GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members

Molecular Brain Research 81 (2000) 109–117 www.elsevier.com / locate / bres

Research report

GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members Wei He

a ,1 2

b ,2

b

a

b

, Sakae Ikeda , Roderick T. Bronson , Grace Yan , Patsy M. Nishina , ¨ Michael A. North a ,1 , Jurgen K. Naggert b , * b

a AXYS Pharmaceuticals, La Jolla, CA 92037, USA The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA

Accepted 5 July 2000

Abstract The tubby gene family consists of four members, TUB, TULP1, TULP2 and TULP3, with unknown function. However, a splice junction mutation within the mouse tub gene leads to retinal and cochlear degeneration, as well as maturity onset obesity and insulin resistance. Mutations within human TULP1 have also been shown to co-segregate in several cases of autosomal recessive retinitis pigmentosa (RP) and TULP1 deficiency in mice leads to retinal degeneration. The primary amino acid sequences of the tubby family members do not predict a likely biochemical function. As a first step in defining their function, we present a detailed characterization of the cellular and subcellular localization of the human (TUB) and mouse (tub) homologous gene products. We report the isolation of TUB splice variants which have different subcellular localizations (nuclear versus cytoplasmic) and which define a nuclear localization signal. In addition, using green fluorescent protein (GFP) tags, we observe a nuclear localization for TULP1, similar to TUB splicing forms TUB 561 and TUB 506. Finally, we report tubby expression in mouse brain by in situ hybridization and by immunohistochemistry with polyclonal antibodies. Protein was found in both the hypothalamic satiety centers and in a variety of other CNS structures including the cortex, cerebellum, olfactory bulb and hippocampus. Both nuclear and cytoplasmic signals were detected with a series of independently generated polyclonal antibodies, consistent with the presence of multiple alternatively spliced isoforms within the CNS.  2000 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Genetic models Keywords: Mouse models; Sensory loss / obesity syndromes; Tubby gene family; Immunohistochemistry; Subcellular localization

1. Introduction Tubby and tubby-like proteins (TULPs) are encoded by members of a small, novel gene family and share no significant regions of sequence similarity to other reported proteins [20,22]. Currently four mammalian tubby family members (TUB, TULP1, TULP2 and TULP3 ) and numerous tubby-like genes in different species (e.g. C. elegans, *Corresponding author. Tel.: 11-207-288-6382; fax: 11-207-2886077. E-mail address: [email protected] (J.K. Naggert). 1 Present address: Agouron Pharmaceuticals, La Jolla, CA 92037, USA. 2 Denotes equal contribution to the work.

Drosophila, maize, Arabidopsis) have been identified. They all share a highly conserved carboxy terminus, the putative functional domain of these genes. The amino terminal half of the proteins are less well conserved and may impart functional specificity. Phylogenetically, TUB and TULP3 are most closely related and have a wider tissue expression than TULP1 and TULP2, which are expressed mainly in the retina and testis, respectively [20,22]. Although the function of this gene family is unknown, the members presumably play an important role in cellular function as mutations within two of the family members, tub and Tulp1, are known to lead to disease phenotypes. Mice homozygous for a splice junction mutation in the tubby gene (resulting in the replacement of the carboxy

0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00164-9

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terminal 44 amino acids with 24 intron-derived amino acids) develop early onset retinal and cochlear degeneration as well as maturity onset obesity associated with insulin resistance. Similarly, several mutations in TULP1 have been shown to segregate with autosomal recessive retinitis pigmentosa in human families, and mice with a Tulp1 deficiency develop retinal degeneration [2,8–10,15]. These findings support the idea that members of this gene family play an essential role in the normal function of the photoreceptor cell in both mouse and man. The primary site of action of tubby in bodyweight regulation is more difficult to predict. Early studies reported that the major site of tub mRNA expression within the CNS was the hypothalamus [18], widely regarded as a critical center for integrating afferent signals reflecting peripheral energy storage and expenditure. More recently, studies (including this one) indicate that tubby is widely and abundantly expressed throughout the brain [25]. As an initial step in establishing the likely biochemical function of tubby and related family members, we have carried out a detailed study of cellular and subcellular protein expression patterns of tub and related family members.

tion. Constructs containing TUB cDNA fragments were electroporated into the Cos-7 cells under standard conditions for mammalian cells (Bio-Rad). After electroporation cells were cultured in chamber slides (LAB-TEK; Nalge Nunc, Rochester, NY) for 8 to 17 h. Transfected cells were then fixed with 4% paraformaldehyde for 10 min and mounted with coverslips using anti-fade mounting medium (Vector Laboratories, Burlingame, CA) with 200 ng / ml DAPI (Sigma, St. Louis, MO) for nuclear staining.

2.3. Antibody production

2. Materials and methods

6X-his tagged TUB-N (exons 1 to 6) or TUB-C (exons 7 to 12) fusion proteins were expressed in E. coli and purified using Ni-NTA agarose resin (Qiagen, Santa Clarita, CA). To produce anti-TUB-N, |0.6 mg of purified TUB-N protein in Tris buffer was injected into a rabbit (Pocono Rabbit Farm and Laboratory, Candensis, PA). The rabbit antiserum was collected and purified against TUB-N (exon 3 to 6) coupled to Affi-Gel 10 (Bio-Rad, Richmond, CA). To produce anti-TUB-C, 0.4 mg of purified protein in Tris buffer mixed with TiterMax Gold (CytRX Corporation, Norcross, GA) was injected into a rat. The rat antiserum was collected and purified against the antigen coupled to Affi-Gel 10 (Bio-Rad).

2.1. 59 RACE and RT PCR

2.4. Western blotting

Four different RACE experiments were performed using mRNA samples from human brain, human testis, and total RNA samples from cell lines 293 and Y79. Tubby specific primers (from exon 9) were used for first strand cDNA synthesis. Synthesis of the second strand cDNA and the adapter ligation were carried out according to the manufacturer’s protocol (Marathon cDNA Amplification Kit; Clontech, Palo Alto, CA). Nested PCR was performed using primers from exon 3 or 4 and primers from adaptor sequences (AP1 or AP2) and the resulting products were cloned into the TA cloning vector (Invitrogen, Carlsbad, CA). Prior to sequencing, clones were further selected using the tubby specific internal primers and the adaptor primer AP2. For RT-PCR, first strand synthesis was carried out using 1 mg mRNA from human brain, heart, or skeletal muscle (Clontech), or 3.5 mg total RNA from human retina. After reverse transcription, PCR reactions were performed using primers specific for each variant, designed to span at least one intron in order to avoid amplification of products from trace amount of genomic DNA in the sample.

Recombinant TUB proteins were expressed in Cos-7 cells and whole cell lysates were subjected to SDS–PAGE. Separated proteins were transferred to PVDF membrane (Novex, San Diego, CA). The blots were blocked according to Amersham’s ECL detection procedure, incubated with primary and peroxidase-conjugated secondary antibodies, and detected using chemiluminescent reagents (Amersham, Uppsala, Sweden). The anti-TUB-N antibody was used to detect TUB protein.

2.2. GFP/TUB fusion protein expression in Cos-7 cells Human TUB cDNA fragments were cloned into the pEGFP expression vector (Clontech). Cos-7 cells were cultured in DMEM high glucose medium with 10% FBS. Cells were harvested and washed in cold PBS for transfec-

2.5. Immunohistochemistry Three-month-old mice (three C57BL6-tub /tub and four C57BL6-1 / 1) were anesthetized with tribromoethanol and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS as fixative. Brains were post-fixed in the same fixative for 1–3 h, dehydrated and embedded in paraffin. Serial sections of 6 mm thickness were cut and mounted on slides pretreated with poly-L-lysine (Sigma, St. Louis, MO). Sections were deparaffinized in xylene and rehydrated through a graded series of alcohol and PBS. A microwave procedure was used for antigen retrieval (8 min in citrate buffer, pH 6–6.5). Slides were then incubated in 0.3% hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. After incubation with blocking solution (3% normal goat serum), slides were incubated overnight with rabbit polyclonal antiserum against the amino-terminal half

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of the tubby protein [14]. Antibody binding was detected using biotinylated secondary antibodies (Vector Laboratories) and Vectastain Elite ABC Kit (Vector Laboratories) using 3,3-diaminobenzidine tetrahydrochloride (DAB) as the enzyme substrate (Vector Laboratories). Some of the sections were counterstained with hematoxylin. The specificity of the antibody binding was confirmed using negative controls in which the primary antibody was omitted, as well as in which the primary antibody had been neutralized with an excess amount of recombinant TUB-N protein.

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appears to involve the production of transcripts encoding different amino termini. As shown in Fig. 1, abundant isoforms present in brain are TUB561, TUB506 and TUB460, whereas TUB506 does not appear to be expressed in retina. Using primers spanning the TUB coding region, we did not detect any other variation in exon usage, other than alternative splicing of exon 5 (data not shown), which has been previously reported [18]. In contrast, no alternative splice variants for TULP1 were detected in a retinal cDNA library.

3.2. Subcellular localization TUB and TULP1 2.6. In situ hybridization In situ hybridization was accomplished on 7-mm-thick paraffin embedded sections with digoxigenin-dUTP labeled riboprobes. To achieve specificity, we selected a 330-bp fragment from exons 3 to 5 of the tubby gene. The selected fragments were first amplified by PCR and cloned into a TA cloning vector in both directions. Anti-sense and sense riboprobes were generated using T7 RNA polymerase. Deparaffinized sections were first treated with proteinase K in Tris–HCl (50 ng / ml) for 30 min at 378C and then dipped into TEA / acetic anhydride solution for 10 min. After dehydration, slides were incubated with 1–2 ng / ml digoxigenin labeled riboprobe in hybridization buffer (50% formamide, 43 SSC, 10% dextran sulfate and 13 Denhart’s solution) overnight at 608C. On the second day, slides were washed twice in 0.1% SSC for 45 min each at 658C and developed with an anti-digoxigenin alkaline phosphatase conjugate and NBT / BCIP as substrate.

3. Results

3.1. Alternative splice variants Two alternatively spliced forms of mRNA derived from the human TUB homologue, encoding predicted proteins of 561 and 506 amino acids, were identified through screening of a human adult brain cDNA library with probes derived from the mouse tubby cDNA. The deduced proteins, TUB 561 and TUB 506, differ at their amino termini as shown in Fig. 1 [18,21]. To identify additional amino terminal splice variants in the CNS and in peripheral tissues, 59 RACE (rapid amplification of cDNA ends) experiments were carried out using RNA from brain, retina, heart and skeletal muscle. Seven different splice variants (coding for five different proteins with different amino termini) were identified and are aligned in Fig. 1. Using RT-PCR with primers designed specifically for each splice variant, several tissue RNA samples were tested for the approximate abundance of each splice form. Primers were chosen to span at least one intron to avoid artifacts arising from trace amounts of genomic DNA in the samples. Most alternative exon usage in the TUB gene

Analysis of the amino terminal sequence of TUB561 indicated the presence of a potential nuclear localization signal (NLS), KKKR, encoded in exon 3 (highlighted in Fig. 1). The putative NLS is present in the splice forms TUB561 and TUB506, but absent from TUB460. To determine the subcellular localization of the alternatively spliced variants and whether the putative NLS functions to direct the transcripts to the nucleus, a series of transient transfection assays in Cos-7 cells using the green fluorescent protein (GFP) tag was carried out. Full length cDNAs for TUB561, TUB506 and TUB460 or cDNAs for the N-terminal half and C-terminal half of TUB561 were cloned into a GFP fusion vector, to produce a C-terminal GFP fusion protein. Cos-7 cells were transfected with each of the fusion constructs and analyzed for the green fluorescence signal 17 h post-transfection. As shown in Fig. 2, expression constructs with GFP alone, or GFP fused either with TUB460 or with only the C-terminal domain of TUB561 produced a cytoplasmic signal. Fusions of GFP with TUB561 or its N-terminal domain as well as with TUB506 were predominately localized in the nucleus. These localization results are consistent with an NLS present in TUB561 / 506 but absent from TUB460. Although generally present throughout the nuclear compartment, both TUB561 and TUB506 were not evenly dispersed. By viewing the same image under phase contrast, we determined that the GFP signal localized within the nucleolus of the nucleus. To eliminate the possibility that the GFP tag caused an artifactual nucleolar localization, untagged TUB561 was expressed in Cos-7 cells and detected with a polyclonal antibody made against the N-terminal half of TUB561 (anti-TUB-N, described further below, and see Materials and methods). As shown in Fig. 2, native TUB561 is detected in the nucleus with a predominant concentration of signal in the nucleolus. A GFP expression construct was also made using the full length TULP1 coding sequence. Transient transfection assays again indicated a general nuclear concentration and a strong nucleolar localization, similar to the observed TUB561 / 506 pattern. Sequence comparison between the N-terminal halves of TULP1 and TUB show that exon 2 and the amino terminus of exon 3, including the putative NLS, are moderately well conserved (Fig. 3).

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Fig. 1. N-terminal splice variants of TUB. Shown are the TUB genomic structure (a) and the amino acid sequences of the different splice forms (b). The first exon encodes the amino terminus of TUB561. The variants TUB518, TUB512, and TUB506 are generated through differential splicing of alternate exons, located upstream of exon 1, onto the common exon 2. TUB460 arises from an internal start site in exon 3. The conserved nuclear localization signal is shown in bold. (c) RT PCR analysis of brain, retina, heart, and skeletal muscle RNA using primers specific for the five splice variants.

Fig. 2. GFP-TUB or -TULP1 fusion protein expression in Cos-7 cells. GFP fusion proteins were examined 17 h after transfection into Cos-7 cells, unless otherwise indicated. (A) GFP alone. (B) GFP TUB-C. (C) GFP/ TUB460. (D) GFP/ TUB-N. (E) GFP/ TUB 561. (F) GFP/ TUB506. (G) GFP/ TULP1, 8 h after transfection. (H) GFP/ TUB561 detected with the anti-TUB-N antibody.

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Fig. 3. Alignment of the conserved common exon 2 and adjacent nuclear localization signal in exon 3. Sequences shown are from human (prefix h), mouse (m), rat (r), dog (d), C. elegans (c.eleg.), and Drosophila (dros.). The conserved putative nuclear localization signal is in bold type.

3.3. Comparison of wild type and mutant tubby distribution To confirm and extend the results from the transient transfection assays above, we carried out direct immunohistochemical studies with mouse brain sections. Due to the high amino acid sequence similarity between mouse and human TUB protein (.90%), we were able to use antibodies raised against human TUB on mouse brain sections. Polyclonal antibodies were produced against two recombinant TUB fusion proteins: the N-terminal half of TUB561 (TUB-N, exons 1 to 6) and the C-terminal half of TUB561 (TUB-C, exons 7 to 12). After purification, those antibodies were examined on Western blots to evaluate their specificity. Both antibodies were able to identify TUB protein expressed in Cos-7 cells with high specificity. The results obtained with anti-TUB-N are shown in Fig. 4, in which isoforms, TUB561, 506 and 460, are detected as one major band with this antibody. No common bands were observed among the three lanes containing Cos-7 lysates, excluding the possibility that the antibody recognized Cos7 derived proteins. Although similar results were obtained with both antibodies, we used the N-terminal antibody because the other tubby family members currently reported have been shown to share very little sequence similarity across this domain. We confirmed the specificity of the antibody immunohistochemically by preincubating the antibody with excess recombinant TUB protein. As shown in Fig. 5, strong immunoreactivity in Purkinje cells of the cerebellum (Fig. 5A) was completely abolished after the adsorption of the antibody (Fig. 5B). We also compared antibody stained sections with adjacent sections hybridized with a riboprobe from a unique N-terminal portion of the tubby gene (exons 3 to 5). Our in situ hybridization (Fig. 6B) detects essentially the same pattern as observed by immunostaining (Fig. 6A). We have previously described a splice junction mutation

in the mouse tubby gene which causes a carboxy terminal 44 amino acid truncation of the protein and addition of 24 amino acids from the intronic sequence [18,21]. Comparison of the immunohistochemical staining patterns of brain sections from 3-month-old C57BL6 / J-tub /tub mice with those from wild type C57BL6-1 / 1 reveals no gross differences in TUB protein distribution or amount (Fig. 7A and B). Our immunostaining results generally agree with the RNA in situ hybridization results published previously [18]. Tubby protein was detected in all major brain areas including the olfactory bulb, cerebral cortex, hypothalamus, hippocampus, and cerebellum. Close examination of the histological slides shows that TUB protein is present in all neurons examined throughout the entire brain.

Fig. 4. Western blot probed with rabbit anti-TUB-N antibody. Human TUB proteins were expressed in Cos-7 cells and whole cell lysates were subjected to SDS–PAGE. The molecular weight for each protein is: 62 kD for TUB561, 56 kD for TUB506, and 50 kD for TUB460.

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Fig. 5. Specificity of the anti-TUB-N antibody. Specificity was demonstrated by preincubating anti-TUB-N antibody with recombinant TUB-N protein before immunohistochemical analysis. Strong staining of Purkinje cells observed in the cerebellum (A) was completely abolished when preincubation of the antibody was carried out (B). Sections were counterstained with hematoxylin.

Fig. 6. Comparison of expression patterns for tub mRNA and TUB protein. In the cerebellar cortex, as well as in other brain regions, the same staining patterns were observed for immunohistochemistry using the anti-TUB-N antibody (A) and in situ hybridization (B) using an antisense probe for the N-terminal sequence of tub.

Fig. 7. Immunostaining of wild-type (A) and tubby (B) brain sections. Staining with the anti-TUB-N antibody is observed throughout the coronal section from both animals. No staining was observed in the section in which the primary antibody was omitted (C). Scale bar51 mm.

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3.4. Subcellular localization by immunohistochemistry Our findings from the transient transfection assays indicated a nucleolar localization for most TUB splice variants and a cytoplasmic localization for at least one splice variant (TUB460). The immunohistochemical analysis is in agreement with these predictions. The subcellular localization of TUB is characterized by high concen-

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trations in the nucleolus of the nucleus and much lower protein levels in the cytoplasm of the neurons in which it is expressed (Fig. 8). There appear to be some cell-specific differences in expression, in that TUB is mainly found in the nucleolus of the neurons in the paraventricular, arcuate, ventromedial, and dorsomedial nuclei of the hypothalamus (Fig. 8E and F). On the other hand, within the cerebellar cortex, TUB is highly expressed both in the nucleolus as

Fig. 8. Brain sections from wild-type mice immunostained with anti-TUB-N antibody. Staining of Purkinje cells in the cerebellum was observed both in the cytoplasm and in the nucleolus (A). Neurons in the cerebral cortex (B), hippocampus (C) and hypothalamus (E, F) were stained mainly in the nucleolus. Strong immunoreactivity was observed in the nucleus of cells in the lateral ventricle subependyma (arrows in D). Gr, granular layer; ML, molecular layer; CA3, CA3 pyramidal cell layer; CP, choroid plexus; LV, the lateral ventricle; 3V, third ventricle; PVN, paraventricular nucleus; ARC, arcuate nucleus; VMH, ventromedial hypothalamus. Scale bar550 mm in A, 100 mm in B–F.

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well as the cytoplasm of the Purkinje cells and staining extends into the dendrites of these cells (Fig. 8A). It is also interesting to note that TUB is very highly expressed in the cells of the subependymal zone (arrows in Fig. 8D).

4. Discussion In B6-tub /tub mice, a mutation in the tubby gene leads to early onset vision and hearing loss as well as progressive retinal and cochlear degeneration [13,14,23]. In early adulthood (12 to 16 weeks of age) the animals begin to gain weight and eventually become obese [5]. Tub is a member of a novel small gene family [20,22] with currently unknown function. Mutations in one other family member, TULP1, have been associated with retinal degeneration in both humans [2,8,10,19], and mice [9,15]. Despite the demonstrated importance of the tubby gene family for normal cellular function, basic molecular, histological and physiological information on the tubby proteins is still missing. Here we report the existence of splice variants, the distribution of tubby protein in the brain, and its subcellular localization. Several isoforms of the tubby protein can be synthesized through the use of alternate 59 exons encoding a translation initiation site and an initiation codon-containing amino terminus. Most of these splice variants utilize the second coding exon in common. Exceptions are TUB460, which utilizes an internal start codon, and a previously reported alternate splice variant in mouse, which lacks exon 5 [18]. The purpose for the existence of these isoforms is currently not known. Splice variants have been shown to confer tissue specificity, as for the human retinal phosphoinositide-specific phospholipase-C beta 4 [1]; developmental regulation, as observed in the differential expression of laminin receptor in embryogenesis [28]; and transcriptional regulation, as evidenced by splice variants of E-selectin, which respond differently to induction by lipopolysaccharides [3]. The presence of TUB protein in both the cytoplasm, and in the nucleolus suggests that the tubby protein may require certain stimulation and / or conformational changes to be transported into the nucleus. On the other hand, the different localization could also be due to differential splicing. The results from our transfection studies showing that TUB460 lacks a functional nuclear localization signal and is found in the cytoplasm may indicate that cytoplasmic vs. nuclear localization is at least in part regulated through alternate splicing. Whether the different isoforms may also be involved in cell-specific expression of the TUB protein remains to be tested using epitope-specific antibodies. In our study, we observe no difference in the distribution or the level of TUB protein between wild type and tub /tub mutant mice, indicating that the tub mutation does not lead to instability or increased degradation of the protein, but rather that this mutation appears to impair the function of

the protein, a conclusion confirmed by a recent analysis of mice carrying a targeted deletion of the tub gene [27]. Our finding that tub /tub mice do not differ from control animals in their TUB protein expression is in contrast to a report that tubby mice lack TUB protein [17]. We have previously shown that both our N- as well as our Cterminal TUB antibodies recognize the wild type protein, but that only the N-terminal antibody detects protein in tub /tub mice [14]. It is, therefore, possible that the antibody used by Kappeller et al. recognized an epitope at the C-terminus of the protein which is missing in the mutant protein. Recent studies demonstrate that tub mRNA is highly expressed in the retinal ganglion cell layer and photoreceptor layer. Loss of function of tubby in the retina triggers photoreceptor apoptosis in a manner similar to that reported in other cases of retinitis pigmentosa (RP) [29]. However, this may not be directly related to tubby’s function since apoptosis appears to be a default pathway in the retina that is triggered by mutations in a wide variety of functionally unrelated genes [30]. Also, apoptosis has not yet been observed in the hypothalamus to explain the progressive obesity observed in tubby mice. Several possible functions for TUB have been recently proposed. Based on in vitro transfection experiments, Kappeller et al. suggest that TUB is an intermediate in the insulin signaling cascade [17]. In addition Zheng et al. had previously found that TUB associates in a yeast two hybrid assay with a novel protein that shows homology to kinases that are part of the stress / mitogen activated protein kinase pathway [31]. More recently the conserved C-terminal domain of TUB has been crystallized and shown to possess a unique barrel structure with a long positively charged putative DNA binding groove [4]. In addition, the same group showed that the TUB C-terminal domain can bind double stranded DNA and that the N-terminal domains of TUB and TULP1, when fused to the GAL4 DNA binding domain, can activate transcription from a GAL4 promoter in vitro [4]. Based on these results, Boggon et al. suggested that TUB is a member of a novel class of transcription factors. Our findings, that TUB is localized in the nucleolus, and TULP1 in the perinucleolar cap and coiled bodies [15], prompts us to speculate that the tubby gene family might be involved in ribosomal and RNA synthesis and / or trafficking. The nucleolus and other nuclear bodies are known to be important in these processes [24,26]. More recently, however, evidence has been obtained that these nuclear organelles may also play a role in the transcription of mRNA [7]. Further experiments will determine whether the different views of tubby function can be reconciled. It is tempting to speculate that the tubby gene family members might be transcription factors that respond to growth signals and mediate transcription of RNAs that are important for ribosome synthesis, such as snoRNAs. This would be consistent with TUB expression in all neurons, not just

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those important in energy metabolism. In addition, insulin receptor expression is widespread in the brain, and insulin has been implicated in the growth of cultured neurons [12] and in the regulation of ribosomal DNA transcription [11]. In this respect, it is interesting that we observe the strongest staining for TUB in the subependymal zone (Fig. 8D), a region of active proliferation in the brain [6,16].

[14]

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Acknowledgements [16]

We are grateful to Drs Susan L. Ackerman, Timothy P. O’Brien and Barbara K. Knowles for careful review of the manuscript. This work was supported by NIH grant DK46977 and a grant from AXYS Pharmaceuticals Inc. Institutional shared services are supported by National Cancer Institute Support grant CA-34196.

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