomical defects in ttx-3 mutant animals. These defects included inappropriate termination of the main axon and sprouting of additional neurites (Hobert et al., 1997; see also Fig. 8). Recently, ectopic neurite sprouting in C. elegans was described as a secondary consequence of defects in neuronal activity of sensory neurons and motoneurons (Peckol et al., 1999; Zhao and Nonet, 2000). In contrast to their capacity to induce sprouting in motoneurons (Zhao and Nonet, 2000), we found that the synaptic transmission mutants unc-18(e81) and unc-13(e51), and the neurotransmitter transport mutant unc-17(e245) have no effect on AIY axon morphology (Table 3). Similarly, several other tested neurotransmission mutants also do not cause any neuroanatomical defects in AIY (Table 3). We additionally tested the outcome of electrical silencing of AIY by individually expressing two gain-of-function K+ channel mutant subunits, EGL-36 (E142K, P489S) (Johnstone et al., 1997) and EGL-2 (A478V) (Weinshenker et al., 1999) under the control of an AIY-specific ttx-3 promoter fragment and found no effect on AIY neuroanatomy (data not shown).
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Taken together, unlike the situation in defined sensory and motoneurons, the absence of synaptic transmission is unlikely to account for the neurite sprouting defects in ttx-3 mutant animals. We favor the possibility that the axonal defects observed in ttx-3 mutant animals represent developmental defects during the phase of axonal outgrowth. To test this notion, we compared the qualitative appearance and penetrance of axonal sprouts observed in several axon pathfinding mutants with those seen in ttx-3(ks5) mutants (ks5 is the only allele that allows visualization of AIY neuroanatomy; Fig. 3). We found that mutations in almost all of the intracellularly acting outgrowth/pathfinding proteins, which are thought to act throughout the nervous system (Antebi et al., 1997; Culotti, 1994; Hedgecock et al., 1985; McIntire et al., 1992), show defects that resemble those seen in ttx-3 mutants (Fig. 8; Table 3). Furthermore, we found that mutations in the cell-specific axon guidance cues unc-6/Netrin (Hedgecock et al., 1990), sax-3/Robo (Zallen et al., 1998) and ina-1/Integrin (Baum and Garriga, 1997), but not in vab-1, the only ephrin receptor in C. elegans (George et al., 1998), or in lin-12/Notch (Giniger et al., 1993; Greenwald et al., 1983) cause axonal defects in the AIY interneurons (Table 3). This is an intriguing finding, as unc-6, sax-3 and ina-1 have not been previously implicated in axon outgrowth into the nerve ring. unc-6 null mutants show normal guidance of amphid sensory axons in the nerve ring (Hedgecock et al., 1990), ina-1 hypomorphic mutants show fasciculation defects, but no apparent outgrowth defects of sensory axons in the nerve ring (Baum and Garriga, 1997) and sax-3 null mutants were reported to show displacements of the nerve ring, but only subtle and cell type-specific premature termination of nerve ring axons (Zallen et al., 1999). The impact of unc-6, sax-3 and ina-1 on AIY axon outgrowth in the nerve ring is thus unlikely, owing to the disruption of nerve ring pioneer neurons because that would presumably cause more pleiotropic axon outgrowth defects in the nerve ring. The netrin, robo and integrin guidance systems may act directly in the AIY interneuron class. Whether components of these guidance systems are under direct control of the TTX-3 homeodomain protein remains to be investigated.
Given the similarity in phenotypes observed between axon outgrowth/pathfinding mutants and ttx-3 mutants, we hypothesize that ttx-3 is required to regulate correct axon outgrowth and pathfinding of the AIY interneurons. As ttx-3 appears to have no effect on the expression of pan-neuronal characteristics (Fig. 6), we hypothesize that cell-type specific aspects of AIY axon outgrowth, such as the expression of a guidance receptor, rather than pan-neuronal aspects of axogenesis, are affected in ttx-3 mutants.
Role of ttx-3 in the differentiation of other neuron classes
In contrast to its pronounced effect on AIY interneuron differentiation, ttx-3 does not appear to influence the adoption of several subtype-specific differentiation features of the ttx-3-expressing sensory neuron classes ADL and ASI. The expression of five differentiation markers of the ASI sensory neurons (daf-7::gfp, osm-10::gfp, sre-1::gfp, zig-2::gfp and zig-3::gfp) was unaffected in ttx-3(mg158) mutants (Table 2). Similarly, the ADL sensory neurons still expressed the putative odorant receptors srb-6 and sre-1 in the absence of ttx-3 activity (Table 2). Both ADL and ASI are also still capable of being filled with the vital dye DiI in ttx-3 mutant animals, suggesting that these sensory neurons do not require ttx-3 to assemble intact, exposed sensory endings.
We examined the fate of the AIA interneuron class in ttx-3 mutants and found that expression of the only two known marker genes of AIA fate are partially affected. sra-11::gfp expression from the otIs62 array can be observed in 64% of ttx-3(+) animals (n=45) and 38% (n=70) of ttx-3(mg158) animals (Fig. 5); UNC-17 antibody staining in AIA can be observed in 94% of wild-type animals (n=32) and 66% (n=58) of ttx-3(ks5) mutant animals (a ttx-3(ks5); mgIs18 strain was used, which in contrast to the stronger ttx-3 alleles permits an unambiguous identification of the AIA neurons by co-staining with anti-GFP antibodies). Additionally, the AIA interneurons appear to display neuroanatomical defects (data not shown). Further cell fate markers and functional assays for the AIA interneurons need to be established in order to fully assess the impact of ttx-3 on AIA differentiation and function.
Regulatory relationship between ceh-10, ttx-3 and ceh-23 in other neurons
Based on reporter gene analysis, the AIY interneuron class appears to be the only neuron class in which the expression of the three homeobox genes ceh-10, ttx-3 and ceh-23 overlaps. The otherwise largely non-overlapping expression pattern of these genes illustrates that ceh-10 is neither sufficient to turn on ttx-3 expression in other neuron classes (RMED, RID, CEP, ALA, AVJ, AIN or CAN) nor necessary to turn on ttx-3 expression in the ADL, ASI or AIA neuron classes. In addition, ttx-3 is neither sufficient to turn on ceh-23 expression in ASI or AIA nor necessary to turn on ceh-23 expression in several other sensory neuron classes (Fig. 1C). However, there are two neuron classes in which the expression of two of the three homeobox genes overlaps (ADL, ttx-3 and ceh-23; CAN, ceh-10 and ceh-23), thus raising the question whether the regulatory relationship between these genes, observed in the AIY interneuron, also exists in ADL and CAN. We found that ceh-23 expression in ADL was unaffected in ttx-3(mg158) mutant animals (data not shown). Thus, the regulatory relation between ttx-3 and ceh-23 is not conserved in neurons other than the AIY interneuron class. In contrast, in the CAN neurons, in which ceh-10 expression overlaps with ceh-23 expression, ceh-10 regulates ceh-23 expression (Forrester et al., 1998). However, this regulation is independent of ttx-3 which is not expressed in CAN. Accordingly, ceh-23 expression in CAN is unaffected in ttx-3(mg158) mutant animals (data not shown). Thus, a regulatory relationship between these three homeobox genes exists in one neuron class (AIY), partially in another neuron class (CAN) and not at all in another neuron class (ADL).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ttx-3 is a key regulator of AIY interneuron differentiation
The understanding of the function of Lhx genes in neuronal development has in many instances been hampered by a lack of molecular markers, which precluded the precise assessment of the consequences of absence of Lhx gene function. By establishing a set of AIY differentiation markers and by visualizing axon morphology using a single neuron class-specific marker, we have carried out a detailed characterization of the consequences of loss of ttx-3 function. In ttx-3 mutants, expression of all known AIY cell fate markers is severely affected; moreover, the neuroanatomical defects of AIY in ttx-3 mutants resemble the defects seen in axonal pathfinding mutants. We conclude that ttx-3 is required to determine most, if not all subtype-specific aspects of the developmental program of the AIY interneurons, including the expression of its neurotransmitter phenotype (unc-17 expression), potential neurotransmitter receptors (ser-2), secreted signaling or adhesion proteins (kal-1, C36B7.7), proteins of unknown function (sra-11) and aspects of axonal outgrowth (Fig. 9). The maintained expression of ttx-3 throughout the life of an animal suggests that ttx-3 is continuously required to maintain differentiated features of the AIY interneurons and it is thus possible that unc-17, ser-2, sra-11, kal-1 and C36B7.7 are direct targets of the TTX-3 protein. We are in the process of testing TTX-3 binding to the promoters of these genes using in vitro DNA binding assays.
In spite of its prominent role in determining subtype-specific neuronal features, TTX-3 does not determine pan-neuronal characteristics. This can be inferred from the neuron-like appearance of AIY in ttx-3 mutant animals (e.g. the AIY cell body extends an axon projection towards the nerve ring) and from the observation that the expression of three pan-neuronal markers is unaffected in ttx-3 mutants. These findings show that adoption of pan-neuronal features is separable from the adoption of more cell-type restricted features, and show that ttx-3 genetically separates these two aspects of neuronal differentiation.
Although ttx-3 is required in AIY to adopt all of its known cell-type specific characteristics, ttx-3 is not sufficient to impose a complete AIY differentiation program onto other neurons. Although ectopic ttx-3 expression can induce the adoption of several AIY-like characteristics, it can only do so in two neuron classes (RID and CAN) and even in those cases ttx-3 is not capable of conferring AIY-like structural features (cell position, axonal anatomy) onto these neurons. It is presumably the absence of an appropriate transcription factor ‘code’ (discussed below) that does not allow TTX-3 to confer AIY-like features to more than two other neuron classes and to more completely convert RID and CAN to an AIY-like fate. Such context-dependent restrictions of Lhx gene action have also been observed for the Drosophila Islet gene and the C. elegans mec-3 gene. Islet is necessary for expression of the dopaminergic and serotonergic phenotype of several ventral cord neurons, yet it is not sufficient to induce serotonergic phenotypes upon ectopic expression and is sufficient to induce dopaminergic phenotypes in only a small set of neurons (Thor and Thomas, 1997). Ectopic expression of the Lhx gene mec-3, which is required to determine touch neuron fate, induces expression of a touch neuron marker in only one to 16 extra cells, all of which appear to contain a homeodomain protein, UNC-86, which acts together with MEC-3 in touch neurons (Duggan et al., 1998). The presence of UNC-86 may thus impose a requirement for ectopic MEC-3 activity that is comparable to the requirement of CEH-10 for ectopic TTX-3 activity.
ttx-3 does not appear to play a central role in the differentiation programs of other neurons besides AIY and possibly AIA. The expression of all tested differentiation markers of two types of sensory neurons that normally express ttx-3 is intact in ttx-3 mutants. Moreover, these sensory neurons appear structurally unaffected by loss of ttx-3 function. This is in striking contrast to AIY, in which the expression of all tested differentiation markers as well as axonal anatomy is severely affected in ttx-3 mutants. We thus consider it most likely that ttx-3 exerts a different regulatory impact in the cell fate specification of these sensory cell types. Owing to the absence of molecular markers as well as functional assays, the role of ttx-3 in AIA interneuron differentiation is unclear at this point. As the expression of the sra-11 and unc-17 fate markers is partly affected and as the AIA neurons appear to display anatomical defects in ttx-3 mutants, it is possible that ttx-3 is as crucial a differentiation regulator in AIA as it is in AIY.
Function of ceh-23 in AIY differentiation
sra-11, an orphan seven-transmembrane-receptor of unknown function, is most strongly expressed in the AIY and AVB neuron classes in L1 larvae, yet consistent and strong expression persists throughout adulthood in the AIY, but not the AVB neuron class. We have shown here that this AIY-specific maintenance mechanism requires ceh-23 gene activity. In contrast, the initiation of sra-11 expression requires ttx-3 but not ceh-23 activity. It is tempting to speculate that separable initiation and maintenance mechanisms introduce an additional level of regulation that could be subject to dynamic control. For example, CEH-23 protein activity could be post-transcriptionally regulated to allow for subtle and environmentally controlled changes in sra-11 expression during the life time of an animal.
The analysis of the role of ceh-23 and sra-11 in regulating
