The Drosophila melanogaster Translational Repressor Pumilio Regulates Neuronal Excitability

Corresponding author: Brett A. Schweers, 6100 Main St., Rice University, Houston, TX 77005., schweers{at}bioc.rice.edu (E-mail)

Communicating editor: T. W. CLINE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Maintenance of proper neuronal excitability is vital to nervous system function and normal behavior. A subset of Drosophila mutants that exhibit altered behavior also exhibit defective motor neuron excitability, which can be monitored with electrophysiological methods. One such mutant is the P-element insertion mutant bemused (bem). The bem mutant exhibits female sterility, sluggishness, and increased motor neuron excitability. The bem P element is located in the large intron of the previously characterized translational repressor gene pumilio (pum). Here, by several criteria, we show that bem is a new allele of pum. First, ovary-specific expression of pum partially rescues bem female sterility. Second, pum null mutations fail to complement bem female sterility, behavioral defects, and neuronal hyperexcitability. Third, heads from bem mutant flies exhibit greatly reduced levels of Pum protein and the absence of two pum transcripts. Fourth, two previously identified pum mutants exhibit neuronal hyperexcitability. Fifth, overexpression of pum in the nervous system reduces neuronal excitability, which is the opposite phenotype to pum loss of function. Collectively, these findings describe a new role of pum in the regulation of neuronal excitability and may afford the opportunity to study the role of translational regulation in the maintenance of proper neuronal excitability.

NEURONAL excitability is regulated by the balance of Na⁺ and K⁺ currents. Mutations that cause an increase in the ratio of Na⁺ currents to K⁺ currents cause neurons to become hyperexcitable, whereas mutations that decrease this ratio reduce neuronal excitability. For example, duplication of the Na⁺ channel gene paralytic (para) or loss-of-function mutations in K⁺ channel genes Shaker (Sh) and Hyperkinetic (Hk) each increase the ratio of Na⁺ channels to K⁺ channels and thereby result in hyperexcitable motor neurons (JAN et al. 1977 ; KAMB et al. 1988 ; LOUGHNEY et al. 1989 ; STERN and GANETZKY 1989 ; STERN et al. 1990 ). Application of quinidine, which inactivates the delayed rectifier K⁺ channel, causes a similar increase in excitability (WU et al. 1989 ; STERN and GANETZKY 1992 ). Alternatively, loss-of-function mutations in para reduce the Na⁺ channel/K⁺ channel ratio and thus decrease motor neuron excitability (SUZUKI et al. 1971 ; GANETZKY and WU 1982 ; LOUGHNEY et al. 1989 ; STERN et al. 1990 ).

At one synapse, the Drosophila larval neuromuscular junction (nmj), the most readily observable effect of increases in neuronal excitability is an increased rate of onset of a phenomenon termed either long-term facilitation (LTF) or augmentation (JAN and JAN 1978 ; WANG et al. 1994 ). LTF at the nmj is a phenomenon in which repetitive nerve stimulation at a sufficient frequency and duration causes subsequent nerve stimulation to evoke a prolonged release of neurotransmitter. This prolonged neurotransmitter release results from prolonged Ca²⁺ sensitivity of the presynaptic nerve terminal and causes a corresponding significant increase in the amplitude and duration of the response of the muscle cell (JAN and JAN 1978 ). For example, flies overexpressing either the para Na⁺ channel or the guanylate cyclase activator frequenin (frq) or carrying loss-of-function mutations of the K⁺ channel gene Hk each increase the rate of LTF onset at the nmj and hence are hyperexcitable (STERN and GANETZKY 1989 ; STERN et al. 1990 ; RIVOSECCHI et al. 1994 ).

Another mutant that exhibits an increased rate of LTF onset at the nmj is bemused (bem; STERN et al. 1995 ). The bem mutation is caused by insertion of a single P-lacW element into region 85D1, 2 of the polytene chromosome (BIER et al. 1989 ; STERN et al. 1995 ). More precise analysis (described below) revealed that this P element is located within the pum transcription unit. The pum gene has been studied in great detail and elucidation of the molecular mechanism by which pum functions in Drosophila embryogenesis has been well characterized (BARKER et al. 1992 ; MACDONALD 1992 ). Pum protein binds directly to specific sequences in the 3' untranslated region (UTR) of maternally supplied hunchback (hb) mRNA (known as nanos-response elements or NREs) and then recruits at least two other proteins, Nanos (Nos) and Brain Tumor (Brat), to the mRNA (MURATA and WHARTON 1995 ; SONODA and WHARTON 1999 , SONODA and WHARTON 2001 ). The resulting complex results in repression of hb translation via deadenylation of the hb message (WREDEN et al. 1997 ).

Whereas the mechanism by which pum functions in embryogenesis is the best understood, pum has been shown to affect other cellular systems as well. For example, members of the ovarette (ovt) class of pum alleles exhibit defects in germ-line stem cell development and maintenance (LIN and SPRADLING 1997 ; FORBES and LEHMANN 1998 ). These studies have also shown that pum plays a role in larval ovary development and that somatic expression of pum is important to the process of oviposition (PARISI and LIN 1999 ). Furthermore, several findings that suggest a neuronal role for pum have been reported. First, the Pum protein has been found to be expressed and able to repress translation in the neuronal tissue of the Drosophila eye (WHARTON et al. 1998 ). Also, another pum allele known as pumuckel (pkl) was shown to be defective in the process of optic nerve pathfinding (SCHMUCKER et al. 1997 ).

Here we show that bem is a new allele of pum, which demonstrates that pum is involved in maintaining proper neuronal excitability. We also show that previously isolated and characterized pum alleles exhibit the same neuronal hyperexcitability observed in bem mutants and that overexpression of pum in the nervous system leads to a decrease in neuronal excitability. We suggest that pum regulates neuronal excitability by regulating the translation of Na⁺ or K⁺ channels directly or via an upstream component of a pathway that regulates these channels.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
All fly stocks were maintained on standard cornmeal/agar Drosophila media at room temperature (20–22°). The pum^ovt and pum^revertant lines (LIN and SPRADLING 1997 ) were kindly provided by the lab of Dr. Haifan Lin (Duke University, Durham, NC). The nos-pum rescue construct (BARKER et al. 1992 ) and pum⁺ parental control lines were kindly provided by the lab of Dr. Ruth Lehmann (New York University, New York). The Bloomington Stock Center provided pum³ and pum¹³, and the Umeå Stock Center provided pum⁷ and pum⁹. The pum^ovt mutants are all caused by P-element mutations within the pum transcription unit (LIN and SPRADLING 1997 ). The pum¹⁶⁸⁸ P element is located in the intron between exons 3 and 4, whereas the P element in all of the other pum^ovt alleles is inserted into the large intron between exons 8 and 9 (PARISI and LIN 1999 ). The pum^revertant is a pum¹⁶⁸⁸ line produced by precise excision of the P element. The pum⁷ allele is an ethyl methanesulfonate (EMS)-induced A-to-T mutation at nucleotide 3890, which causes a premature stop codon at amino acid 949 and encodes a Pum protein product without an RNA-binding domain (TEARLE and NUSSLEIN-VOLHARD 1987 ; FORBES and LEHMANN 1998 ). Similarly, pum⁹ is an EMS-induced deletion of nucleotides 4224–4498, resulting in production of a Pum protein product that lacks the RNA-binding domain (TEARLE and NUSSLEIN-VOLHARD 1987 ; FORBES and LEHMANN 1998 ). Finally, pum¹³ is an EMS-induced point mutation within the pum RNA-binding domain that results in a single amino acid substitution, G1330D. The encoded protein is able to bind hb mRNA but is unable to repress translation (TEARLE and NUSSLEIN-VOLHARD 1987 ; WHARTON et al. 1998 ). No molecular information has been reported on pum³. The isogenic wild-type strain from which bem was produced is referred to as bem⁺. The isogenic wild-type strain from which pum⁷, pum⁹, and pum¹³ were produced is referred to as pum⁺. The bem⁺ and pum⁺ chromosomes served as controls in the genetic experiments presented.

Fertility analyses:
Single females of the appropriate genotypes were crossed to three wild-type males. The parental flies were removed from the vial 10 days later. Offspring were counted and cleared from the vials as they eclosed. Counting was terminated and offspring number was totaled once adults ceased eclosing.

Behavioral analyses:
Negative gravitaxis experiments were performed by placing single 6-day-old males of the appropriate genotype in an empty vial. The fly was banged to the bottom of the vial and we recorded the time required for the fly to right itself and climb 5 cm. Flight experiments were performed by emptying single flies from vials onto a flat and clean tabletop. When necessary, flies were encouraged to fly by prodding with a paintbrush. Any fly that was unable to achieve flight after 5 sec of prodding was deemed a nonflier.

Electrophysiological analyses:
Dissections and muscle recordings from third instar larvae were performed as described previously (JAN and JAN 1976 ; STERN et al. 1995 ). For measurement of LTF onset rates, larvae were bathed in saline containing 0.1 mM quinidine and 0.15 mM Ca²⁺. LTF onset rate was measured following nerve stimulation at the indicated frequencies. For measurement of failure rates, the nerves were stimulated for 10 sec at a frequency of 1 Hz. The number of stimuli per 10-sec stimulation train that failed to evoke any muscular response was recorded. Failure rate analysis was performed in the absence of quinidine and at a Ca²⁺ concentration of either 0.15 mM or 0.10 mM.

The pum and bem mutations were balanced over a TM6 balancer chromosome marked with the dominant Tubby (Tb) marker, which allowed larvae of the desired genotype to be recognized.

Expression studies:
Northern blots were performed using mRNA extracted from 40 flies or 800 heads of each genotype. Flies were decapitated by freezing in liquid nitrogen and subsequent vigorous shaking through a U.S. standard sieve no. 25, which has an opening size of 710 µm. Preparation of mRNA was accomplished with the QIAGEN (Valencia, CA) Oligotex mRNA kit, according to the manufacturer's instructions. The probe used was a PCR product produced from nucleotides 3600–4400 of the pum cDNA (BARKER et al. 1992 ), obtained from the Lehmann lab. Results were visualized using a phosphorimaging system and Fuji Mac-BAS software.

Western blots were performed on total protein extracted from the heads of 40 flies. The flies were decapitated as described above and protein was extracted by standard methods and probed with a 1:3000 dilution of a rabbit polyclonal antibody, called anti-PUM-I, which is specific for the Pum protein (ZAMORE et al. 1997 ). Rabbit anti-PUMI was produced from the internal portion of the protein (FORBES and LEHMANN 1998 ) and was kindly provided by the lab of Dr. Ruth Lehmann. A sheep-anti-rabbit IgG conjugated to horseradish peroxidase from Boehringer Mannheim (Indianapolis) was used as the secondary antibody. Results were visualized using a Pierce Bioluminescent 3,3'-diaminobenzidine substrate kit and subsequent exposure of the membrane to film.

Production of transgenic flies:
The pum coding sequence was removed from the pNB40/R7-1 plasmid (BARKER et al. 1992 ) via digestion with NheI and XbaI. The pum sequence was then cloned into the XbaI site of pUAST (BRAND and PERRIMON 1993 ) to create pUAS-pum. Transgenic flies were then produced using the standard embryo pole plasm injection technique. The transformant line chosen for further experimentation had the integration site on the second chromosome and this stock was named UAS-pumII.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Location of the bem P element:
DNA flanking the bem P element was compared to the entire Drosophila genome database using the Berkeley Drosophila Genome Project Blast program. We found that the bem P element is inserted into the large intron between exons 8 and 9 of the pum transcription unit (Fig 1), 54 kb from pum exon 8 and 75 kb from pum exon 9. This information raised the possibility that the bem mutant phenotypes are caused by improper transcriptional initiation or aberrant splicing of the pum transcript.

View larger version (10K): In this window In a new window Download PPT slide   Figure 1. The bem P-lacW element is located in the large intron between pum exons eight and nine. The map shows the pum transcription unit and intron exon boundaries and is adapted from BARKER et al. 1992 and PARISI and LIN 1999 . Transcription occurs from right to left, distal to proximal on chromosome 3. Open boxes represent alternatively spliced exons that correspond to the 5' UTR, whereas all other exons, representing the ORF, are indicated by solid boxes. Intron and exon sizes are approximate. The location of the bem P-lacW element is designated by the black flag and the locations of previously characterized pumovt alleles, caused by insertion of the PZ-element, are designated by the white flags. The locations of the pertinent EMS alleles (pum7, pum9, and pum13) are also indicated.

Complementation of bem fertility defects:
First, a group of P-element-induced pum^ovt mutants (PARISI and LIN 1999 ) was tested for their ability to complement the bem female sterility defect (Table 1). Fertility tests with the appropriate heterozygous females revealed that pum³²⁰³, but not pum¹⁶⁸⁸, fails to complement the bem female fertility defect (Table 1). These observations support the hypothesis that bem female sterility is due to disruption of Pum protein function.

Additional complementation experiments were performed with the chemically induced mutants pum⁷ and pum⁹, which each express Pum lacking a functional RNA-binding domain (TEARLE and NUSSLEIN-VOLHARD 1987 ; FORBES and LEHMANN 1998 ), and pum¹³, which expresses Pum that is able to bind to the NRE sequence and recruit Nos but is unable to repress translation of hb mRNA (TEARLE and NUSSLEIN-VOLHARD 1987 ; WHARTON et al. 1998 ; SONODA and WHARTON 2001 ). We found that pum⁷, pum⁹, and pum¹³ are each recessive for this fertility defect (data not shown), and all fail to complement bem female sterility (Table 1). These data strongly support the conclusion that bem female sterility is caused by improper pum function. Also, these results show that the Pum protein function in female fertility requires a functional RNA-binding domain.

Rescue of bem female sterility:
If sterility in bem females is due to loss of pum function, then this phenotype should be rescued by expression of pum⁺ in transgenic flies. We found that fertility was significantly, but not completely, restored in bem females expressing a pum transgene under the transcriptional control of nos (BARKER et al. 1992 ), which is expressed in the ovary (Fig 2). The difference in fertility seen between the rescued bem mutants and wild-type females might be an effect of insufficient Pum protein in the ovaries due to the presence of only one copy of the nos-pum rescue construct. Alternatively, this difference might reflect a role for pum in female fertility in a tissue that does not express nos. These results, in addition to the complementation results described above, demonstrate that the fertility defects seen in bem mutant females are indeed caused by improper pum function. These observations led us to investigate the possibility that the behavioral and electrophysiological defects seen in bem mutants are caused by disruption of Pum function as well.

View larger version (17K): In this window In a new window Download PPT slide   Figure 2. Ovarian expression of pum is able to rescue bem female sterility. The means and standard errors (unpaired t-test) of offspring number from single females of the indicated genotypes are presented. For bem/bem, n = 6 and for bem/bem; nos-pum, bem+/bem+ and bem/bem+, n = 7. (*) P < 0.0001 vs. bem/bem.

Complementation of bem behavioral defects:
We tested several pum alleles to determine if they failed to complement the behavioral phenotypes of bem. Flies heterozygous for the bem P element and pum³²⁰³, pum⁷, and pum⁹ were produced and we found that, in each case, the pum allele was unable to complement the negative gravitaxis and flight defects observed in bem (Fig 3; data not shown). These experiments demonstrate that the behavioral defects exhibited by bem mutants are caused by disruption of pum function. In contrast, pum¹⁶⁸⁸ was able to complement bem behavioral defects (Fig 3A), similar to its ability to complement the fertility defects. pum¹⁶⁸⁸ might exhibit different properties from other pum^ovt alleles because it is located in a different region of pum.

View larger version (16K): In this window In a new window Download PPT slide   Figure 3. Several pum alleles fail to complement bem behavioral defects. The means and standard errors (unpaired t-test) of climbing times from the indicated genotypes are presented. (A) Results obtained from tests with pumovt mutants. (*) P = 0.0007, (**) P < 0.0001 vs. bem+/bem+. The sample sizes were as follows: bem+/bem+ (n = 23), bem/bem (n = 12), pum3203/bem+ (n = 33), pum3203/bem (n = 27), pum1688/bem+ (n = 28), pum1688/bem (n = 15), pumrev/bem (n = 34). (B) Results obtained from tests with EMS-induced pum alleles. (*) P < 0.0001 vs. bem+/bem+. The sample sizes were as follows: bem+/bem+ (n = 23), bem/bem (n = 12), pum7/bem+ (n = 29), pum7/bem (n = 27), pum9/bem+ (n = 41), pum9/bem (n = 37), pum+/bem (n = 17).

Neuronal excitability defects of pum^ovt mutants:
To investigate the possibility that previously characterized pum mutants exhibit a neuronal hyperexcitability similar to what is observed in bem mutants, we tested two homozygous pum^ovt lines (pum²⁰⁰³ and pum⁴⁸⁰⁶) for increased rates of LTF onset. As shown in Fig 4B, both pum²⁰⁰³ and pum⁴⁸⁰⁶ exhibit increased LTF onset rate. In fact, the LTF onset rates of pum²⁰⁰³ and pum⁴⁸⁰⁶ are virtually indistinguishable from the rate observed in bem mutants. These findings support the possibility that bem neuronal hyperexcitability is due to improper Pum function.

View larger version (33K): In this window In a new window Download PPT slide   Figure 4. pum alleles exhibit neuronal defects and fail to complement bem neuronal hyperexcitability. LTF onset rates were determined in the presence of 0.1 mM quinidine and at an external Ca2+ concentration of 0.15 mM. (A) Representative traces showing LTF onset at the nmj for the indicated genotypes. Black arrowheads indicate LTF onset. (B–D) Means and standard errors of LTF onset rates for the indicated genotypes at the indicated stimulation frequencies (hertz). (B) The neuronal excitability defects of pumovt mutants: For bem+/bem+, n = 22; for bem/bem, n = 12; for pum2003/pum2003, n = 8; for pum4806/pum4806, n = 6. (C) The inability of pum7 to complement bem neuronal hyperexcitability: For bem/pum7, n = 8; for bem+/pum7, n = 8; for bem+/pum+, n = 8; for bem/pum+, n = 7. (D) The inability of pum9 to complement bem neuronal hyperexcitability: For bem/pum9, n = 6; for bem+/pum9, n = 5; for bem+/bem+, n = 8; for bem/pum+, n = 7.

Complementation of bem neuronal excitability defects:
Next, we investigated the ability of pum⁷ and pum⁹ to complement bem neuronal hyperexcitability. Third instar larvae of genotypes bem/pum⁷ and bem/pum⁹ were produced and the rate of LTF onset was determined. We found that these larvae exhibited a rate of LTF onset that was significantly faster than that of wild-type control larvae as well as that of larvae heterozygous for pum⁷, pum⁹, and bem (Fig 4C and Fig D). The observation that pum⁷ and pum⁹ are unable to complement the hyperexcitability defect seen in bem neurons shows that bem hyperexcitability is due to improper Pum function.

Expression of pum mRNA in bem and pum^ovt mutants:
We found pum transcripts in both males and females (Fig 5A), which is predicted on the basis of the observation of bem mutant phenotypes in both males and females. Also, improper transcription of pum was found in bem mutants. In particular, the larger transcript in bem adults appeared to be smaller than the largest transcript in wild-type control adults. We also observed a striking difference between the transcripts present in bem and wild-type heads (Fig 5B). In particular, wild-type heads express four different pum mRNA species corresponding to the apparent molecular sizes of 9.0 and 6.7 kb as well as a doublet at 8.1 kb. We found that the 9.0-kb transcript and the upper band of the 8.1-kb doublet are absent from bem heads (Fig 5B). A final Northern blot was performed to investigate pum mRNA expression in the heads of previously generated and characterized pum^ovt mutants. pum³²⁰³, which was shown to be unable to complement bem behavioral defects, and pum¹⁶⁸⁸, which complements these defects, are each missing the same 9.0-kb head transcript that is absent from bem heads (Fig 5C). Although these findings show that the bem P element is indeed affecting pum expression in adult flies and especially in heads, they do not allow determination of which pum transcripts are needed for proper neuronal function. However, these results do show that the 9.0-kb transcript is not necessary in heads for proper behavior.

View larger version (45K): In this window In a new window Download PPT slide   Figure 5. pum mRNA is expressed improperly in bem mutant flies. Northern blots were performed with mRNA extracted from 40 flies (A) or 800 heads (B and C) of each genotype. For A and B, the bottom blot is the same membrane shown in the top probed with the ribosomal protein gene L29a to serve as a loading control.

Pum protein expression in bem and pum^ovt mutants:
Western blot analysis was performed to determine the effect of bem and pum^ovt mutations on Pum protein levels. We found that Pum protein is greatly reduced in abundance in heads from bem mutants as well as some pum^ovt mutants, when compared to bem⁺ parental and pum^ovt revertant controls (Fig 6). The parental wild-type and revertant control heads abundantly express three different protein isoforms corresponding to the apparent molecular sizes of 156, 130, and 93 kD (resolution of these bands was greatly enhanced upon shorter exposure times, data not shown), whereas greatly reduced amounts of all three isoforms are present in the bem heads (Fig 6, also see PARISI and LIN 1999 ). Also, pum¹⁶⁸⁸ is missing the 156-kD isoform whereas pum³²⁰³ and pum⁶⁸⁹⁷ are missing both the 156- and 130-kD isoforms (Fig 6). Because pum¹⁶⁸⁸ is able to complement bem behavioral defects while pum³²⁰³ is not, these results demonstrate that the 156-kD isoform is not necessary for Pum function in controlling behavior (Fig 3). The genetic and molecular data presented here show that all of the bem mutant phenotypes are due to improper pum function. Therefore, the bem mutation is allelic to pum and we recommend that bem should henceforth be referred to as pum^bem.

View larger version (47K): In this window In a new window Download PPT slide   Figure 6. Pum protein is expressed improperly in bem and pumovt mutant heads. Western blots were performed on total protein extracted from the heads of 40 flies from the indicated genotypes. Results were visualized using a bioluminescent DAB substrate kit and subsequent exposure of the membrane to film.

Overexpression of pum in the nervous system:
We utilized the UAS/GAL4 system to examine the effect of overexpressing Pum protein in the nervous system. Transgenic flies carrying UAS-pum were generated and crossed to flies carrying an elav-GAL4 transgene, which produces Gal4 protein specifically in postmitotic neurons. From electrophysiological analysis of third instar larvae, we found that overexpression of pum prevents LTF onset in motor neurons even after 90 sec of 10-Hz stimulation. In contrast, larvae that express either UAS-pum or elav-GAL4 alone exhibit normal LTF onset rates (data not shown).

During these studies we noted that larvae overexpressing neuronal pum responded to nerve stimulation with a higher frequency of failure of excitatory junctional potentials (ejp's) than did controls in saline containing low Ca²⁺ (0.15 mM). In particular, we found that nerve stimuli were successful in eliciting an ejp only 20% of the time in the pum overexpression lines, whereas nerve stimuli elicited an ejp 80% of the time at nmj's from wild-type and control lines (Fig 7B). Thus, pum overexpression reduces transmitter release at low Ca²⁺. In contrast, we found that nmj's from the hyperexcitable pum^bem loss-of-function mutants respond to nerve stimulation with an ejp 95% of the time. This result suggests that loss of pum in neurons increases transmitter release at low Ca²⁺ (Fig 7B). The difference between pum^bem and wild-type failure rates is even more evident at lower external Ca²⁺ (0.10 mM) at which stimulations to wild-type neurons successfully elicit an ejp following only 35% of the stimuli, whereas stimulations to pum^bem neurons elicit an ejp following 85% of the stimuli (Fig 7C). Flies overexpressing pum in their nervous system were also tested for behavioral defects and temperature-sensitive paralysis but no differences from wild type were evident (data not shown). The data collected from neurons overexpressing pum support the conclusion that the level of Pum protein expression in neurons regulates neuronal excitability. In particular, insufficient neuronal Pum causes hyperexcitability, whereas excess neuronal Pum reduces excitability.

View larger version (13K): In this window In a new window Download PPT slide   Figure 7. Overexpression of pum in the nervous system causes an increased failure rate in motor neurons. The number of stimuli (stimulus frequency 1 Hz for 10 sec) that evoke an ejp is indicated. The bath contained a Ca2+ concentration of either 0.15 mM (A and B) or 0.1 mM (C). (A) Representative traces of failures and successes from nerves of the indicated genotypes. Black arrowheads indicate failures. (B and C) Means and standard errors (unpaired t-test) of failure rates from nerves of the indicated genotypes are presented. Six larvae from each genotype were tested. (*) P < 0.0005 and <0.0001 vs. bem+/bem+ for B and C, respectively.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Discovery of a new pum allele:
We have shown by several criteria that bem is a new allele of pum. First, several pum alleles failed to complement the pum^bem fertility defects. Second, female fertility in pum^bem mutants was significantly restored by the expression of pum under the transcriptional control of the nos promoter. Third, several pum alleles failed to complement both the behavioral defects and defective neuronal excitability of pum^bem mutants. Fourth, both pum mRNA and protein are expressed improperly in the heads of pum^bem adults. Finally, we showed that previously identified pum mutants exhibit the same increased motor neuron excitability as seen in pum^bem and that overexpression of pum in larval neurons decreases motor neuron excitability. Therefore, we have uncovered a new role of pum in regulating neuronal excitability.

We have also begun to assign specific functions to individual Pum protein isoforms. In particular, we found that pum¹⁶⁸⁸ eliminates only the 156-kD isoform from heads and retains expression of both the 130- and 95-kD Pum isoforms, whereas expression of all three Pum isoforms in pum^bem heads is greatly reduced. Since pum¹⁶⁸⁸ complements pum^bem behavioral defects we can conclude that the 156-kD Pum isoform is not required for Pum function in controlling behavior.

Mechanisms of pum action:
The Pum protein is a member of the PUF-domain-containing protein family, which is an evolutionarily conserved family of RNA-binding proteins found in organisms from yeast to humans (COGLIEVINA et al. 1995 ; MIOSGA and ZIMMERMANN 1996 ; PURNELLE and GOFFEAU 1997 ; ZAMORE et al. 1997 ; KRAEMER et al. 1999 ; NAKAHATA et al. 2001 ; TADAUCHI et al. 2001 ). Whereas orthologs of pum have been isolated in several organisms, the molecular mechanism by which pum functions has been characterized in most detail in the process of posterior pattern formation during Drosophila embryogenesis. In this system, Pum binds to specific sequences (NREs) that are present in the 3'-UTR of hb mRNA (WHARTON and STRUHL 1991 ; SONODA and WHARTON 1999 ; ZAMORE et al. 1999 ). The hb-bound Pum then recruits Nos and Brat to the mRNA, forming a quaternary complex that results in repression of hb mRNA translation (SONODA and WHARTON 1999 , SONODA and WHARTON 2001 ). The mechanism of repression results in part from deadenylation of the hb transcript (WREDEN et al. 1997 ). This repression is required for the formation of posterior structures in the developing embryo (BARKER et al. 1992 ). In pum mutants, hb mRNA is translated throughout the embryo, resulting in abrogation of posterior development and the absence of posterior abdominal segments (BARKER et al. 1992 ).

Pum has effects in Drosophila in addition to controlling the formation of posterior structures. In particular, certain pum alleles that exhibit defects in germ-line stem cell differentiation have been identified (LIN and SPRADLING 1997 ). In this process, pum and nos might act together to inhibit pole cell division by binding sequences similar to NREs present in cyclin B mRNA thereby repressing its translation (SONODA and WHARTON 2001 ). An unidentified pole cell-specific factor (possibly similar to brat) is also implicated by the observation that pum and nos do not affect cyclin B translation in somatic cells (RICHTER and THEURKAUF 2001 ). In addition, pum has a role in optic nerve pathfinding and is also able to repress translation in photoreceptors in a nos-dependent manner (SCHMUCKER et al. 1997 ; WHARTON et al. 1998 ). However, neither the pum target mRNA in developing optic nerves nor the adult ocular target has been identified. These results taken together suggest that improper translational regulation of unknown target mRNA(s) during neuronal development or in the adult nervous system results in the increased motor neuron excitability seen in pum^bem and pum^ovt mutants.

Effects of translational regulation on neuronal excitability:
Much data concerning the role of translational regulation in neuronal function have been collected. Both translational machinery and mRNA are located in certain dendrites and axons as well as the cell body of neurons (STEWARD et al. 1996 ; TIEDGE and BROSIUS 1996 ). In addition, it was shown in both Aplysia and rats that translation is required for learning, memory, and proper synaptic plasticity (KANG and SCHUMAN 1996 ; MARTIN et al. 1997 ). Furthermore, recent studies have shown that translation at the Drosophila nmj affects both the anatomy and physiology of these synapses (SIGRIST et al. 2000 ). Our studies suggest that Pum might regulate translation in the cell body, dendrites, or axons and that this translational regulation is important in maintaining proper neuronal excitability. For example, maintenance of proper neuronal excitability may be achieved by translational regulation of ion channel mRNAs directly or through regulation of an upstream ion channel regulator. Therefore, isolation of the pum^bem mutation allows the opportunity to study with genetic methods the role played by translational regulation in maintaining proper neuronal excitability.

	FOOTNOTES

² Present address: UT Southwestern Medical School/Parkland Memorial Hospital, Dallas, TX 75390.

	ACKNOWLEDGMENTS

We are grateful to the Bloomington and Umeå Stock Centers for the provision of fly lines, Paul MacDonald for providing pum anti sera, Ruth Lehmann for providing the pum rescue construct fly line and pum anti sera, and Haifan Lin for providing pum^ovt fly lines and pum antisera. We also thank present and former members of the Mike Stern and Kate Beckingham labs for stimulating and insightful discussions, especially Stephen Richards for providing suggestions that guided the direction of the project. We also thank Vik Vaz for help in performing behavioral tests and we are grateful to Bonnie Bartel and Mike Gustin for their help in revision of this manuscript. This work was supported by National Institutes of Health grant GM-46566 (to M.S.), a Grant-In-Aid from the American Heart Association (to M.S.), a Houston Livestock Show and Rodeo Scholarship (to B.A.S.), and a National Science Foundation predoctoral fellowship (to K.J.W.).

Manuscript received December 11, 2001; Accepted for publication April 12, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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