Differential Activation of Pituitary Hormone Genes by Human Lhx3 Isoforms with Distinct DNA Binding Properties

Kyle W. Sloop, Bradley C. Meier, JeAnne L. Bridwell, Gretchen E. Parker, Amy McCutchan Schiller and Simon J. Rhodes

Department of Biology Indiana University-Purdue University Indianapolis Indianapolis, Indiana 46202-5132


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lhx3 is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. We identified two isoforms of human Lhx3, hLhx3a and hLhx3b, which differ in their ability to trans-activate pituitary gene targets. These factors are identical within the LIM domains and the homeodomain, but differ in their amino-terminal sequences preceding the LIM motifs. Both isoforms are localized to the nucleus and are expressed in the adult human pituitary, but gene activation studies demonstrate characteristic functional differences. Human Lhx3a trans-activated the {alpha}-glycoprotein subunit promoter and a reporter construct containing a high-affinity Lhx3 binding site more effectively than the hLhx3b isoform. In addition, hLhx3a synergized with the pituitary POU domain factor, Pit-1, to strongly induce transcription of the TSHß-subunit gene, while hLhx3b did not. We demonstrate that the differences in gene activation properties between hLhx3a and hLhx3b correlate with their DNA binding to sites within these genes. The short hLhx3b-specific amino-terminal domain inhibits DNA binding and gene activation functions of the molecule. These data suggest that isoforms of Lhx3 may play distinct roles during development of the mammalian pituitary gland and other neuroendocrine systems.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular mechanisms that regulate the development of complex endocrine organ systems are poorly understood. The pituitary gland represents an important model system to study how regulatory genes direct the determination and differentiation of the individual cell lineages that ultimately comprise a mature organ. During pituitary development, neural ectoderm cells from the diencephalon, destined to form the posterior lobe of the gland, associate with a fold of oral ectoderm known as Rathke’s pouch to form the primordial pituitary. Six distinct cell types emerge from Rathke’s pouch during organogenesis to populate the mature anterior and intermediate lobes of the pituitary (see Refs. 1, 2, 3 for reviews). These cells release characteristic protein hormones that regulate growth, lactation, metabolism, reproduction, and the response to stress.

Proper formation of Rathke’s pouch and the subsequent differentiation of pituitary cell types require the secretion of inductive signaling molecules such as BMP-2, BMP-4, and FGF-8. These molecules are secreted from the ventral diencephalon and the developing pituitary to initiate cellular differentiation cascades within embryonic pituitary cells in a spatial and temporal fashion (4, 5, 6). The protein signals induce subsequent pituitary cellular specification by activating multiple transcription factors. Based on their expression patterns, gene knockout experiments, and the analysis of naturally occurring mutants, many transcription factors have been implicated in pituitary organogenesis. They include Lhx3 (also known as P-Lim or LIM3), Lhx4/Gsh-4, Isl-1, Krox-24, T/ebp/TTF-1, Hesx1/Rpx, Pitx-1/P-Otx, Pitx2, Prop-1, Pit-1/GHF-1, Brn-4, Six-3, SF-1, Pax-6, Msx-1, Nkx 3.1, GATA-2, P-Frk, thyrotrope embryonic factor (TEF), and Zn-16 (Ref. 5 ; reviewed in Refs. 1, 2, 3).

Mutations in pituitary transcription factors have been shown to cause pituitary disease. Humans with mutations in the Pit-1 or Prop-1 genes exhibit combined pituitary hormone deficiency (CPHD). Pit-1 is a well characterized POU domain pituitary transcription factor that directly activates the GH, PRL, and TSHß genes, as well as its own gene (reviewed by Refs. 1, 2, 3, 7). Patients with mutated Pit-1 genes have deficiencies in GH, PRL, and TSH. Many types of Pit-1 gene mutations have been identified, including those with autosomal dominant and recessive patterns of inheritance (see Refs. 7, 8 for review). The Prop-1 (or Prophet of Pit-1) transcription factor is upstream of Pit-1 in the pituitary developmental cascade (9). In addition to deficiencies of GH, PRL, and TSH, patients with mutations in the Prop-1 gene may lack LH and FSH (8). Much effort continues to be devoted to the goal of characterizing additional genetic lesions associated with human pituitary disease.

During pituitary development, the actions of the LIM homeodomain transcription factors Lhx3, Lhx4, and Isl-1 are essential for the establishment of Rathke’s pouch and the subsequent differentiation of specialized hormone-secreting cell types. LIM homeodomain proteins contain two amino-terminal LIM motifs and interact with DNA using a characteristic homeodomain. The LIM domain is a conserved, zinc finger-like structure that mediates interactions with other proteins, and LIM homeodomain proteins have been demonstrated to be critical to many developmental pathways (reviewed in Refs. 10, 11, 12). During early embryogenesis, the Lhx3 gene is expressed in several regions of the brain and spinal cord, and then it becomes restricted to the primordial pituitary cells of Rathke’s pouch and their descendents in the adult gland (13, 14, 15, 16, 17). In the nervous system, the Lhx3, Lhx4, and Isl-1 gene products are required for the specification of motor neurons that emerge from the developing neural tube (e.g. Refs. 18, 19, 20, 21). Elegant studies of mice with ablated Lhx3 genes have revealed that pituitary development is arrested after the formation of Rathke’s pouch and that Lhx3 is required for differentiation of the hormone-secreting cells of the pituitary (22, 23). Lhx4 also is required for complete development of Rathke’s pouch; but unlike Lhx3, this factor is not essential for the determination and specification of differentiated pituitary cell types (23). Mice lacking both Lhx3 and Lhx4 genes do not develop a rudimentary Rathke’s pouch, indicating that at least one of these genes is required during the initial stages of pituitary development (23). Our laboratory and others have demonstrated that Lhx3 can activate pituitary trophic hormone genes, acting both alone and with other pituitary transcription factors such as Pit-1 and Pitx1/P-Otx (15, 24, 25). Additional functional analyses are required to fully understand the role of products of the Lhx3 gene in the developing nervous system, the early establishment of Rathke’s pouch, and in later pituitary gland function and maintenance.

To further characterize the molecular actions of Lhx3, we identified two alternate forms of human Lhx3, hLhx3a and hLhx3b, which share the LIM domains, homeodomain, and carboxyl terminus of Lhx3, but possess distinct amino-terminal protein sequences. Both isoforms are nuclear proteins and are detected in the pituitary gland, but trans-activation assays revealed different abilities to activate anterior pituitary hormone gene regulatory regions. Human Lhx3a activated a reporter gene containing the {alpha}-glycoprotein subunit ({alpha}GSU) promoter and a minimal reporter gene containing consensus Lhx3 binding sites. Further, Lhx3a synergized with Pit-1 in induction of the TSH ß-subunit (TSHß) gene promoter. By contrast, Lhx3b was either inactive or only weakly activated trophic hormone genes. We demonstrate that the differences in trans-activation ability between hLhx3a and Lhx3b correlate with their DNA binding to sites within these target genes. Our results indicate that the amino terminus of hLhx3b inhibits the ability of this factor to bind DNA and trans-activate target genes compared with Lhx3a. To our knowledge, this is the first description of different functional properties for alternate forms of a LIM homeodomain class transcriptional regulator. The hLhx3a-specific and hLhx3b-specific amino-terminal domains may represent novel functional motifs derived throughout evolution to confer properties unique to Lhx3 that are important in mammalian development. These findings suggest that Lhx3 isoforms may perform distinct roles in the development of the pituitary gland and during motor neuron differentiation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of Human Lhx3 cDNAs
The PCR was used to amplify a region encoding the carboxyl terminus of human Lhx3 (hLhx3) from pituitary cDNA (see Materials and Methods). Rapid amplification of cDNA ends (5'-RACE) then was performed to obtain longer cDNAs. Finally, partial hLhx3 cDNAs were used as probes in screens of a pituitary cDNA library to obtain full-length clones. Sequencing of hLhx3 clones obtained from RACE and library screening revealed multiple, distinct hLhx3 cDNAs. Two predominant species encoded hLhx3 proteins with identical LIM domains, homeodomains, and carboxyl termini, but with distinct amino-terminal protein sequences (Fig. 1AGo). We named these protein isoforms hLhx3a and hLhx3b. The GenBank accession numbers of the reported sequences are: hLhx3a (AF156888), hLhx3b (AF156889). Some hLhx3b cDNAs contained an approximately 200-bp insert in the 3'-untranslated region (nucleotide sequences deposited in GenBank).



View larger version (54K):
[in this window]
[in a new window]
 
Figure 1. Deduced Protein Sequences of Human Lhx3 Isoforms

A, Schematic depiction of the domain structures of hLhx3a and hLhx3b. Hatched regions represent domains unique to each form; L, LIM domain; HD, homeodomain; LSD, Lhx3/LIM3-specific domain. B, Alignment of mammalian Lhx3 proteins. Comparison of human Lhx3a/b (Ha, Hb), murine Lhx3 (Ma, accession number L38249; Mb, L38248), and porcine Lhx3 (P, AF063245). The LIM domains, homeodomain, and Lhx3/LIM3-specific domain are boxed/reversed. Dots indicate identity; dashes denote gaps introduced to optimize alignment. C, Similarity of Lhx3/LIM3 family proteins. Numbers are percentage identity to hLhx3 for individual regions. C, Chicken (accession number 1708828); X, Xenopus laevis (547856); z, zebrafish (2497671); mLhx4, mouse Lhx4 (AF135415); d, Drosophila melanogaster (AF109306).

 
The unique amino-terminal region of hLhx3b is very similar to an alternate form of murine Lhx3 (16) (94% identity, Fig. 1BGo). By contrast, the shorter amino-terminal sequence of hLhx3a is only 58% similar to the amino terminus of murine Lhx3a (Fig. 1BGo). The a- and b-specific domains of hLhx3 do not significantly match other sequences in the databases. The common regions of the hLhx3 proteins display strong overall similarity to murine (94% identity) and porcine (95% identity) Lhx3 sequences (Fig. 1BGo). The LIM and DNA binding domains of Lhx3 are strongly conserved in the mammalian species, with complete identity in the LIM 2 domain and the homeodomain (Fig. 1BGo). Comparison of mammalian Lhx3 sequences with Lhx3/LIM3 family proteins from nonmammalian species also reveals conservation of the LIM and DNA-binding domains and the carboxyl-terminal LIM3-specific domain (LSD) noted by Glasgow et al. (17) and Thor et al. (18) (Fig. 1CGo). The amino termini of nonmammalian Lhx3/LIM3 proteins display more similarity to the amino terminus of hLhx3a than to that of hLhx3b, but overall are diverged in length, composition, and sequence.

Analysis of the Human Lhx3 Gene and Its Products
To determine whether the identified hLhx3 cDNAs represented a single genomic locus, Southern analyses were performed. Human Lhx3 cDNA probes hybridized to single DNA fragments (Fig. 2AGo; and data not shown), indicating that hLhx3 is encoded by a single gene. In Northern analyses, hLhx3 cDNA probes hybridized to a rare mRNA of approximately 2.4 kb in samples of adult human pituitary poly A+ RNA (Fig. 2BGo). Signals were not detected in samples of total pituitary RNA (Fig. 2BGo). As controls, RNA blots also were hybridized to a human Pit-1 cDNA probe (Fig. 2BGo). In vitro transcription/translation was performed using rabbit reticulocyte lysates to generate radiolabeled hLhx3a and hLhx3b proteins from the identified cDNAs. Analysis of these proteins by electrophoresis revealed apparent molecular masses of approximately 60 kDa for both isoforms (Fig. 2CGo). As we have previously described for porcine Lhx3 (25), this apparent molecular mass is slightly larger than that predicted from the hLhx3a/b open reading frames, suggesting modification of the Lhx3 protein in these preparations or aberrant migration during electrophoresis due to composition.



View larger version (62K):
[in this window]
[in a new window]
 
Figure 2. Analysis of the Human Lhx3 Gene and Its Products

A, Southern analysis demonstrates that hLhx3 is encoded by a single gene. Human genomic DNA was digested with EcoRI and blotted to a nylon membrane. The blot was probed with a hLhx3 cDNA probe at high stringency. The migration position of mol wt standards (kb) is given. B, Northern analysis of hLhx3 gene expression. Human total (20 µg) or poly A+ RNA (1 µg) was separated on a denaturing gel, transferred to a nylon membrane, and probed with the indicated radiolabeled cDNA probes. The migration positions of ribosomal RNAs are shown. Arrowhead indicates hLhx3 RNA. C, Human Lhx3a and hLhx3b proteins. Radiolabeled hLhx3a and hLhx3b isoforms and myc epitope-tagged derivatives (a-myc, b-myc) were generated by in vitro transcription/translation. Proteins were separated by SDS electrophoresis, and dried gels were visualized by fluorography. The migration positions of protein standards (in kilodaltons) are shown.

 
To determine the intracellular localization of the hLhx3 isoforms, vectors for expression of hLhx3 isoforms as fusions with a multimerized green fluorescent protein (4xGFP) (26) were constructed. The 4xGFP protein localized exclusively to the cytoplasm of transfected cells (Fig. 3Go, A and B). By contrast, constructs containing hLhx3a or hLhx3b were detected in the nucleus (Fig. 3Go, C–F), indicating that they are nuclear proteins.



View larger version (114K):
[in this window]
[in a new window]
 
Figure 3. Nuclear Localization of hLhx3 Proteins

Human 293T cells were transiently transfected with expression vectors for either multimerized GFP as a control (4xGFP, panels A and B), hLhx3a-4xGFP (panel C and D), or hLhx3b-4xGFP (panel E and F). Cells were observed under phase contrast (panels A, C, and E), or fluorescence was visualized by krypton-argon laser scanning confocal microscopy (panels B, D, and F). 4xGFP is restricted to the cytoplasm, whereas hLhx3a-4xGFP and hLhx3b-4xGFP are detected in the nuclei of transfected cells. Bar = 10 µm.

 
Expression of Lhx3 RNAs in the Pituitary
To precisely measure expression levels of hLhx3 gene transcripts in human pituitary samples, a "real time" quantitative PCR assay was developed (see Materials and Methods). RNA was isolated from human pituitary tissue, and cDNA was transcribed with a hLhx3-specific primer. Quantitative PCR amplification reactions then were performed using three hLhx3-specific oligonucleotides: two to prime amplification of a region within the 3'-end of the mRNA and a fluorescent internal oligonucleotide that hybridized to PCR products to allow monitoring of the reaction. Serial dilutions of hLhx3 cDNA were amplified simultaneously with patient samples to generate a standard curve (Fig. 4AGo, inset). The standard curve was linear in a range from 100 to 10 million copies of hLhx3. Seven normal adult pituitary samples were assayed: hLhx3 was present at levels ranging from 2,000 to 11,000 copies/150 ng RNA with a mean value of 7,200 copies/150 ng RNA (Fig. 4AGo). This value is consistent with hLhx3 being a rare message. In previous studies, we have demonstrated that ubiquitously expressed genes such as ß-actin are expressed at approximately 500,000 copies/150 ng RNA (27). In specialized tissues such as adipose, we have shown that mRNAs for hormones such as leptin are found at approximately 300,000 copies/150 ng RNA, and transcription factors such as CCAAT/enhancer binding protein-{alpha} (C/EBP{alpha}) are found at about 150,000 copies/150 ng RNA (27).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 4. Assay of Lhx3 Expression in Human Pituitary Glands and Mouse Pituitary Cell Lines

A, A quantitative fluorescent assay for human Lhx3 gene expression was developed. RNA was extracted from adult human pituitaries and cDNA generated using a 3' gene-specific primer. PCR was performed using gene-specific primers and reactions monitored in real time using an internal fluorescent hLhx3-specific probe. Results are the means of four independent experiments ± SEM. Inset shows standard curve using hLhx3 cDNA as input. Ct, PCR cycle number where the reporter dye fluorescent emission increases above baseline emission (49 ). B, Pituitary expression of transcripts of the hLhx3 gene encoding hLhx3a and hLhx3b isoforms. RT-PCR was used to amplify the approximately 1.2-kb coding regions of hLhx3a and hLhx3b using pituitary gland cDNA from two representative patients. Reaction products were separated by agarose gel electrophoresis. M, Mol wt markers; Control, negative control. C, Expression of Lhx3 isoforms in pituitary cell lines. RT-PCR was used to amplify specific regions of Lhx3a (139-bp product, a) and Lhx3b (165-bp product, b) using cDNA from the indicated cell lines. Reaction products were separated by acrylamide gel electrophoresis. Negative control reactions (-) were performed in parallel in the absence of reverse transcriptase.

 
To examine whether hLhx3 RNAs encoding the hLhx3a and hLhx3b isoforms are expressed in human pituitary samples, oligonucleotide primers were designed to specifically amplify either the hLhx3a or hLhx3b protein coding regions. RT-PCR experiments indicated that both forms are found in the adult pituitary (Fig. 4BGo). To investigate whether the Lhx3 isoforms were expressed in specific cell types, we determined expression of Lhx3 isoforms in established mouse pituitary cell lines. In the {alpha}-TSH thyrotrope tumor cell line (28), both mLhx3a and mLhx3b isoforms were readily detected (Fig. 4CGo). In AtT20 corticotrope cells, low levels of mLhx3b were detected, and in some preparations, trace levels of mLhx3a were also observed. Neither isoform was detected in the GHFT-1 cell type: this Pit-1-expressing cell line represents an intermediate stage of pituitary development before differentiation and expression of trophic hormone genes (29). These data are consistent with observations of the overall levels of Lhx3 gene expression by RNA analyses of these cell types (Refs. 14, 15, 16 ; K. W. Sloop and S. J. Rhodes, unpublished data).

Differential Activation of Pituitary Hormone Genes by Human Lhx3 Isoforms
We and others have previously demonstrated that Lhx3 can activate anterior pituitary trophic hormone gene promoters (15, 24, 25). For example, we have demonstrated that Lhx3 can induce transcription from the {alpha}GSU gene by specifically binding to a site located at -350 to -323 bp (known as the pituitary glycoprotein basal element, PGBE) within the proximal region of the promoter (15, 25). This gene encodes the common subunit of the LH, FSH, and TSH anterior pituitary hormones. Other laboratories have shown that the Lhx2/LH-2 LIM homeodomain transcription factor also can recognize this element (30), and that this element is required to correctly restrict expression of the {alpha}GSU gene to pituitary gonadotropes and thyrotropes in transgenic mice (31). To test the ability of the hLhx3 isoforms to activate the {alpha}GSU gene, we transiently cotransfected human embryonic 293 cells with {alpha}GSU luciferase reporter genes and expression vectors containing full-length hLhx3a and hLhx3b cDNAs. 293 cells were used because they are of human origin, efficiently transfected, and do not express Lhx3. In these assays, hLhx3a activated the {alpha}GSU promoter (Fig. 5AGo). Surprisingly, hLhx3b did not induce transcription from this promoter (Fig. 5AGo). Similar experiments using expression vectors encoding hLhx3a or hLhx3b with carboxyl myc epitope tags gave the same results: hLhx3a-myc activated the {alpha}GSU promoter and hLhx3b-myc was inactive (Fig. 5AGo). In these experiments, the epitope-tagged constructs were generally more active: this is likely due to the fact that, in these constructs, the 3'-untranslated region of the cDNA is absent. The murine Lhx3 3'-untranslated region contains ATTTA sequence motifs that may confer instability to the RNA (32). These motifs are conserved in the human Lhx3 sequences (data not shown), and the levels of both isoforms are therefore likely to be somewhat lower in the full-length cDNA experiments. An alternate explanation is that the single myc epitope confers an activation function to the Lhx3 molecules. This has been noted in experiments where multimers of five myc epitope tags are used (33). It is important to note, however, that the relative activities observed for the two Lhx3 isoforms are consistent for all types of expression constructs. To confirm expression of the hLhx3 proteins in the transfected cells, Western analysis was performed using a specific anti-myc antibody. Both isoforms were detected at similar levels as protein species of approximately 60 kDa (Fig. 5BGo). This apparent molecular mass is similar to that of the in vitro translated hLhx3 proteins described above. Western blots were quantified to determine relative expression levels of the hLhx3 isoforms. The observed expression levels of the two isoforms were similar; on average, the hLhx3b isoform was expressed at slightly higher levels (1.2-fold the level observed for hLhx3a).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 5. Differential Activation of the {alpha}-Glycoprotein Subunit ({alpha}GSU) Gene Promoter by hLhx3 Isoforms

A, Human 293 cells were transiently transfected with a mouse {alpha}GSU luciferase reporter gene and the indicated expression vectors. hLhx3-myc, Lhx3 fused to a myc-epitope. Promoter activity was assayed by measurement of luciferase activity after 48 h. Activities are mean [light units/10 sec/µg total protein] of triplicate assays ± SEM. A representative experiment of at least seven experiments is depicted. B, Western analysis using an anti-myc monoclonal antibody of cells transfected with control and myc epitope-tagged hLhx3 expression vectors confirmed expression of hLhx3a and hLhx3b proteins. The migration positions of protein standards (in kilodaltons) are shown.

 
In addition to its ability to trans-activate the {alpha}GSU promoter, we and others have demonstrated that Lhx3 can induce pituitary trophic hormone gene promoters in synergy with pituitary transcription factors such as Pit-1 and Pitx1/P-Otx (15, 24, 25). To examine whether the hLhx3 isoforms also had distinct abilities to cooperate with other factors in transcriptional induction, we transfected a TSHß reporter gene with hLhx3a and hLhx3b expression vectors. Pit-1 and hLhx3a moderately activated the TSHß promoter and together strongly induced transcription (Fig. 6AGo). Human Lhx3b only weakly activated the TSHß promoter and did not effectively synergize with Pit-1 (Fig. 6AGo). As controls, similar experiments were performed with TEF. TEF is a PAR-bZIP transcription factor that strongly induces the TSHß promoter (34). In our experiments, TEF activated expression from the TSHß promoter, and expression of hLhx3a or hLhx3b did not affect this activity (Fig. 6BGo), demonstrating that the observed synergy between hLhx3a and Pit-1 was specific.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 6. Induction of the TSHß Promoter by hLhx3 Isoforms and Other Pituitary Transcription Factors

Human 293 cells were transiently transfected with a mouse TSHß promoter reporter gene plasmid and hLhx3a, hLhx3b, Pit-1, and/or TEF expression vectors. Luciferase activity was assayed after 48 h. Values are mean [light units/10 sec/µg total protein] of triplicate assays ± SEM. A representative experiment of at least four experiments is depicted.

 
The {alpha}GSU and TSHß promoters are complex regulatory structures containing recognition sites for multiple transcription factors. For example, the {alpha}GSU PGBE Lhx3 binding site appears to cooperate with other elements in gene regulation (31, 35). We therefore extended our studies by comparing the abilities of hLhx3a and hLhx3b to activate a defined synthetic luciferase reporter gene containing three copies of a consensus Lhx3 DNA binding element cloned upstream of a minimal promoter. Again, expression vectors for hLhx3a and hLhx3a-myc activated transcription from this reporter more effectively than the corresponding hLhx3b vectors (Fig. 7Go). These data further support the hypothesis that hLhx3a is a more potent activator of transcription than hLhx3b for target genes containing this type of recognition element.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 7. Transactivation of a Luciferase Reporter Gene Containing Lhx3 Binding Sites by hLhx3 Isoforms.

Human 293 cells were transiently transfected with a Lhx3 reporter gene containing three Lhx3 consensus sites and hLhx3a or hLhx3b expression vectors. Reporter gene activity was measured 48 h after transfection. Activities are mean [light units/10 sec/µg total protein] of triplicate assays ± SEM. A representative experiment of at least five experiments is depicted.

 
DNA Binding Properties of hLhx3 Isoforms
The different abilities of hLhx3 isoforms to activate transcription from the tested reporter genes indicated that the hLhx3a- and hLhx3b-specific domains conferred distinct transcriptional properties upon the hLhx3 protein. Our data show that the two isoforms were both expressed in the nucleus at similar levels (Figs. 3Go and 5Go). Mechanisms for the different activities include the possibilities that the a-specific and b-specific domains are trans-activation or trans-repression domains, respectively; or that the a- and b-specific domains may mediate interaction with distinct regulatory factors; or that the a- and b-specific domains may differently modulate the DNA binding properties of hLhx3. We have previously demonstrated that the LIM domains of Lhx3 repress DNA binding by the homeodomain (15, 25). To test the hypothesis that the a- and b-specific domains conferred distinct DNA binding properties upon the hLhx3 molecule, we performed electrophoretic mobility shift assays (EMSAs) to test binding to the {alpha}GSU -350/-323 bp Lhx3 site and to the Lhx3 consensus binding site. Experiments using in vitro translated native hLhx3 proteins revealed that hLhx3a bound to these sites more effectively than hLhx3b (Fig. 8BGo). Similar results were obtained with hLhx3 proteins containing carboxyl-terminal myc epitope tags (Fig. 8BGo).



View larger version (78K):
[in this window]
[in a new window]
 
Figure 8. Expression of Recombinant hLhx3 Proteins and Analysis of Binding to DNA Target Sequences

A, Coomassie brilliant blue stain of a SDS-polyacrylamide gel analysis of GST fusion proteins containing either hLhx3a or hLhx3b. The migration of mol wt standards is indicated. B, Electrophoretic mobility shift assay using Lhx3 consensus binding site (lanes 1–12) or {alpha}GSU gene -350/-323 binding site (lanes 13 and 14) oligonucleotide probes. Radiolabeled probes were incubated with the indicated proteins and competitor DNAs and the resulting complexes were separated from free probe (F) by electrophoresis. Lane 1, Unprogrammed rabbit reticulocyte lysate as a negative control (lysate); lanes 2–5, reactions contained in vitro translated hLhx3 proteins. A nonspecific band is noted by an asterisk. Human Lhx3 protein/DNA complexes are indicated by an arrow. Lane 6, GST as a negative control; lanes 7–14: recombinant hLhx3 proteins expressed in E. coli. Comp, Reactions containing approximately 1000-fold molar excess of unlabeled binding site as a competitor.

 
To confirm this result, we expressed recombinant hLhx3 proteins in E. coli as fusions to the carboxyl terminus of glutathione-S-transferase (GST). This approach allowed the use of affinity-purified proteins of known concentration (Fig. 8AGo). EMSA reactions using the recombinant purified proteins again indicated that hLhx3a bound more effectively than hLhx3b to both DNA sites (Fig. 8BGo). Addition of excess unlabeled binding site DNA to hLhx3a EMSA reactions reduced the amount of bound complex, whereas it abolished the binding of hLhx3b (Fig. 7BGo; and data not shown). These experiments demonstrate that the b-specific domain inhibits the binding of hLhx3b to this class of DNA site. Further, the b-specific domain can perform this function when positioned at the amino terminus of the native protein or when placed in an internal context such as in the GST fusion proteins.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Through evolution, Lhx3-type proteins appear to have been largely conserved in structure, expression pattern, and function. The Lhx3/LIM3 gene is transiently expressed in the developing nervous systems of Drosophila, Xenopus, zebrafish, chickens, and mammals (13, 14, 15, 16, 17, 18, 19), and it has been demonstrated to play important roles in the specification of motor neuron subtypes in some of these species (18, 19, 20, 21). In addition, Lhx3 is critical for both early development and terminal differentiation of the mammalian pituitary (22, 23). Expression also is detected in other developing endocrine structures including the pineal gland (13, 14, 15, 16, 17) and the Drosophila ring gland (18), the major site of production of developmental hormones in higher dipterans. As summarized in Fig. 1Go, the LIM domains, the homeodomain, and the Lhx3/LIM3-specific domain are conserved in Lhx3-type proteins from Drosophila to humans.

In this study, we have demonstrated that proteins with distinct functional properties are generated from the human Lhx3 gene. During evolution, the increased complexity of organisms has required an extended number of regulatory factors with sufficient capacity to control the development of organ systems comprised of multiple, differentiated cell types. This increased molecular diversity has been accomplished by mechanisms such as the formation of additional genes by gene duplication; the generation of multiple proteins from single genes by the use of distinct promoters; and posttranscriptional processes. Posttranscriptional mechanisms include the use of alternate translation initiation codons, alternative RNA splicing, the production of protein isoforms from single RNAs by RNA editing, and proteolytic processing. For Lhx3-type proteins, it appears that both gene expansion (to produce genes encoding related proteins such as Lhx4) and RNA-mediated mechanisms generating isoforms such as hLhx3a and hLhx3b have been used to provide a battery of proteins with important roles in the development of both neural and endocrine structures, including the anterior pituitary.

Alternate isoforms of LIM homeodomain proteins, nonhomeodomain LIM proteins, and several other types of transcription factors are known. For example, the LIM homeodomain transcription factors, Lhx6.1a and Lhx6.1b, appear to be generated by an alternative splicing event that generates proteins with different carboxyl terminal amino acids (36). These factors are expressed at different times in the developing mouse brain, but unique functional properties of the isoforms have not been demonstrated (36). Alternate forms of LMO7 and LIM-kinase proteins lacking either the LIM domain or the kinase domain, respectively, have also been described (37, 38). The isoforms of these proteins have different expression patterns and distinct functional properties. Interestingly, Skn-1a and Skn-1i are alternatively spliced POU domain factors expressed in the epidermis that, like Lhx3, have different amino-terminal domains (39). Similar to the hLhx3b-specific domain, the i-specific domain of Skn-1 inhibits DNA binding and gene trans-activation by this factor. However, primary amino acid sequence comparison between these inhibitory domains reveals no similarity. Alternate isoforms with distinct functions and/or expression patterns are also seen in the nuclear receptor protein superfamily. Examples include the distinct expression patterns of the isoforms of the peroxisome proliferator-activated receptor {gamma} developmental regulatory protein (40, 41) and the unique transcriptional properties of the ß2 thyroid receptor isoform (42).

In the mouse, isoforms of Lhx3 also have been described (16, 32). We suggest that the human Lhx3b clone described in this study is equivalent to murine Lhx3b, because the two amino-terminal domains are nearly identical (Fig. 1Go). The human and murine Lhx3a amino-terminal domains are much less alike, but some sequence features are similar (Fig. 1Go). We hypothesize that the Lhx3a forms are functionally related, but this function does not require conservation of the Lhx3a-specific domain. To date, other LIM homeodomain proteins with alternate functional domains have not been described. The hLhx3a- and hLhx3b-specific amino-terminal motifs may be novel functional domains derived throughout evolution to confer properties unique to Lhx3a and Lhx3b that allow specific roles in the development of the pituitary gland and neural structures.

We have demonstrated that hLhx3a is able to activate specific target genes, whereas hLhx3b is inactive and that these functional differences correlate with a reduced binding of hLhx3b to DNA elements within these genes ( Figs. 4–8GoGoGoGoGo). These data, and the observation that the Lhx3a-specific domain is poorly conserved in sequence and length (Fig. 1Go), together suggest that the Lhx3b-specific domain confers intramolecular inhibition of DNA binding and trans-activation functions. We have previously demonstrated that the LIM domains of mammalian Lhx3 proteins inhibit DNA binding (15, 25), and others have made similar observations for several LIM homeodomain proteins (reviewed in Ref. 12). Therefore, the Lhx3b-specific domain may inhibit DNA binding by configuring the LIM domains such that they exert a greater inhibitory function upon the homeodomain. Alternatively, the Lhx3b-specific domain may function independently of the LIM domains, or it may exert both LIM-dependent and independent effects.

Our data suggest that the alternate hLhx3 isoforms may have distinct functions within the developing pituitary gland and neural structures such as the embryonic spinal cord. Our results suggest that the described Lhx3 isoforms are critical to thyrotrope differentiation and maintenance because both are expressed in the {alpha}-TSH cell line, and hLhx3a activated the {alpha}GSU and TSHß genes. The low level of Lhx3 expression in AtT20 cells is consistent with observations of Lhx3 knockout mice that have some corticotropes, but lack other pituitary cell types (22). It is tempting to speculate that the more abundant Lhx3b isoform in AtT20 cells plays a unique role in differentiation of this cell type.

The described hLhx3 isoforms may have distinct target genes or, dependent on its expression profile, hLhx3b may play a direct or indirect dominant negative role. Experiments in the mouse have suggested that in this species Lhx3a may be expressed earlier than Lhx3b during development (32). Specification of differentiated pituitary cell phenotypes appears to be controlled by the combinatorial actions of multiple tissue-specific transcription factors and signaling proteins (reviewed in Refs. 1, 2, 3). The differential activities or expression patterns of alternate forms of essential members of this program, such as Lhx3 and Pit-1, may play critical roles in the determination of pituitary cell fates. Indeed, we and others have described isoforms of Pit-1 that have distinct properties and expression patterns (e.g. Refs. 43, 44 ; reviewed in Ref. 7). In addition, the described Lhx3 isoforms may interact differently with broadly expressed regulatory proteins or transcriptional coactivators/corepressors. For example, a conserved family of nuclear LIM domain-interacting proteins, known as NLI/Ldb1/CLIM/Chip, has been characterized (Refs. 45, 46, 47 and references therein). These proteins appear to be regulatory partners for LIM homeodomain factors and can mediate homo- and heterodimerization of these factors. Recently, a RING-H2 zinc finger protein, RLIM, was identified and also described as a regulatory partner for LIM homeodomain factors (48). This coregulatory protein appears to recruit the Sin3a/histone deacetylase corepressor complex to function as a LIM-associated inhibitory factor (48). Coregulators such as NLI proteins and RLIM may differentially modulate the ability of Lhx3 isoforms to regulate target genes.

The detection of hLhx3 in the pituitary gland and the demonstration of its ability to regulate pituitary trophic hormone gene promoters suggest a continued role for Lhx3 in the adult human pituitary gland in maintenance of hormone gene expression. This study also provides tools for future investigations examining the role of hLhx3 in pituitary diseases such as combined pituitary hormone deficiency and pituitary tumor disease. Further studies of hLhx3 and related factors will extend our understanding of the developmental program that establishes the hormone-releasing cells of the human pituitary gland and may facilitate future protocols designed to treat pituitary diseases.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RNA Extraction/cDNA Synthesis
Adult human pituitaries were obtained from the National Hormone and Pituitary Program or were obtained at autopsy at the Indiana University School of Medicine. As could best be assessed, pituitary samples were normal and did not have adenomas. Patients ranged in age from 36 to 65 yr, and weighed from 75 to 136 kg. Tissues were frozen in liquid nitrogen and ground to a powder on dry ice/liquid nitrogen, and total RNA was extracted using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) and bromochloropropane as a chloroform substitute. cDNA was synthesized using Superscript II reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) and oligo d(T), random hexamer, or gene-specific primers as required.

DNA Cloning/DNA Sequencing/Plasmid Construction
Database searches revealed that a human expressed sequence tag (EST) sequence displayed similarity to murine and porcine Lhx3. The bacterial culture containing this plasmid could not be retrieved by any of the distributors of EST clones. PCR was then used to amplify this sequence from adult human pituitary cDNA using the following primers: (5'-cggaattctacaacacctcgcccaagccgg-3', 5'-cggaattcggaacgaggggcccttgac-3'). RACE was then performed using the 5'-RACE 2.0 system (Life Technologies, Inc.) and adult human pituitary cDNA. Finally, partial hLhx3 cDNAs were used as high stringency probes (final wash = 0.5x SSC, 65 C) of a human pituitary cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA). From 1 million plaques, 12 positive bacteriophage were identified, and cDNAs were subcloned into pBluescript KS II (-) (Stratagene, La Jolla, CA) as described (25). Seven clones were completely sequenced on both strands by automated DNA sequencing using a Perkin Elmer Corp. (Norwalk, CT) DNA sequencer (Biochemistry Biotechnology Facility, Indiana University School of Medicine). Sequence analysis was performed using Wisconsin Genetics/GCG and DNASIS (Hitachi, South San Francisco, CA) software.

Expression vectors for hLhx3a and hLhx3b were generated by directly cloning full-length cDNAs into pcDNA3 (Invitrogen). Myc epitope-tagged hLhx3 expression vectors (hLhx3a-myc, hLhx3b-myc) were made by cloning compatible cDNA fragments into pcDNA3.1/Myc-His(-)C (Invitrogen). DNA fragments were generated by PCR using the following primers: 5'-cgggatccgatcgcttcggcagcagctg-3' (5', hLhx3a-myc); 5'-cgggatccttgatatttaccccggaggc-3' (5', hLhx3b-myc); and 5'-gcgaagcttggaactgagcgtggtctacctca-3' (3', hLhx3a/b-myc). An expression vector containing four copies of GFP was constructed by removing the 4xGFP cassette from plasmid p713 (Ref. 26 ; a generous gift of Dr. Ursula Stochaj, McGill University, Montreal, Quebec, Canada) by digestion with KpnI and EcoRI and cloning of this fragment into pcDNA3 to generate pcDNA3–4xGFP. Compatible hLhx3 DNA fragments were generated by PCR using the following primers: 5'-tacaagcttcgcgatgctgctggaaacgg-3' (5', hLhx3a); 5'-tacaagcttaccatggaggcgcgcgggga-3' (5', hLhx3b); and 5'-cccggtaccaactgagcgtggtctacctc-3' (3', hLhx3a/b). These fragments then were digested with HindIII and KpnI and cloned into pcDNA3–4xGFP to generate vectors expressing hLhx3a-4xGFP and hLhx3b-4xGFP.

Southern and Northern Analyses
Human genomic DNA was extracted from peripheral blood using a QIAamp Blood Maxi Kit (Qiagen, Chatsworth, CA). DNA was digested to completion with restriction enzymes, electrophoresed through 0.7% agarose gels, and transferred to nylon membranes (Hybond-N+, Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were hybridized to radiolabeled hLhx3 cDNA probes in ExpressHyb buffer (CLONTECH Laboratories, Inc.) at 1–2 x 106 cpm/ml for 2–4 h. DNA fragments were labeled by random priming using the Klenow fragment enzyme (Life Technologies, Inc.) and 32P-dCTP (Amersham Pharmacia Biotech) to a specific activity of >1 x 109 cpm/µg. After hybridization, membranes were washed in 0.5x SSC at 65 C, followed by exposure to MR film (Eastman Kodak Co., Rochester, NY) with intensifying screens.

Total human pituitary RNA was extracted as described above. Poly A+ RNA was purchased from CLONTECH Laboratories, Inc. RNA was separated on denaturing, formaldehyde agarose gels followed by transfer to Nytran Plus membranes using the Turboblotter system (Schleicher & Schuell, Inc., Keene, NH). Probes were a 0.4 kb hLhx3 cDNA fragment encoding the LIM domains of the protein and a 1.2 kb cDNA encoding human Pit-1. Membranes were hybridized to radiolabeled cDNA probes as described above.

In Vitro Transcription/Translation
Radiolabeled hLhx3 proteins were synthesized in vitro from pcDNA3 expression vector substrates using T7 RNA polymerase, TnT rabbit reticulocyte lysates (Promega Corp., Madison, WI), and 35S-methionine (Amersham Pharmacia Biotech, Arlington Heights, IL). Proteins were analyzed using SDS-PAGE followed by treatment with Amplify fluorography reagent (Amersham Pharmacia Biotech) and exposure to MR film (Eastman Kodak Co.) at -80 C.

Confocal Microscopy
Human 293T cells (1 x 105) were grown in chamber slides (Nunc) and transfected with 4xGFP, hLhx3a-4xGFP, or hLhx3b-4xGFP expression vectors as described below. After 48 h, cells were washed with 1x PBS, and then either directly visualized live or fixed with 2% paraformaldehyde, washed with 70% ethanol/30% PBS, and stored in 1x PBS before visualization. Fluorescence and trans-illumination images were collected using a MRC 1024 laser scanning confocal microscope (Bio-Rad Laboratories, Inc., Richmond, CA) with a 60x water immersion objective (Nikon, Melville, NY) and a krypton-argon laser. Images were captured with Metamorph software (Universal Imaging, West Chester, PA).

Quantitative Assay of Gene Expression
"Real time" quantitative PCR utilizing the ABI PRISM 7700 Sequence Detection System (Perkin Elmer Corp.) was performed as previously described (27, 49). Total RNA was isolated from human pituitary tissue as described above. Reverse transcription of total RNA with a hLhx3-specific reverse primer (5'-ctcccgtagaggccattg-3') was performed using SuperScript II reverse transcriptase (Life Technologies, Inc.). Quantitative PCR amplification reactions were performed in triplicate and included: 2 µl of cDNA synthesis reaction, 1x TaqMan Buffer A, 300 nM dATP, dCTP, dGTP, and 600 nM dUTP, 3.5 mM MgCl2, 1.25 U AmpliTaq Gold DNA polymerase (Perkin Elmer Corp.), 0.5 U of AmpErase uracil N-glycosylase, 300 nM forward primer (5'-ggacaaggacagcgttcag-3') and reverse primer, and 200 nM hLhx3 fluorogenic probe (5'-ttccccgatgagccttccttggcggaa-3'). Reaction parameters were as follows: 50 C, 2 min; 94 C, 10 min; and then 35 cycles of 94 C, 30 sec; 60 C, 1 min. Serial dilutions of hLhx3 cDNA were amplified simultaneously with patient samples to generate a standard curve. Values reported are averages of four independent experiments.

RT-PCR Analysis
cDNA was synthesized from adult pituitary RNA as described above using oligo-d(T) as a primer. PCR then was performed using the following primers: 5'-cgggatccatgctgctggaaacggggct-3' (5', hLhx3a); 5'-cgggatccatggaggcgcgcggggagct-3' (5', hLhx3b); and 5'-cggaattctcagaactgagcgtggtcta-3' (3', hLhx3a/b). Cycling parameters were as follows: 94 C, 30 sec; 60 C, 30 sec; 72 C, 1 min; for 30 cycles. Reaction products were analyzed on 1% agarose Tris-borate gels. For analysis of mouse pituitary cell lines, cDNA was synthesized from total RNA using 5'-tggtcacagcctgcacacat-3'. PCR then was performed using the following primers: 5'-aaccactggattagtgactg-3' (5', mLhx3a); 5'-gaagttcagggtcggaggg-3' (5', mLhx3b); and 5'-tggtcacagcctgcacacat-3' (3', mLhx3a/b). Cycling parameters were as follows: 94 C, 30 sec; 60 C, 30 sec; 72 C, 30 sec; for 30 cycles. Reaction products were analyzed on 11% acrylamide Tris-borate gels.

Cell Culture, Transfection Assays, Statistical Analysis
Human embryonic kidney 293 and 293T cells were cultured in DMEM (Life Technologies, Inc.) with 10% FBS (Irvine Scientific, Santa Ana, CA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Irvine Scientific); 1.5 x 105 cells per 60-mm dish were transfected with calcium phosphate/DNA precipitates using the CalPhos system (CLONTECH Laboratories, Inc.). Reporter plasmid (0.5 µg) and expression vector (0.1–1.0 µg of expression vector) were added per 60-mm dish, and all groups received equal final DNA concentrations. The murine {alpha}GSU promoter luciferase plasmid (30) was a generous gift of Dr. Richard Maurer (Oregon Health Sciences University, Beaverton, OR). The murine TSHß -1.2-kb promoter luciferase plasmid was as described (34). The Lhx3 consensus binding site reporter gene was constructed by cloning three copies of 5'-cagaaaattaattaattgtaa-3' upstream of a minimal PRL (-36 bp) promoter luciferase reporter gene (J. L. Bridwell, J. R. Price, G. E. Parker, K. W. Sloop, A. McCutchan Schiller, and S. J. Rhodes, in preparation). Control cultures received empty expression vector DNA. Luciferase activity was measured 48 h after transfection as described (25). All assays were performed in triplicate. Total cell protein was determined by the Bradford method (Bio-Rad Laboratories, Inc.) and luciferase activity was normalized to protein concentration. Data points were compared using a one-tailed Student’s t test for paired samples using Sigma Plot 2.0 (Jandel Scientific, San Rafael, CA). Values were considered significantly different when P < 0.05.

Western Analysis
Western analysis of 293 or 293T cells transfected with hLhx3a-myc, hLhx3b-myc, or control expression vectors was performed as we described previously (25). The mouse anti-myc monoclonal antibody 9E10, developed by Evan et al. (50), was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). Ascites fluid was used at a 1:5000 dilution. The secondary antibody was a goat antimouse/horseradish peroxidase (Sigma, St. Louis, MO) at 1:15,000. Results were visualized using Supersignal chemiluminescence reagents (Pierce Chemical Co., Rockford, IL) and MR film (Eastman Kodak Co.). Data were quantified using a Bio-Rad Laboratories, Inc. imaging densitometer and Molecular Analyst software (Bio-Rad Laboratories, Inc.).

Recombinant Protein Preparation/EMSA
Bacterial expression vectors for GST-hLhx3a and GST-hLhx3b fusion proteins were generated by cloning BamHI/EcoRI compatible fragments of the hLhx3a and hLhx3b cDNAs into pGEX-KT (51). cDNA fragments were generated by PCR using the following oligonucleotides: 5'-cgggatccatgctgctggaaacggggct-3',5'-cggaattctcagaactgagcgtggtcta-3' (hLhx3a) and 5'-cgggatccatggaggcgcgcggggagct-3',5'-cggaattctcagaactgagcgtggtcta-3' (hLhx3b). Recombinant proteins were expressed in E. coli BL21 (DE3) pLysS and affinity-purified as we have previously described (25). Proteins were analyzed on 12% SDS-PAGE gels. EMSAs were performed as described (25). Oligonucleotides representing the -350 to -323 bp region of the murine {alpha}GSU promoter (5'-acattaggtacttagctaattaaatgtg-3' and 5'-cacatttaattagctaagtacctaatgt-3') were used or the Lhx3 consensus binding site described above. In competition experiments, 1000-fold molar excess of unlabeled binding site DNA was added to EMSA reactions.


    ACKNOWLEDGMENTS
 
We are very grateful to Drs. B. Azarelli, D. Crowell, R. Maurer, L. Slieker, U. Stochaj, and the National Hormone and Pituitary Program for materials and useful advice. We also thank T. Lahr, R. Sandoval, and A. Showalter for assistance.


    FOOTNOTES
 
Address requests for reprints to: Simon J. Rhodes, Ph.D., Department of Biology, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, Indianapolis Indiana 46202-5132.

This work was supported by grants to S.J.R. from the National Science Foundation, the National Research Initiative Competitive Grants Program/US Department of Agriculture, and the Indiana University-Purdue University Indianapolis Office of Faculty Development. We dedicate this paper to the memory of Dr. Raymond Russo.

Received for publication June 28, 1999. Revision received August 24, 1999. Accepted for publication September 8, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Watkins-Chow DE, Camper SA 1998 How many homeobox genes does it take to make a pituitary gland? Trends Genet 14:284–290[CrossRef][Medline]
  2. Kioussi C, Carriere C, Rosenfeld MG 1999 A model for the development of the hypothalamic-pituitary axis: transcribing the hypophysis. Mech Dev 81:23–35[CrossRef][Medline]
  3. Sheng HZ, Westphal H 1999 Early steps in pituitary organogenesis. Trends Genet 15:236–240[CrossRef][Medline]
  4. Ericson J, Norlin S, Jessell TM, Edlund T 1998 Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary. Development 125:1005–1015[Abstract/Free Full Text]
  5. Treier M, Glieberman AS, O’Connell SM, Szeto DS, McMahon JA, McMahon AP, Rosenfeld MG 1998 Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 12:1691–1704[Abstract/Free Full Text]
  6. Takuma N, Sheng HZ, Furuta Y, Ward JM, Sharma K, Hogan BL, Pfaff SL, Westphal H, Kimura S, Mahon KA 1998 Formation of Rathke’s pouch requires dual induction from the diencephalon. Development 125:4835–4840[Abstract/Free Full Text]
  7. Rhodes SJ, Rosenfeld MG 1996 Molecular involvement of the pit-1 gene in pituitary cell commitment. J Anim Sci 74:94–106
  8. Procter AM, Phillips III JA, Cooper DN 1998 The molecular genetics of growth hormone deficiency. Hum Genet 103:255–272[CrossRef][Medline]
  9. Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, O’Connell SM, Gukovsky I, Carrière C, Ryan AK, Miller, AP, Zuo L, Gleiberman AS, Anderson B, Beamer WG, Rosenfeld MG 1996 Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 384:327–333[CrossRef][Medline]
  10. Dawid IB, Breen JJ, Toyama R 1998 LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet 14:295–306[CrossRef][Medline]
  11. Jurata LW, Gill GN 1998 Structure and function of LIM domains. Curr Top Microbiol Immunol 228:75–113[Medline]
  12. Curtiss J, Heilig JS 1998 Delimiting development. Bioessays 20:58–69[CrossRef][Medline]
  13. Taira M, Hayes WP, Otani, Dawid IB 1993 Expression of LIM class homeobox gene Xlim-3 in Xenopus development is limited to neural and neuroendocrine tissues. Dev Biol 159:245–256[CrossRef][Medline]
  14. Seidah NG, Barale JC, Marcinkiewicz M, Mattei MG, Day R, Chrétien M 1994 The mouse homeoprotein mLIM-3 is expressed early in cells derived from the neuroepithelium and persists in adult pituitary. DNA Cell Biol 13:1163–1180[Medline]
  15. Bach I, Rhodes SJ, Pearse IIRV, Heinzel T, Gloss B, Scully KM, Sawchenko PE, Rosenfeld MG 1995 P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 92:2720–2724[Abstract]
  16. Zhadanov AB, Bertuzzi S, Taira M, Dawid IB, Westphal H 1995 Expression pattern of the murine LIM class homeobox gene Lhx3 in subsets of neural and neuroendocrine tissues. Dev Dynam 202:354–364[Medline]
  17. Glasgow E, Karavanov AA, Dawid IB 1997 Neuronal and neuroendocrine expression of lim3, a LIM class homeobox gene, is altered in mutant zebrafish with axial signaling defects. Dev Biol 192:405–419[CrossRef][Medline]
  18. Thor S, Andersson SGE, Tomlinson A, Thomas JB 1999 A LIM-homeodomain combinatorial code for motor neuron pathway selection. Nature 397:76–80[CrossRef][Medline]
  19. Tsuchida T, Ensini M, Morton SB, Baidassare M, Edlund T, Jessell TM, Pfaff SL 1994 Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79:957–970[Medline]
  20. Sharma K, Sheng HZ, Lettieri K, Li H, Karavanov A, Potter S, Westphal H, Pfaff SL 1998 LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons. Cell 95:817–828[Medline]
  21. Osumi N, Hirota A, Ohuchi H, Nakafuku M, Imura T, Kuratani S, Fujiwara M, Noji S, Eto K 1997 Pax-6 is involved in the specification of hindbrain motor neuron subtype. Development 124:2961–2972[Abstract/Free Full Text]
  22. Sheng HZ, Zhadanov AB, Mosinger Jr B, Fujii T, Bertuzzi S, Grinberg A, Lee EJ, Huang EJ, Huang SP, Mahon KA, Westphal H 1996 Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 272:1004–1007[Abstract]
  23. Sheng HZ, Moriyama K, Yamashita T, Li H, Potter SS, Mahon KA, Westphal H 1997 Multistep control of pituitary organogenesis. Science 278:1809–1812[Abstract/Free Full Text]
  24. Bach I, Carrière C, Ostendorff HP, Anderson B, Rosenfeld MG 1997 A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes Dev 11:1370–1380[Abstract]
  25. Meier BC, Price JR, Parker GE, Bridwell JL, Rhodes SJ 1999 Characterization of the porcine Lhx3/LIM-3/P-Lim LIM homeodomain transcription factor. Mol Cell Endocrinol 147:65–74[CrossRef][Medline]
  26. Chatterjee S, Javier M, Stochaj U 1997 In vivo analysis of nuclear protein traffic in mammalian cells. Exp Cell Res 236:346–350[CrossRef][Medline]
  27. Sloop KW, Surface PL, Slieker LJ 1998 Changes in leptin expression are not associated with corresponding changes in CCAAT/enhancer binding protein-{alpha} (C/EBP-{alpha}). Biochem Biophys Res Commun 251:142–147[CrossRef][Medline]
  28. Akerblom IE, Ridgway EC, Mellon PL 1990 An alpha-subunit-secreting cell line derived from a mouse thyrotrope tumor. Mol Endocrinol 4:589–596[Abstract]
  29. Lew D, Brady H, Klausing K, Yaginuma K, Theill L, Stauber C, Karin M, Mellon PL 1993 GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice. Genes Dev 7:683–693[Abstract]
  30. Roberson MS, Schoderbek WE, Tremml G, Maurer RA 1994 Activation of the glycoprotein hormone {alpha}-subunit promoter by a LIM-homeodomain transcription factor. Mol Cell Biol 14:2985–2993[Abstract]
  31. Brinkmeier ML, Gordon DF, Dowding JM, Saunders TL, Kendall SK, Sarapura VD, Wood WM, Ridgway EC, Camper SA 1998 Cell-specific expression of the mouse glycoprotein hormone-{alpha} subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells. Mol Endocrinol 12:622–633[Abstract/Free Full Text]
  32. Zhadanov AB, Copeland NG, Gilbert DJ, Jenkins NA, Westphal H 1995 Genomic structure and chromosomal localization of the mouse LIM/homeobox gene Lhx3. Genomics 27:27–32[CrossRef][Medline]
  33. Ferreiro B, Artinger M, Cho KWY, Niehrs C 1998 Antimorphic goosecoids. Development 125:1347–1359[Abstract/Free Full Text]
  34. Drolet D, Scully K, Simmons DM, Swanson LW, Rosenfeld MG 1991 TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev 5:1739–1753[Abstract]
  35. Heckert LL, Schultz K, Nilson JH 1995 Different composite regulatory elements direct expression of the human {alpha} subunit gene to pituitary and placenta. J Biol Chem 270:26497–26504[Abstract/Free Full Text]
  36. Kimura N, Ueno M, Nakashima K, Taga T 1999 A brain region-specific gene product Lhx6.1 interacts with Ldb1 through tandem LIM-domains. J Biochem 126:180–187[Abstract]
  37. Putilina T, Jaworski C, Gentleman S, McDonald B, Kadiri M, Wong P 1998 Analysis of a human cDNA containing a tissue-specific alternatively spliced LIM domain. Biochem Biophys Res Commun 252:433–439[CrossRef][Medline]
  38. Edwards DC, Gill GN 1999 Structural features of LIM kinase that control effects on the actin cytoskeleton. J Biol Chem 274:11352–11361[Abstract/Free Full Text]
  39. Andersen B, Schonemann MD, Flynn SE, Pearse RVII, Singh H, Rosenfeld MG 1993 Skn-1a and Skn-1i: two functionally distinct Oct-2-related factors expressed in epidermis. Science 260:78–82[Medline]
  40. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8:1224–1234[Abstract]
  41. Mukherjee R, Jow L, Croston GE, Paterniti Jr JR 1997 Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPAR{gamma}2 versus PPAR{gamma}1 and activation with retinoid X receptor agonists and antagonists. J Biol Chem 272:8071–8076[Abstract/Free Full Text]
  42. Langlois MF, Zanger K, Monden T, Safer JD, Hollenberg AN, Wondisford FE 1997 A unique role of the ß-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone. J Biol Chem 272:24927–24933[Abstract/Free Full Text]
  43. Haugen BR, Wood WM, Gordon DF, Ridgway EC 1997 A thyrotrope-specific variant of Pit-1 transactivates the thyrotropin ß promoter. J Biol Chem 268:20818–20824[Abstract/Free Full Text]
  44. Diamond SE, Chiono M, Gutierrez-Hartmann A 1999 Reconstitution of the protein kinase A response of the rat prolactin promoter: differential effects of distinct Pit-1 isoforms and functional interaction with Oct-1. Mol Endocrinol 13:228–238[Abstract/Free Full Text]
  45. Jurata LW, Gill GN 1997 Functional analysis of the nuclear LIM domain interactor NLI. Mol Cell Biol 17:5688–5698[Abstract]
  46. Jurata LW, Pfaff SL, Gill GN 1998 The nuclear LIM domain interactor NLI mediates homo- and heterodimerization of LIM domain transcription factors. J Biol Chem 273:3152–3157[Abstract/Free Full Text]
  47. Breen JJ, Agulnick AD, Westphal H, Dawid IB 1998 Interactions between LIM domains and the LIM domain-binding protein Ldb-1. J Biol Chem 273:4712–4717[Abstract/Free Full Text]
  48. Bach I, Rodriguez-Esteban C, Carrière C, Bhushan A, Krones A, Rose DW, Glass CK, Andersen B, Belmonte JCI, Rosenfeld MG 1999 RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex. Nat Genet 22:394–399[CrossRef][Medline]
  49. Heid CA, Stevens J, Livak KJ, Williams PM 1996 Real time quantitative PCR. Genome Res 6:986–994[Abstract]
  50. Evan GI, Lewis GK, Ramsay G, Bishop JM 1985 Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616[Medline]
  51. Hakes DJ, Dixon JE 1992 New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 202:293–298[Medline]