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Originally published In Press as doi:10.1074/jbc.M000655200 on April 4, 2000

J. Biol. Chem., Vol. 275, Issue 25, 19106-19114, June 23, 2000
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Pancreatic Homeodomain Transcription Factor IDX1/IPF1 Expressed in Developing Brain Regulates Somatostatin Gene Transcription in Embryonic Neural Cells*

Petra T. SchwartzDagger §, Beatriz Pérez-VillamilDagger , Alicia Rivera||, Rosario Moratalla||, and Mario VallejoDagger **DaggerDagger

From the Dagger  Reproductive Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, the || Instituto Ramón y Cajal, Consejo Superior de Investigaciones Científicas, 28002 Madrid, Spain, and the ** Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas/Universidad Autónoma, Calle Arturo Duperier 4, 28029 Madrid, Spain

Received for publication, January 25, 2000, and in revised form, March 27, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hox-like homeodomain proteins play a critical role during embryonic development by regulating the transcription of genes that are important for the generation of specific organs or cell types. The homeodomain transcription factor IDX1/IPF1, the expression of which was thought until recently to be restricted to the pancreas and foregut, is required for pancreas development and for the expression of genes controlling glucose homeostasis. We report that IDX1/IPF1 is also expressed in embryonic rat brain at a time coincident with active neurogenesis. Electrophoretic mobility shift assays with nuclear extracts of embryonic brains indicated that IDX1/IPF1 binds to two somatostatin promoter elements, SMS-UE-B and the recently discovered SMS-TAAT3. The requirement of these elements for IDX1/IPF1 transactivation of the somatostatin gene in neural cells was confirmed in transfection studies using embryonic cerebral cortex-derived RC2.E10 cells. Immunohistochemical staining of rat embryos showed IDX1/IPF1-positive cells located near the ventricular surface in germinative areas of the developing central nervous system. Cellular colocalization of IDX1/IPF1 and somatostatin was found in several areas of the developing brain, including cortex, ganglionic eminence, hypothalamus, and inferior colliculus. These results support the notion that IDX1/IPF1 regulates gene expression during development of the central nervous system independent of its role on pancreas development and function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homeodomain genes encode a large family of transcription factors that are grouped in different classes, including, for example, Hox-, POU-, Pax-, LIM-, and Dlx-like proteins, depending on the relative degree of conservation of the homeodomain. During gestation, expression of individual homeodomain genes is critical for proper regional development of the central nervous system (CNS)1 and peripheral tissues of the embryo.

IDX1/IPF1 (also known as STF-1 and PDX-1) is a transcription factor encoded by a Hox-like homeodomain gene, the expression of which was initially described to be restricted to the pancreas and duodenum, and it has been demonstrated that pancreas development requires IDX1/IPF1 in mice (1, 2). This requirement was confirmed in the human by the identification of an individual with pancreatic agenesis and a homozygous mutation in the IPF-1 gene (3). In addition, IDX1/IPF1 is essential for normal pancreatic islet function by regulating the expression of a number of pancreatic genes, including insulin, somatostatin, islet amyloid polypeptide, and glucose transporter type 2 (4-8). Importantly, in humans, heterozygous mutations of the IPF-1 gene are linked to a type of autosomal dominant diabetes mellitus known as maturity onset diabetes of the young (MODY4) (9).

In the developing neural tube, different homeodomain genes that control mechanisms of regional and cellular determination are selectively expressed in the primordia of the three major regions of the CNS: forebrain, midbrain, and hindbrain (10). In the hindbrain, certain Hox-like homeodomain genes specify positional information utilized by neural precursor cells to develop into appropriate types of neurons or glial cells (11). A similar type of code involving LIM and Pax homeodomain genes operates in the spinal cord (12). In the midbrain, proper development requires the expression of engrailed genes (13, 14).

In the forebrain, expression of most Hox genes is not readily detectable. However, other classes of mammalian homeobox genes, including Nkx, Otx, Emx, and Dlx, show restricted patterns of expression preferentially located in the forebrain (15) and are required for the development of forebrain structures (16-18) or for the differentiation of specific cell types in different regions of the brain (18, 19). In addition, homeodomain transcription factors of two other classes (POU and Pax) are important for forebrain development (20-23).

In a preliminary study, we found that expression of IDX1/IPF1 during embryonic development occurs in the CNS as well as in the pancreas (24). In the present study, we provide evidence that the somatostatin gene is a target for regulation by IDX1/IPF1 in neural cells and that neural expression of IDX1/IPF1 is spatially and temporally regulated during CNS development. Thus, our data reinforce the notion that the transcriptional functions of IDX1/IPF1 are broader than previously anticipated, as they indicate the likelihood that IDX1/IPF1 plays a role in neural development.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RT-PCR/Southern Blot Hybridization Analyses-- Developing brains from rat embryos removed at different gestation times from timed-pregnant Harlan Sprague-Dawley rats were dissected under the microscope, and the meningeal membranes were carefully removed. Brains of embryonic day 16 (E16) and E19 embryos were further dissected into two parts by a transverse section made at the level of the caudal edge of the cerebral cortex. The piece rostral to this cut was labeled as forebrain, and the piece caudal to this cut was labeled as hindbrain. In addition, brains were removed from adult rats, and several brain regions were dissected as described (25). Total RNA (10 µg) from individual samples was incubated with (dT)15 and avian myeloblastosis virus reverse transcriptase to synthesize cDNA.

For PCR amplification of IDX1/IPF1, two different sets of primers were used in independent experiments, yielding identical results. In the first set, a forward primer that corresponds to nucleotides 90-117 (5'-CGGCTGCAACCATGGATAGTGAGGAGCAG-3') and a reverse primer that anneals to nucleotides 280-296 (5'-AAGTCCCCCGGACATCT-3') of the published cDNA (6) were used. In the second set, a forward primer that corresponds to nucleotides 96-118 (5'-CCACCATGAATAGTGAGGAGCA-3') and a reverse primer that anneals to nucleotides 305-322 (5'-GGCGAGCGGGGGCACTTC-3') were used. In both cases, PCR conditions were as follows: 98 °C for 1 min; 30 cycles of 96 °C for 30 s, 56 °C for 30 s, and 75 °C for 1 min; and a 5-min incubation at 75 °C. After PCR, an aliquot of the reaction was resolved in a 1% agarose gel, blotted onto a nylon membrane, probed with a 32P-labeled internal primer that anneals to nucleotides 216-236 (5'-GCCGCCAGCCCCCACCTCCGC-3'), and autoradiographed at -70 °C.

Primers for PCR amplification of Emx-1 and Dlx-2 were designed based on the published sequences of the rat cDNAs (26). The Emx-1 forward primer (nucleotides 10-31) was 5'-CCTGGCTGGCTGGGTGCACACC-3', and the reverse primer (nucleotides 278-289) was 5'-CCACTCACGAAGGCCGCCTCG-3'. PCR conditions were as follows: 94 °C for 5 min; 30 cycles of 96 °C for 30 s, 65 °C for 30 s, and 75 °C for 1 min; and a 5-min incubation at 75 °C. As a probe to detect the PCR product after blotting onto nylon, the following 32P-labeled internal primer was used: 5'-CACTCCTCTTCGGCGGCAGCG-3'. The Dlx-2 forward primer (nucleotides 1-23) was 5'-ACAGCCATGTCTGCTTAGACCAG-3', and the reverse primer (nucleotides 298-321) was 5'-TTCACGCCGTGATACTGATACTGG-3'. PCR conditions were as follows: 98 °C for 1 min; 30 cycles of 96 °C for 30 s, 60 °C for 30 s, and 75 °C for 1 min; and a 5-min incubation at 75 °C. The 32P-labeled internal primer used as a probe to detect the PCR product was 5'-ACTTCCAAGCTCCGTTCCCGA-3'.

For PCR amplification of actin, the primers used were as follows: forward, 5'-GACGATATGGAGAAGATTTGGCA-3', which anneals to exon 2; reverse, 5'-CCATCTCTTGCTCGAAGTCTAGG-3', which anneals to exon 3. The following protocol was used: 94 °C for 5 min; 20 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; and a 5-min incubation at 72 °C. As a probe to detect the PCR product after blotting onto nitrocellulose, the following 32P-labeled internal primer was used: 5'-CACACGCAGCTCATTGTAGAAAGT-3'.

In all cases, controls were carried out in which samples were treated with RNase A prior to cDNA synthesis to rule out genomic DNA contamination. In preliminary experiments, different numbers of PCR cycles were tested to ensure that the resulting signal in most samples does not increase to saturation.

Western Immunoblots-- Brain samples or cell lines were lysed in buffer containing 125 mM Tris-HCl (pH 6.8), 4% SDS, 15% glycerol, 10% beta -mercaptoethanol, and 10 mM dithiothreitol. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. Immunoreactivity was detected with the alpha 253 antiserum (1:10,000 dilution) (3, 27) and visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

DNA-Protein Binding Assays-- Electrophoretic mobility shift assays were carried out with nuclear extracts of rat developing forebrains or RC2.E10 cells exactly as described (28). For competitions, wild type oligonucleotides, mutated TAAT oligonucleotides (6, 28), or nonspecific oligonucleotides of unrelated sequences were used. The sequences of all the oligonucleotides used have been published previously (6, 28-30).

Cell Lines and Transfections-- Neural RC2.E10 cells derived from rat E16 embryonic cortex (28), and rat pancreatic islet somatostatin-producing RIN-1027-B2 cells (31) were used and cultured as described. All plasmids and transient transfections of RC2.E10 cells using Lipofectin (Life Technologies, Inc.) have been described (6, 28).

Immunohistochemistry-- Rat fetuses of different ages were removed from timed-pregnant Harlan Sprague-Dawley rats and fixed overnight in 4% paraformaldehyde. After fixation, they were washed with phosphate-buffered saline, transferred to phosphate-buffered saline containing 20% sucrose, and kept at 4 °C for at least 24 h. Sagittal cryostat sections (10 µm) were cut and kept at -80 °C until used. Sections were brought to room temperature, permeabilized with methanol for 2 min at -20 °C, treated with 5% normal goat serum, and then incubated overnight with the alpha 253 anti-IDX1/IPF1 antiserum (1:1500 dilution) at 37 °C. Immunodetection was carried out with a secondary biotinylated goat anti-rabbit antiserum (Bio-Rad) using nickel-intensified immunoperoxidase staining with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA).

Two-color dual antigen immunostaining was carried out serially as described (32). First, somatostatin was detected with a specific monoclonal rat antiserum (33) diluted 1:100, using immunoperoxidase staining enhanced with nickel ammonium, which yields a dark blue color. Sections were then washed, and IDX1/IPF1 immunohistochemistry proceeded as described above, using immunoperoxidase staining without nickel ammonium, yielding a brown color.

Degenerate RT-PCR-- Three regions of the rat brain (cortex, hippocampus, and hypothalamus/thalamus) were dissected essentially as described (25, 34), with the exceptions that only parietal cortex was taken and that the hypothalamus/thalamus was dissected as a single block of tissue. Total RNA (10 µg) purified by CsCl gradient centrifugation from each one of these regions was primed with (dT)15 and incubated with avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals) to synthesize cDNA. One-tenth of this cDNA preparation was used for PCR, using degenerate oligonucleotides corresponding to conserved residues found in helices 1 and 3 of Hox-like homeodomains. A control sample that lacked RNA was processed in parallel. The resulting PCR products were ligated into pBluescript (KS) (Stratagene, La Jolla, CA); after transforming Escherichia coli JM109, 25-30 individual colonies corresponding to each brain region were randomly picked, and plasmid minipreps were prepared and sequenced using the dideoxy chain termination method (Sequenase kit, United States Biochemicals, Cleveland, OH). The degenerate oligonucleotide sequences, PCR conditions, and subcloning procedure were exactly as described (6, 35). Sequences were compared with those in the GenBankTM data base by using the BLAST Network Service provided by the National Center for Biotechnology Information.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of IDX1/IPF1 in the Developing CNS-- To confirm that IDX1/IPF1 is expressed in the brain, we carried out RT-PCR/Southern blot hybridization analysis with specific primers to IDX1/IPF1, using embryonic as well as adult rat brains. As a control to monitor developmental changes in homeodomain gene expression, we determined, in parallel experiments using the same template for RT-PCRs, the expression of Emx-1 and Dlx-2, two homeodomain genes known to be expressed restrictively in telencephalic areas of the developing brain (18, 19, 36-38). As expected, Emx-1 and Dlx-2 transcripts were present in the brains of E14 and E15 rats, enriched in the forebrain relative to the hindbrain (Fig. 1, E16 and E19). We found that IDX1/IPF1 transcripts are present in the brains of E14 and E15 rats (Fig. 1). In contrast to Emx-1 and Dlx-2, there was no difference in the levels of expression of IDX1/IPF1 between forebrain and hindbrain at E16. Finally, at E19, expression of Emx-1 and Dlx-2 was robust and restricted to the forebrain, but IDX1/IPF1 levels had decreased both in the forebrain and in the hindbrain (Fig. 1).


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  		Fig. 1.   Expression of IDX1/IPF1 transcripts in embryonic and adult central nervous system as assessed by RT-PCR/Southern blotting. For E14 and E15 embryos, the whole brain was used. For E16 and E19 embryos, the brain was divided into forebrain (F) and hindbrain (H). Expression of Emx-1 and Dlx-2, two unrelated homeodomain genes preferentially expressed in the forebrain, was used for comparison. For each lane, IDX1/IPF1, Emx-1, Dlx-2, and actin were amplified using aliquots of the same cDNA template. The bottom panel shows the results of densitometric analyses carried out to quantify the relative intensity of IDX1/IPF1 bands. The results of two independent experiments are depicted: black bars correspond to the experiment shown in the top panel, and white bars correspond to a different experiment (not shown). Results are expressed as percentage of increment of densitometry measurements of IDX1/IPF1 bands (in arbitrary units) relative to the intensity of the corresponding actin bands. Densitometry of scanned autoradiographs was carried out using NIH Image 1.62 software. MB, midbrain; HYP, hypothalamus; HIPP, hippocampus; STR, striatum; BSTM, brainstem; CER, cerebellum; CTX, cortex.

In the adult brain, IDX1/IPF1 transcripts appeared to be of relatively low abundance in most regions examined, with the exception of the cerebellum (Fig. 1). As expected, Emx-1 and Dlx-2 were found to be preferentially expressed in telencephalic areas (hippocampus, striatum, and cortex) (Fig. 1).

Immunochemical detection of IDX1/IPF1 protein in neural cells was carried out initially by Western immunoblotting using an antiserum (alpha 253) that recognizes specifically the C-terminal 12 amino acids of IDX1/IPF1 (3, 27). Western immunoblot analysis confirmed that this antiserum recognizes both IDX1/IPF1 synthesized in vitro, and IDX1/IPF1 constitutively expressed in pancreatic islet-derived RIN-1027-B2 cells (Fig. 2), a cell line from which IDX1/IPF1 was originally cloned (6). In addition, we detected immunoreactive IDX1/IPF1 in nuclear extracts of E15 and E17 rat brains, as well as in nuclear extracts of RC2.E10 cells (Fig. 2), a neural precursor cell line derived from rat embryonic cortex (28, 73).


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  		Fig. 2.   Detection of IDX1/IPF1 protein by Western immunoblotting using the alpha 253 antiserum. Arrow indicates immunoreactive IDX1/IPF1. RL, unprogrammed rabbit reticulocyte lysate; IDX-1, IDX1/IPF1 synthesized in rabbit reticulocyte lysate; RIN-B2, extract from pancreatic islet RIN-1027-B2 cells; RC2.E10, extract from developing cortex-derived RC2.E10 cells. Brain extracts of E15 and E17 rat embryos included only the forebrain. Similar experiments were repeated at least three times.

The finding of IDX1/IPF1 in RC2.E10 cells, which express the somatostatin gene in a neural-specific manner (28), prompted us to explore the possibility that IDX1/IPF1 regulates somatostatin gene expression in neural cells.

IDX1/IPF1 Present in Neural Cells Binds to Somatostatin Gene DNA-regulatory Elements-- The promoter region of the somatostatin gene contains at least four homeodomain-binding DNA regulatory elements with a common TAAT core motif, named SMS-UE-B, SMS-TAAT1, SMS-TAAT2, and SMS-TAAT3, some of which appear to act as targets for regulation of somatostatin gene expression by IDX1/IPF1 in pancreatic cells (5, 6, 28, 30, 39). We sought to investigate whether IDX1/IPF1 expressed in developing neural tissue is able to bind to these elements.

Initially, we determined by electrophoretic mobility shift assays that nuclear extracts prepared from embryonic rat brains contain proteins that recognize synthetic oligonucleotides corresponding to these TAAT sites. Several specific DNA-protein complexes, distributed in three different clusters of slow (cluster 1), medium (cluster 2), and fast (cluster 3) relative electrophoretic mobility, were found to bind to SMS-TAAT1 (Fig. 3A). No appreciable differences were observed between brain nuclear extracts prepared from E14 or E16 embryos (Fig. 3A). A similar pattern was found when the SMS-TAAT2 probe was used, with the exception that the bands corresponding to complex 1 were not detected (Fig. 3B).


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  		Fig. 3.   Electrophoretic mobility shift assays indicating the binding of nuclear proteins prepared from E14 or E16 rat brains to SMS-TAAT1 (A), SMS-TAAT2 (B), SMS-TAAT3 (C), and SMS-UE-B (D). Oligonucleotide probes used in each assay are indicated at the bottom. Clusters of protein complexes with different electrophoretic mobility bound to SMS-TAAT1 and SMS-TAAT2 are numbered. Nuclear extracts were incubated in the absence (-) or presence of competing oligonucleotides (10- or 100-fold molar excess) of identical probe sequence or in the presence of the corresponding mutated TAAT oligonucleotide (Mut), used in a 100-fold molar excess. One representative example of at least five independent experiments is shown.

When the SMS-TAAT3 probe was used, only two major protein-DNA complexes with relatively slow electrophoretic migration were detected in brain nuclear extracts from E14 or E16 embryos (Fig. 3C). Finally, two complexes were found to bind to SMS-UE-B in brain nuclear extracts from E14 embryos, but only one was detected when brain nuclear extracts were prepared from E16 embryos (Fig. 3D).

To investigate whether any of the complexes observed with embryonic brain nuclear extracts contain IDX1/IPF1, we carried out electrophoretic mobility shift assays in the presence of the alpha 253 antiserum or in the presence of a different anti-IDX1/IPF1 antiserum (alpha 251), which specifically recognizes the N-terminal 12 amino acids of IDX1/IPF1 (3, 27). The addition of either of these antisera to the binding reaction did not disturb the protein complexes bound to SMS-TAAT1 and SMS-TAAT2 (not shown). However, we found that the presence of either of these antisera in the binding reaction results in the disappearance of the upper band detected with the SMS-TAAT3 (Fig. 4A) or with the SMS-UE-B probes (Fig. 4B). Similar results were obtained when nuclear extracts from neural RC2.E10 cells, which express the somatostatin gene (28), were used (not shown). Thus, these experiments identify IDX1/IPF1 as a homeodomain transcription factor that binds somatostatin gene regulatory elements in neural cells.


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  		Fig. 4.   Electrophoretic mobility shift assays showing the binding of IDX1/IPF1 present in nuclear extracts of E14 and E16 rat brains to oligonucleotide probes corresponding to the somatostatin SMS-TAAT3 and SMS-UE-B elements. Binding reactions were carried out in the presence of either normal rabbit serum (NRS) or anti-IDX1/IPF1 alpha 251 or alpha 253 antisera. Arrows indicate bands corresponding to protein-DNA complexes containing IDX1/IPF1. One representative example of three independent experiments is shown.

IDX1/IPF1 Activates Somatostatin Gene Expression in Neural Cells-- We carried out transient transfection experiments to evaluate the transactivational activity of IDX1/IPF1 on the somatostatin gene. First, we confirmed that transfection of RC2.E10 cells with pBJ5-IDX-1 (6), an expression plasmid encoding IDX1/IPF1, results in elevated levels of IDX1/IPF1 expression (Fig. 5A). We then cotransfected increasing amounts of the pBJ5-IDX-1 plasmid with a fixed amount of plasmid SMS900, a chloramphenicol acetyltransferase (CAT) reporter plasmid that contains a fragment of the somatostatin gene spanning nucleotides -900 to +54 (40). Expression of SMS900 after transient transfections in RC2.E10 cells has been characterized previously (28). Cotransfection of pBJ5-IDX-1 and SMS900 resulted in a concentration-dependent increase in the levels of CAT activity (Fig. 5C).


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  		Fig. 5.   IDX1/IPF1 transactivates the somatostatin gene promoter in embryonic cerebral cortex-derived RC2.E10 cells. A, Western immunoblotting carried out with the anti-IDX1/IPF1 alpha 253 antiserum demonstrating over-expression of IDX1/IPF1 in RC2.E10 cells transfected with pBJ5-IDX-1. B, schematic representation of wild type (SMS900) and mutated (M) somatostatin CAT reporter plasmids used to transfect RC2.E10 cells. TAAT-containing elements are depicted as open boxes, and their mutated versions are depicted as black boxes. The elements are as follows: T1, SMS-TAAT1; T2, SMS-TAAT2; T3, SMS-TAAT3; and UE, SMS-UE-B. C, relative CAT activities elicited by SMS900 (15 µg) in the absence (-) or presence of different amounts of pBJ5-IDX-1 observed after transfections in RC2.E10 cells. D, relative CAT activities elicited by each one of the somatostatin CAT reporter plasmids depicted in B cotransfected in RC2.E10 cells with 2.5 µg of pBJ5-IDX-1 (IDX-1) or pBJ5 with no insert (vector). Values for CAT activities are expressed as percentages of the activities elicited by wild type SMS900 CAT and represent the mean ± S.E. of at least three independent experiments carried out in duplicate.

To determine whether any of the TAAT-containing elements of the somatostatin gene promoter are important for IDX1/IPF1 transactivation in neural cells, we carried out another series of transient cotransfection experiments in RC2.E10 cells, using mutant SMS900 plasmids in which each one of the TAAT motifs had been altered by site-directed mutagenesis in such a way that binding of nuclear proteins is impaired (28).

Disruption of either SMS-TAAT1 or SMS-TAAT2 (Fig. 5D), or of both elements together (not shown), did not alter the transactivation activity of IDX1/IPF1. However, disruption of either SMS-TAAT3 or SMS-UE-B independently inhibited the increase in the levels of CAT activity elicited by cotransfection with pBJ5-IDX-1 (Fig. 5D), indicating that the integrity of these elements is required for the transactivation of the somatostatin gene by IDX1/IPF1 in neural cells.

Distribution of IDX1/IPF1-expressing Cells in the Developing Brain-- To gain information on the regional distribution of cells expressing IDX1/IPF1 in the developing CNS, we carried out immunohistochemistry on sagittal sections of rat embryos. As expected, we observed IDX1/IPF1 immunoreactivity in the duodenum and pancreatic mesenchyme (not shown). In the developing CNS, we found IDX1/IPF1 expressed in several regions.

In the telencephalon, cells with IDX1/IPF1 immunopositive nuclei were readily detectable throughout the depth of the neocortical neuroepithelium of E14 and E15 embryos (Fig. 6A). At E15, a higher density of cells was often found clustering in the subventricular zone (Fig. 6A). The intensity of immunostaining and relative number of immunopositive cells decreased at E16. In the neocortical epithelium of these embryos, IDX1/IPF1-positive cells were restricted to the ventricular and subventricular zones; by E17, IDX1/IPF1 immunoreactivity in the neocortex was practically undetectable (Fig. 6A), and positive nuclei were observed only occasionally. IDX1/IPF1 was also found expressed in the archicortex, which corresponds to the hippocampal primordium (Fig. 6B), as well as in the germinative layers of the medial and lateral ganglionic eminence corresponding to the striato-pallidal primordium that generates the basal ganglia (Fig. 6B). However, at E17, IDX1/IPF1 expression in the hippocampus and basal ganglia was undetectable (not shown).


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  		Fig. 6.   Immunohistochemical localization of IDX1/IPF1-expressing cells in the central nervous system of developing rats. Images depict nuclear localization of IDX1/IPF1 in black. All pictures correspond to parasagittal sections of developing rat brains, prepared at the indicated gestation times (E14 to E17) and are oriented so that anterior is left and dorsal is top. A, decreasing expression in the developing neocortex observed between E14 and E17. At E14, IDX1/IPF1 immunopositive cells are present throughout the entire thickness of the neuroepithelium; at E15, the highest density of immunoreactive cells is found close to the ventricular surface; at E16, weakly immunoreactive cells occupy the ventricular and subventricular zones; and at E17, IDX1/IPF1 cells were undetectable. B, IDX1/IPF1 immunopositive cells in different brain regions at E15. C, IDX1/IPF1-immunopositive cells observed at a late gestation time (E17), when immunoreactivity in other brain areas is no longer detectable. All pictures were taken at the same magnification. Scale bar, 50 µm. A total of 14 immunohistochemical determinations on eight embryos were carried out by two researchers independently. 3V, third ventricle; 4V, fourth ventricle; acx, archicortex (hippocampus); ah, anterior hypothalamic neuroepithelium; ap, area postrema; aq, aqueduct; cb, cerebellar primordium; hyp, hypothalamus; ic, inferior colliculus; iz, intermediate zone; lge, lateral ganglionic eminence; LV, lateral ventricle; mge, medial ganglionic eminence; pt, pretectum; pyr, pyramidal tract; sc, superior colliculus; svz, subventricular zone; teg, tegmental neuroepithelium; tu, tuberal hypothalamus; vz, ventricular zone.

IDX1/IPF1 immunoreactivity was also found in periventricular areas of the hypothalamus of E14 and E15 embryos, in scattered cells surrounding the optic, mammillary, and infundibular recesses (not shown), and in the anterior hypothalamic neuroepithelium and the tuberal hypothalamus, where the highest density of cells with immunopositive nuclei was observed (Fig. 6B). No IDX1/IPF1 immunoreactivity was detected in the hypothalamus of E17 embryos.

In the mesencephalon, IDX1/IPF1 immunopositive cells were found in the pretectum and in the tectum, as well as in the tegmental neuroepithelium (Fig. 6B). In rostral areas (pretectum and superior colliculus), expression decreased as development proceeded, so that in E17 embryos, a relatively smaller number of positive cells were detected. In contrast, IDX1/IPF1 immunoreactivity in the caudal mesencephalon did not decrease, and many immunopositive cells were detected in the inferior colliculus of E17 embryos (Fig. 6C).

More caudally, expression of IDX1/IPF1 was found in the cerebellar primordium of E14 (not shown) and E15 embryos, in cells that were located close to the ventricular surface (Fig. 6B). No immunoreactivity was detected in E17 embryos.

Finally, intensely immunoreactive cells restricted to the area postrema appeared late in development (Fig. 6C). Also, in E17 embryos, immunopositive cells were detected in the developing pineal gland (Fig. 6C).

Coexpression of IDX1/IPF1 and Somatostatin in Neural Cells-- In pancreatic cells, IDX1/IPF1 regulates somatostatin gene expression (5, 6, 39). In the CNS, somatostatin expression first appears at E14, and its developmental pattern of expression coincides with the spatial and temporal pattern that we found for IDX1/IPF1 (41-43). Therefore, we sought to determine whether somatostatin and IDX1/IPF1 are coexpressed in the same cells in the CNS during embryonic development. The presence of somatostatin-immunoreactive cells in different brain regions of E15 embryos, including ganglionic eminence, cortex (Fig. 7), hypothalamus, and mesencephalon (not shown), was confirmed a priori by standard immunohistochemistry. Dual antigen immunohistochemistry revealed the presence of immunopositive cells for both somatostatin and IDX1/IPF1 in these areas (Fig. 7). The vast majority of somatostatin-positive cells stained positively for IDX1/IPF1, strongly supporting the notion that IDX1/IPF1 may also regulate somatostatin gene expression in neural cells. In addition, we detected a number of cells that showed IDX1/IPF1-positive nuclei, but we did not stain for somatostatin (Fig. 7).


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  		Fig. 7.   Coexpression of IDX1/IPF1 and somatostatin demonstrated by dual antigen immunohistochemistry in parasagittal sections of E15 rat brain. Sections were stained only for somatostatin (A and C) or for both somatostatin and IDX1/IPF1 (B and D). Neocortex (A and B) and ganglionic eminence (C and D) are shown. Somatostatin immunoreactivity (depicted in blue) is localized to the cytoplasm, and IDX1/IPF1 immunoreactivity (depicted in brown) is localized to the nucleus. Arrows indicate examples of doubly labeled cells. Arrowheads indicate examples of cells that stained for IDX1/IPF1 but not for somatostatin. Scale bar, 20 µm. LV, lateral ventricle. Coexpression of IDX1/IPF1 and somatostatin was confirmed in four independent experiments.

Expression of Hox-like Genes in Brain Tissue-- To further investigate the expression of IDX1/IPF1 and other Hox genes in the forebrain, we carried out RT-PCR using RNA purified from three different adult brain regions. As PCR primers, we used degenerate oligonucleotides encoding the sequences ELEKE and KIWFQN, conserved in the homeodomains of Hox-like proteins. Sequence analyses of individual clones prepared from the resulting PCR products revealed the presence of several Hox-like genes expressed in each of the different brain regions analyzed (Table I).

                              
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Table I
Sequences encoding Hox-like homeodomains detected by cloning and sequencing of degenerate RT-PCR products obtained from RNAs expressed in three different brain regions

In the hypothalamus/thalamus region, 52% of the clones sequenced encode the homeodomain of Gbx-2, also known as MMox-A (44), and 12% of the clones encode MAB26. Both of these Hox-like homeodomain sequences had been previously found expressed in developing mouse telencephalon using different degenerate primers (44). In addition, Gbx-2 has been shown to be expressed in the diencephalic area that contains the prospective dorsal thalamus in mouse embryos (36, 45). MAB26 was the most frequently found clone in the hippocampus, followed by HoxA1 (Table I). HoxA1 was also found with relatively high frequency (28%) in the cortex. However, in this region, the most frequently found sequence (34%) encoded the homeodomain of IDX1/IPF1.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although expression of the homeodomain transcription factor IDX1/IPF1 has been described as being restricted to the foregut and pancreas, we reported recently evidence indicating that it is also expressed in the developing CNS (24). This study demonstrates that expression of IDX1/IPF1 in neural cells is developmentally regulated in a region- and time-specific manner. In the cortex, ganglionic eminence, and hypothalamus, expression decreases as development proceeds, whereas in other areas, such as the inferior colliculus and area postrema, expression progressively increases or appears later in development, respectively. This dual mode of expression is reminiscent of the pattern of IDX1/IPF1 expression in the pancreas. At early stages, it occurs in proliferative cells of the growing pancreatic primordium in both exocrine and endocrine compartments (1, 2), but subsequently it is restricted to cells in the islets of Langerhans, consistent with two independent functions, one related to the genesis of insulin-producing beta -cells and the other related to the maintenance of their phenotype and of glucose homeostasis (46, 47).

Our data support the hypothesis that IDX1/IPF1 participates in the regulation of the neural-specific expression of the somatostatin gene. In pancreatic delta -cells, IDX1/IPF1 regulates somatostatin gene expression by binding SMS-UE-B (also known as TSE-I) and SMS-TAAT1 (also known as TSE-II) (5, 6, 39, 48, 49). However, in neural cells, SMS-TAAT1 (or SMS-TAAT2) does not appear to be recognized by IDX1/IPF1. A comparison of the effects of both alpha 251 and alpha 253 antisera on the binding of nuclear proteins from neural RC2.E10 cells and from somatostatin-producing pancreatic RIN-1027-B2 cells confirmed that even though IDX1/IPF1 is present in both cell types, it binds to SMS-TAAT1 and SMS-TAAT2 only in pancreatic cells (not shown), suggesting that differences in the protein environment within the nucleus of neural or pancreatic cells can alter IDX1/IPF1 DNA binding specificity. Indeed, the requirement of the integrity of the SMS-UE-B and SMS-TAAT3 elements suggests that the protein complexes bound to each one of them may engage in interactions that are functionally important for IDX1/IPF1 transactivation in neural cells, as it occurs in pancreatic cells (39, 48-52).

In the embryo, the location of IDX1/IPF1-expressing cells close to the ventricular surface in areas of the developing CNS, such as hypothalamus, ganglionic eminence, and cortex, suggests that those cells are proliferative or differentiating neural precursors. In the hypothalamus, IDX1/IPF1 is present at a time of active neurogenesis (reviewed in Ref. 20) in regions that generate neurons for several nuclei, including those with the highest number of somatostatin-expressing cells (periventricular, suprachiasmatic, and arcuate nuclei and anterolateral hypothalamus) (43, 53).

Expression of IDX1/IPF1 in cells of the germinative zone of the ganglionic eminence coincides with a peak of neurogenesis in this structure (54-57). It has been suggested that other homeodomain proteins of different families (POU, Lhx, and Dlx), also expressed in the germinative zone at this time, participate in the initiation of the differentiation program of proliferating precursors (18, 20, 22, 58). Thus, IDX1/IPF1 could act coordinately with some of these transcription factors to generate combinatorial codes for the selective regulation of specific genes. Interestingly, striatal somatostatin-expressing interneurons are generated at this time (56, 57).

In the cortex, IDX1/IPF1 was found in cells of the proliferating neuroepithelium, often close to the ventricular surface (E14 and E15), but not in layers distal to the intermediate zone (E16). Thus, IDX1/IPF1 appears to be down-regulated as differentiating precursors or young neurons migrate through the cortex. In the rat, cortical neurogenesis takes place up until E20 (20, 59), but expression of IDX1/IPF1 was practically undetectable by E17. Therefore, the observed pattern of IDX1/IPF1 expression in the developing cortex is consistent with a role in neural precursors that generate specific types of neurons during a restricted period of time, but not in the maintenance of their mature phenotype. One of these types may correspond to somatostatin-producing neurons. Like IDX1/IPF1, somatostatin gene expression in many cells of the developing brain is transient, so that the relative number of somatostatin-expressing cells in the mature cortex is lower than that present during corticogenesis (41, 43, 60, 61).

We found a number of cells that stain for IDX1/IPF1 but not for somatostatin in all areas examined. Thus, IDX1/IPF1 probably regulates the expression of other genes in the CNS, but their identities are unknown. Nonetheless, that the somatostatin gene is a target for regulation by IDX1/IPF1 in neural cells is further supported by our electrophoretic mobility shift assays and transfection studies using somatostatin-expressing cortex-derived RC2.E10 cells.

One function of IDX1/IPF1 is to participate in the homeostatic control of normoglycemia by coordinately regulating the expression of several genes involved in glucose-sensing mechanisms and glucose utilization in the pancreas (46, 47). The brain acts as a center for the integration of information pertaining to fuel metabolism and energy homeostasis (62). In particular, the area postrema contains glucose-responsive neurons, and the hypothalamus contains glucose-, glucagon-like peptide-1-, and leptin-responsive neurons, which may play a role in the central control of food intake and maintenance of normoglycemia (63-66). Our finding of IDX1/IPF1 in the developing hypothalamus and area postrema suggests that one putative function of IDX1/IPF1 in the embryo may be the development of an integrated system of cells located both in the CNS and in the periphery to coordinately regulate glucose uptake and metabolism.

The effects of the inactivation of IDX1/IPF1 on pancreas development have been assessed in detail in mutant mice (1, 2, 46, 47), but no studies on the development of the CNS in those mice have been reported. IDX1/IPF1 mutant mice exhibit lack of pancreas and early postnatal lethality (1, 2), preventing the finding of any possible neural deficit. However, possible defects due to lack of IDX1/IPF1 in the CNS may be subtle and not readily apparent unless purposely studied in detail, as it is the case for other homeodomain genes (18, 19).

Several genes encoding transcription factors, including Nkx2.2, Isl-1, and BETA2/NeuroD, are restrictively expressed in both the developing pancreas and the developing CNS. Targeted disruption of the Isl-1 gene results in early arrest of pancreatic development and embryonic lethality (67). Like mice lacking IDX1/IPF1, mice with inactivation of the genes encoding BETA2/NeuroD or Nkx2.2 develop severe neonatal diabetes, due to alterations in pancreatic development, and die postnatally (68, 69). In the CNS, lack of BETA2/NeuroD or Nkx2.2 results in alterations in the differentiation of specific populations of neural cells (70, 71). Whether neural alterations in specific areas of the CNS contribute to the observed early postnatal lethality of these mice remains unknown. Thus, the possibility of specific defects in the CNS of IDX1/IPF1 mutant mice merits further detailed investigation.

The finding of a human subject with a homozygote mutation in IDX1/IPF1 and pancreatic agenesis led to the proposal that other genes, such as BETA2/NeuroD or Nkx2.2, may also be candidates for human neonatal diabetes (69). It is noteworthy that although this is a rare condition, a recent survey of reported cases revealed that a significant proportion of patients (at least 20%) exhibit signs of neural defects, including impaired mental and motor development (72). This observation, together with the findings reported in this study, supports the notion that some elements of the transcriptional control of neuronal and pancreatic differentiation are conserved (67), despite the different ontogenic origins of these tissues.

    ACKNOWLEDGEMENTS

We thank Joel Habener for anti-IDX1/IPF1 antisera and for helpful discussions, Ricardo Martinez for the somatostatin antiserum, and Mehboob Hussain for critical reading of the manuscript.

    FOOTNOTES

* Supported in part by United States Public Health Service Grant DK-49670, by a grant from the Whitehall Foundation (to M. V.), by an Acción Especial from the Consejo Superior de Investigaciones Científicas, and by Fondo de Investigaciones Sanitarias Grant 98/1368 (to R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Klinik für Frauenheilkunde und Geburtshilfe, University of Lübeck Medical School, 23538 Lübeck, Germany. Supported by a fellowship from Deutsche Forschungsgemeinschaft.

Present address: Laboratory of Developmental Biology, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129. Partially supported by a fellowship from the Consejo Superior de Investigaciones Científicas, Spain.

Dagger Dagger To whom correspondence should be addressed. Tel.: 91-585-4890; Fax: 91-585-4587; E-mail: mvallejo@iib.uam.es.

Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M000655200

    ABBREVIATIONS

The abbreviations used are: CNS, central nervous system; CAT, chloramphenicol acetyltransferase; E16, embryonic day 16; PCR, polymerase chain reaction; RT, reverse transcription.

    REFERENCES
TOP
ABSTRACT
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RESULTS
DISCUSSION
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