help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2007-0567
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lavoie, P.-L.
Right arrow Articles by Drouin, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lavoie, P.-L.
Right arrow Articles by Drouin, J.
Molecular Endocrinology 22 (7): 1647-1657
Copyright © 2008 by The Endocrine Society

Developmental Dependence on NurRE and EboxNeuro for Expression of Pituitary Proopiomelanocortin

Pierre-Luc Lavoie, Lionel Budry, Aurélio Balsalobre and Jacques Drouin

Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada H2W 1R7

Address all correspondence and requests for reprints to: Jacques Drouin or Aurélio Balsalobre, Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal 110, Avenue des Pins Ouest, Montréal, Québec, Canada H2W 1R7. E-mail: jacques.drouin{at}ircm.qc.ca; or aurelio.balsalobre{at}ircm.qc.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell-specific expression of the pituitary proopiomelanocortin (POMC) gene depends on the combinatorial action of a large number of DNA-binding transcription factors (TFs). These include general and cell-restricted factors, as well as factors that act as effectors of signaling pathways. We have previously defined in the distal POMC promoter a composite regulatory element that contains targets for basic helix-loop-helix TFs conferring cell specificity and for NGFI-B orphan nuclear receptors that are responsive to CRH signaling and to glucocorticoid negative feedback. These factors act on neighboring regulatory elements, the EboxNeuro and NurRE, respectively. Currently, the EboxNeuro is thought to be the target of NeuroD1 during fetal development, but this factor may not account for activity in the adult pituitary; it is also unknown whether the NurRE and NGFI-B-related factors are active before establishment of the hypothalamic-pituitary portal system. In order to assess the importance of these regulatory elements and their cognate TFs throughout pituitary organogenesis and in the adult, we have assessed the activity of mutant POMC promoters in transgenic mice throughout development. These experiments indicate that the EboxNeuro and cognate basic helix-loop-helix factors are required throughout development and in the adult gland, beyond expression of NeuroD1. Similarly, the data reveal sustained importance of the NurRE and its cognate factors throughout pituitary development. These data contrast the sustained dependence throughout development on the same regulatory elements with the highly dynamic patterns of TF expression and the modulation of their activity in response to signaling pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PITUITARY EXPRESSION OF the proopiomelanocortin (POMC) gene depends on multiple transcription factors (TFs) and their cognate DNA-binding sites within the POMC promoter (1). Analysis of this promoter in tissue culture cells, as well as in vivo using transgenic mice, has provided a complex picture involving over 12 different TFs (2, 3, 4). Ubiquitous/general TFs have been implicated, such as SP1, chicken ovalbumin upstream promoter transcription factor, AP1, and class A basic helix-loop-helix (bHLH) factors (2, 5, 6). However, most interest has been on factors that contribute either tissue or cell specificity of expression. These include the pan-pituitary factor Pitx1 that was originally cloned on the basis of its interaction with a single site within the POMC promoter (7). Pitx1 and the related Pitx2 are both expressed in the developing pituitary (8), and they contribute to transcription of all hormone-coding genes of the pituitary (9). In many pituitary lineages, the action of Pitx1/2 on the hormone-coding genes is mediated through protein interactions with cell-restricted TFs, such as Pit1 in somatolactotroph cells, steroidogenic factor 1 in gonadotrophs, and Tpit in corticotrophs and melanotrophs (9, 10, 11, 12). Cell specificity of POMC transcription is largely achieved through the action of the T-box factor Tpit that is highly restricted in its expression to the two pituitary POMC lineages, the corticotrophs and melanotrophs (11). On the POMC promoter, Tpit binds a DNA sequence that is only 5 bp away from the Pitx1-binding site, and transcriptional activation by these two TFs is highly dependent on each other (11). The role of Tpit in pituitary development extends beyond POMC expression, because Tpit gene inactivation showed that it is required for terminal differentiation and maintenance of both corticotrophs and melanotrophs (13); in humans, TPIT mutations have thus been associated with isolated ACTH deficiency (14, 15, 16, 17). Given its highly restricted expression, Tpit is certainly the major determinant for pituitary-specific expression of POMC, but it is not the sole determinant of cell specificity because we also identified the neurogenic bHLH factor NeuroD1 as an important factor for cell-specific expression of POMC (6). NeuroD1 acts on its target, the EboxNeuro, within the distal domain of the POMC promoter by formation of heterodimers with bHLH factors of the general/ubiquitous class A group such as E47 (Pan1), E12 (Pan2), or ITF2. Within the POMC promoter, the EboxNeuro is highly dependent on interaction with the Tpit/PitxRE, and synergism between these regulatory elements was shown to result from direct protein-protein interactions between Pitx1 and the bHLH domain of the NeuroD1 dimerization partner (18). The importance of NeuroD1 and its cognate Ebox within the POMC promoter was previously assessed; mutagenesis of the EboxNeuro was shown to curtail POMC promoter activity in transfected AtT-20 cells, a corticotroph model cell line (18). Analysis of the NeuroD1 knockout, however, indicated that this specific bHLH appears to be required only transiently in early pituitary development between the onset of POMC expression at embryonic d 12.5 (e12.5) and e15.5/16.5 when NeuroD1 expression decreases (19). These analyses never addressed the long-term importance of the EboxNeuro for POMC expression or the putative role of other bHLH factors acting on this regulatory element.

Within the distal POMC promoter, the EboxNeuro is part of a composite regulatory element that includes the neighboring NurRE, which mediates part of the CRH-dependent signals (20). Indeed, CRH action leads to activation of the protein kinase A and MAPK pathways and ultimately activation of the three NGFI-B (Nur77)-related orphan nuclear receptors, also including Nurr1 and NOR1 (21). These orphan nuclear receptors act on the NurRE palindrome by forming dimers, either homodimers or heterodimers, with each other. In contrast to a consensus NurRE sequence that does not have preference for various dimers of these orphan nuclear receptors, the POMC gene NurRE exhibits a marked preference for dimers that contain at least one moiety of NGFI-B (22). In addition to promoting formation of NGFI-B dimers, CRH signaling also leads to recruitment of the coactivator steroid receptor coactivator 2 (SRC2, TIF2) to the NurRE (21). CRH signaling, and in particular the action of SRC2, is also modulated by Rb and the related protein p107; these tumor suppressors form a complex with direct interactions to both NGFI-B and SRC2 (23, 24). As for the EboxNeuro, the action of the NurRE within the context of the native POMC promoter is also dependent on interactions with the Tpit/PitxRE and its cognate proteins; in this instance, direct interactions between NGFI-B and Tpit were documented (25). Finally, the NurRE is also a target for another important regulatory mechanism of POMC expression, namely feedback repression by glucocorticoids (Gc) and their receptors (GRs). Indeed, GR represses NGFI-B-dependent transcription by a mechanism of mutual antagonism, transrepression, which does not involve direct interaction of GR with DNA (26, 27). The importance of GR transrepression of NGFI-B-dependent activity was recently highlighted by its involvement in the pathogenesis of Gc resistance in corticotroph adenomas that cause Cushing disease (28). The distal region of the POMC promoter, and in particular the EboxNeuro and the NurRE, thus appear to be critical for POMC expression in AtT-20 cells. The basis for maintenance of EboxNeuro activity beyond the period of high NeuroD1 expression in corticotrophs (after e16) is an open question, and the activity of the NurRE before establishment of the hypothalamo-pituitary system (at ~e16) is uncertain. On this latter point, Lugo and Pintar (29, 30) have shown that responsiveness of the POMC gene to both CRH and Gc occurs around e16 in the rat, at the time when the hypothalamo-pituitary-portal system is established. However, pituitary POMC is expressed in anterior lobe corticotrophs as early as e12.5 in the mouse: this suggests that the NurRE may not be as critical in early corticotroph cells, before CRH can reach the developing pituitary.

We have used transgenic mice to assess the relative importance of the NurRE and EboxNeuro for POMC expression at different times during pituitary development and in the adult. Prior work had shown that the upstream 480 bp of the rat POMC promoter was sufficient for pituitary-specific expression of this gene, both in intermediate lobe melanotrophs and in anterior lobe corticotrophs (3, 4). It was further shown that deletion of the distal region of the promoter impairs its activity in transgenic mice such that it is barely detectable (31). Using specific mutations of either regulatory element, we found that both elements individually are more important for POMC expression in adult tissues than in the fetal pituitary. The EboxNeuro is critical at all times, even after down-regulation of NeuroD1, thus suggesting that other bHLH factors may take over when NeuroD1 is down-regulated. The dependence on NurRE for expression at e14.5 suggests that this element contributes to POMC promoter activity before CRH action and that NurRE/NGFI-B-dependent transcription is not conditional on CRH signaling. Collectively, this work demonstrates the dynamic dependence of the POMC promoter on various regulatory elements and their cognate TFs during development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression Profiles for NeuroD1 and Nurr1 during Pituitary Development
Pituitary expression of NeuroD1 was studied previously both by in situ hybridization (6) and by immunohistochemistry (18). These analyses showed preferential expression of NeuroD1 in corticotroph cells of the developing anterior pituitary between e12.5 and e16.5 with much lower levels in adult tissues; adult expression was detected by in situ hybridization (6) but not by immunohistochemistry (18). Since then, we have generated new antibodies against NeuroD1 that have a much higher titer than the one used in these original studies. The new antibodies have confirmed the onset of NeuroD1 expression in early corticotrophs at e12.5 of mouse pituitary development (data not shown), but they have also revealed low-level expression in other cells (mostly {alpha}GSU-positive cells) that had not been detected previously (data not shown). Thus, NeuroD1 is expressed in a significant number of cells at e14.5 in the developing anterior pituitary lobe (Fig. 1EGo), and the number of NeuroD1-positive cells is decreased by e16.5 (Fig. 1FGo) and is much weaker by e18.5 (Fig. 1GGo). Throughout development, the bulk of immunoreactive NeuroD1 protein is localized in the nucleus. In the adult anterior lobe, very few NeuroD1-positive nuclei remain, but some cells exhibit cytoplasmic NeuroD1 immunoreactivity (Fig. 1HGo). Thus, the use of a much better antibody has shown that NeuroD1 expression is not as restricted as originally believed, but the onset and down-regulation of NeuroD1 expression in corticotrophs is similar to what was previously described. In addition, there is no evidence for melanotroph expression of NeuroD1.


Figure 1
View larger version (81K):
[in this window]
[in a new window]

  		Fig. 1. Expression of ACTH, NeuroD1, and Nurr1 during Pituitary Development

A–D, ACTH expression was assessed by immunohistochemistry using a mouse monoclonal antibody against ACTH. Cytoplasmic staining (insets) is observed in some cells at e14.5 (A), e16.5 (B), e18.5 (C), and adult (D). E–H, NeuroD1 expression was assessed by immunohistochemistry using a rabbit antibody against NeuroD1. Staining was observed mostly in the nucleus of cells within the developing anterior pituitary at e14.5 (E), e16.5 (F), and e18.5 (G). The adult anterior lobe exhibits weak immunoreactivity, but this label is predominantly cytoplasmic (H); such labeling is not observed with preimmune serum (data not shown). Note that NeuroD1 immunoreactivity is never detected in intermediate lobe cells. I–L, Nurr1 expression during pituitary development assessed by immunohistochemistry with polyclonal Nurr1 antibody. Nuclear staining is observed in most cells of developing pituitary at e14.5 (I), e16.5 (J), and e18.5 (K), and in the adult gland (L).

 
The other factors acting on the distal element of the POMC promoter are the Nur factors, NGFI-B, Nurr1, and NOR1. We have used an antibody against Nurr1 to characterize expression of this factor during pituitary ontogeny. Throughout development, Nurr1 was found to be present in the nucleus of most cells of the pituitary, including cells of the anterior and intermediate lobes as evidenced at e14.5, e16.5, and e18.5 (Fig. 1Go, I–K) and in the adult gland (Fig. 1LGo). We have been unable to find suitable NGFI-B antibodies for immunohistochemistry, and antibodies that were adequate for detection of NOR1 in other tissues did not reveal a significant signal in the pituitary (data not shown). Failure to detect NOR1 protein is in agreement with prior data on the relative expression of the three Nur factors (22).

Specific Mutants for EboxNeuro and NurRE
To assess the relative roles of the EboxNeuro and NurRE in POMC expression during development, we generated specific mutations of these regulatory elements and assessed the loss of function that results from these mutations by gel retardation and reporter activity in transfected cells. The EboxNeuro mutation changed four of six residues within the motif CANNTG as indicated in Fig. 2AGo. This mutant Ebox no longer bound NeuroD1/E47 heterodimers in gel retardation assays (Fig. 2BGo, lane 6 compared with lane 3). When the same mutation was introduced into a simple reporter plasmid that contains two copies of this EboxNeuro, the NeuroD1-dependent activity of the reporter was completely abolished (Fig. 3Go, panel B compared with panel A).


Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 2. Loss of DNA Binding Ability for Mutant EboxNeuro and NurRE

A, Sequence of the EboxNeuro and of the mutant EboxNeuro used in the present work. B, The EboxNeuro probe binds in vitro-translated NeuroD1/E47 heterodimers (lane 3), whereas only nonspecific binding is observed with control in vitro translation extracts (lane 2) or extracts directed to synthesize NeuroD1 on its own (lane 4). The probe is shown in lane 1. The EboxNeuro mutant probe is used on its own in lane 5 and with control in vitro translation extract in lane 7. This probe does not bind NeuroD1/E47 heterodimers (lane 6). C, Sequence of the POMC NurRE and of its single and double mutants. D, Binding of in vitro-translated NGFI-B to NurRE probe (lane 3) shows formation of homodimer and monomer complexes as indicated. The same probe (lane 1) is not bound by control in vitro translation extracts (lane 2). A NurRE probe mutated as shown in C in one half of the palindrome forms monomeric complexes with NGFI-B (lane 6), but a NurRE probe mutated in both halves no longer forms NGFI-B complexes (lane 9). Mut., Mutant.

 

Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Transcriptional Activity of EboxNeuro and NurRE Reporter, and of Reporters with Mutations in Each Regulatory Element

A heterologous cell system was used to assess responsiveness of EboxNeuro and NurRE and their mutants to NeuroD1 and NGFI-B, respectively. HEK293 cells express Pitx1 and E47 but do not express Tpit. For these experiments HEK293 cells were cotransfected with expression vectors for Tpit (RSV-Tpit, 75 ng) and/or NGFI-B (CMX-NGFI-B, 150 ng) and/or NeuroD1 (CMV-NeuroD1, 150 ng), as indicated. A, Activity of reporter plasmid containing the wild-type NurRE and the EboxNeuro upon expression of NGFI-B or NeuroD1. B, Activity of similar reporter containing the same mutations of the EboxNeuro as characterized in Fig. 2Go. C, Similar reporter containing the double mutation for each half-site of the NurRE as Fig. 2Go. Data represent the means ± SEM of three experiments each performed in duplicates.

 
The NurRE is a palindromic regulatory element containing two half-sites for Nur factor binding (Fig. 2CGo). In gel retardation, the NurRE probe therefore forms dimer and monomer complexes with NGFI-B (Fig. 2DGo) or with other members of this subfamily (22). Mutagenesis of one half-site of the NurRE results in the loss of dimer formation but maintains monomer binding (Fig. 2DGo, lane 6). Complete loss of DNA binding requires mutagenesis of both half-sites (Fig. 2Go, C and D, lane 9). The mutagenesis of both half-sites results in loss of NGFI-B responsiveness in cotransfection assays in agreement with the loss of DNA binding (Fig. 3CGo).

Transgenic Analysis of POMC Promoter Activity
Prior work by us and others showed that a –480-bp fragment of the rat POMC promoter is sufficient to confer cell-specific expression in the pituitary of transgenic mice (3, 31). Deletion of this promoter to –380 bp decreases expression in adult transgenic pituitaries by more than 1000-fold (18). To assess the relative contribution of the EboxNeuro and the NurRE for POMC expression during development, we constructed an enhanced green fluorescent protein (EGFP) transgene driven by the –480-bp rat POMC promoter. Expression of this transgene can be assessed with great sensitivity by fluorescence microscopy for EGFP. Three different transgenic lines were established using this transgene and used to validate cell-specific expression of the POMC promoter (Fig. 4Go). Expression of the EGFP transgene was correlated by coimmunofluorescence with expression of POMC at e13.5 and e17.5 and in adult pituitaries. Excellent colocalization of EGFP transgene and ACTH immunofluorescence was observed at all times of development (Fig. 4Go, G–I), both for corticotrophs of the anterior lobe as well as for intermediate lobe melanotrophs. It is noteworthy that the intensity of the EGFP signal per cell increases during development: thus, longer exposures are required to detect transgene expression at earlier times such as e13.5 (Fig. 4AGo) than for adult tissues (Fig. 4CGo), this being accompanied by greater background fluorescence.


Figure 4
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 4. Pituitary Expression of POMC-EGFP Transgene

Analysis of transgene expression by fluorescence microscopy on sections of pituitaries (A–C) is compared with expression of POMC assessed by ACTH immunohistochemistry (D–F). The merge of these two signals (G–I) clearly indicates excellent overlap of the signals (yellow). Transgene expression was analyzed at e13.5 (A, D, and G), e17.5 (B, E, and H) and in the adult gland (C, F, and I). A few cells outside of the pituitary show signal for both EGFP and POMC: this is due to nonspecific signal in red blood cells. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe.

 
To provide quantitative comparison of transgene expression, quantitative RT-PCR (qRT-PCR) was used on RNA isolated from dissected transgenic pituitaries harboring the –480-bp POMC-EGFP transgene. This analysis indicated that transgene expression increases about 2-fold between e14.5 and e17.5 and increases another 50-fold in adult tissues (Fig. 5BGo). Thus, the level of transgene expression mimics expression of the endogenous POMC gene because the POMC mRNA levels increase 3-fold and 300-fold between the same time points (Fig. 5AGo). During the e17.5 and adult interval, the mass (and total number of cells) of the pituitary increases about 10-fold, and the bulk of adult cells are somatotrophs and lactotrophs. This suggests that the large increase in POMC and EGFP mRNA during this interval represents a significant increase in mRNA amount per cell in addition to the increased cell number. This interpretation is consistent with greater histochemistry signal-to-noise ratio in adult (Fig. 4Go, C, F, and I) vs. e13.5 (Fig. 4Go, A, D, and G) or e17.5 (Fig. 4Go, B, E, and H) pituitaries.


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 5. Quantitation by qRT-PCR of POMC and POMC-EGFP Transgene Expression in Pituitary

A, POMC mRNA levels were measured in pituitaries dissected at e14.5 and e17.5 of development as well as from adult pituitaries. rRNA 40S was used as reference in these experiments. The data represent the means ± SEM of nine determinations from three different transgenic lines (three mice each). Similar variation was observed for mice within the same transgenic line as between mice from different transgenic founders. B, Quantitation of EGFP mRNA in transgenic pituitaries by qRT-PCR. Pituitaries were dissected from transgenic mice from three different lines for each of the transgenes as indicated, and qRT-PCR quantitation of EGFP mRNA was calculated relative to 40S rRNA. P values are indicated on the figures for e14.5 and e17.5; for comparison of mutants to control in adult: P ≤ 10–5. The data represent the means ± SEM for nine to 21 mice. C, Relative transgene expression for mice of each founder line. Pituitaries from transgenic lines highlighted in bold were used for further characterization. Mut., Mutant; n.s., nonsignificant.

 
Marked Role of EboxNeuro in Adult Pituitary
The in vivo role of the EboxNeuro was assessed in transgenic mice that harbor a mutant –480-bp POMC promoter carrying the same mutation of the EboxNeuro as characterized in Figs. 2Go and 3Go. A similar proportion of transgenic mouse lines express this transgene (three of six) compared with the wild-type POMC promoter (four of seven) or to the transgene carrying the NurRE mutation (five of 11). Transgene expression was reduced for the EboxNeuro mutant compared with wild-type promoter because qRT-PCR analyses at e14.5, e17.5 (statistical significance is borderline at e17.5; P = 0.06), and adult show EGFP RNA levels that are about 2-, 3-, and 100-fold lower, respectively (Fig. 5BGo). The greatest impact of the mutation is thus observed in adult tissues.

The qRT-PCR assay measures total EGFP mRNA in the gland but does not provide an assessment of cell distribution. Prior work in tissue culture cells had indicated that mutation of cell-specific regulatory elements of the POMC promoter results in greater relative activity in non-POMC cells, including cells of other pituitary lineages. To verify whether expression of the EboxNeuro mutant promoter is affected similarly in POMC lineages as it is in total pituitary RNA, we scored the percentage of ACTH-positive cells in the anterior lobe that are also EGFP positive in representative transgenic lines of wild-type and mutant promoter transgenics (Fig. 6Go). These analyses were thus conducted on pituitaries from the line that showed intermediate transgene expression levels by qRT-PCR in adult mice of each transgenic group (Fig. 5CGo): the transgenic pituitaries analyzed in Fig. 6Go were from lines 862, 914, and 113. At all times, this analysis showed that about 80% of ACTH-positive cells are also EGFP positive in control EGFP-POMC pituitaries. In contrast, the proportion of double positive cells was about 4, 6, and 2% in e14.5, e17.5, and adult EboxNeuro mutant promoter transgenics, respectively (Fig. 6Go). Thus, the loss of activity in adult pituitaries is similar when assessed at the cell level by this approach compared with the qRT-PCR data (Fig. 5BGo). However, significant differences are observed at e14.5 and e17.5 for the two measurements. It was verified that this difference could not be due to contaminating genomic DNA because PCR products of different sizes would be obtained with cDNA or genomic DNA. We therefore suggest that low-level transgene expression in POMC cells may escape detection by fluorescence. It is not possible, however, to exclude the possibility that the mutant transgene may be leaky and have low expression in pituitary cells other than POMC cells. Nonetheless, it is noteworthy that EGFP-positive cells in mutant transgenic pituitaries can be readily identified as shown on the merged signals for e14.5, e17.5, and adult pituitaries (Fig. 6Go). Be that as it may, these data clearly support a critical role for the EboxNeuro at all times examined. In particular, the loss of activity in adult tissues (both corticotrophs and melanotrophs) indicates that this regulatory element is important throughout development, including after down-regulation of NeuroD1 expression (19).


Figure 6
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 6. Coexpression of POMC-EGFP Transgene with POMC (ACTH) at Different Times of Development for Wild-Type POMC Promoter and Promoters Mutated in EboxNeuro or NurRE

A, For each transgene (POMC-EGFP, the EboxNeuro mutant promoter and the NurRE double-mutant promoter), a representative line was used for assessment of transgene expression by fluorescence (green) and for immunofluorescence analysis of ACTH-positive cells (red). The merge picture shows overlap in expression (yellow). A, Representative sections for pituitaries carrying each transgene at e14.5 of pituitary development. B, Quantitation of the percentage ratio of ACTH-positive cells that colabel for EGFP. Data represent the means ± SEM of three independent counts of more than 100 ACTH-positive cells each. Insets show co-labeling of cells. At all developmental times, about 20% of ACTH (red)-positive cells in pituitaries harboring the POMC-EGFP transgene were negative for EGFP (green); for the most part, these cells were weakly positive for ACTH, such that their presence is not readily visible on the Merge pictures. The specificity and significance of this weak signal are presently unclear, but quantitation of double-positive cells included all cells that had signal above background. C, Transgene and ACTH expression in sections from transgenic pituitaries at e17.5 of development. D, Quantitation of the percentage of ACTH-positive cells that are also EGFP-positive for each transgene at e17.5. E, Coexpression of transgene and ACTH in adult gland for each transgene. The U-shaped intermediate lobe is clearly identified on these low magnification sections. For both mutant transgenes, transgene expression is not detectable in the intermediate lobe. F, Quantitation of the percentage ratio of ACTH-positive cells that are also positive for EGFP in the anterior lobe of adult transgenic pituitaries.

 
Increasing Contribution of NurRE during Development
Similar analyses were carried out with transgenic mice carrying the NurRE mutant promoter. This mutation did not affect transgene expression quite as much as the EboxNeuro whether expression was assessed by qRT-PCR (Fig. 5BGo) or EGFP fluorescence (Fig. 6Go). Again, significant reductions (~50-fold) in transgene RNA levels were observed in adult pituitaries, but the mutation had borderline effects in fetal tissues (Fig. 5BGo). When scored at the cell level by coimmunofluorescence, transgene expression was significantly reduced for the NurRE mutant compared with the wild-type promoter (Fig. 6Go) at all times of development. However, the NurRE mutant promoter retained much more relative activity at e14.5 than at e17.5 or in the adult anterior lobe (Fig. 6Go, panel B compared with panels D and F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The purpose of the present work was to test the developmental importance of critical POMC-regulatory elements, the EboxNeuro and NurRE, particularly at developmental times when either TFs or corresponding signaling pathways are not active. Thus, prior work had shown the importance of the EboxNeuro and of the cognate NeuroD1 neurogenic bHLH TF for POMC transcription. However, the expression of NeuroD1 in agreement with the transient phenotype of the NeuroD1 knockout had supported a role for this factor from the onset of POMC expression at e12.5 to about e16 of mouse pituitary development (19). By specifically mutating the EboxNeuro in a transgenic assay, the present work conclusively showed the sustained importance of the EboxNeuro and presumably their cognate bHLH factors, throughout development and in the adult pituitary. Similarly, prior work had shown the importance of the NurRE for responsiveness of CRH signaling and the marked dependence of this signaling for transcriptional activation by the cognate Nur factors, particularly NGFI-B (20, 21, 22). Because the hypothalamo-pituitary portal system is only established around day e16, the importance of the NurRE and cognate factors earlier in development was questionable. The present work clearly showed that this regulatory element is critical for POMC expression before the onset of CRH action.

Developmental Pattern of POMC Expression
The first unexpected finding of this study was the very large increase in POMC mRNA observed in the adult pituitary gland compared with e17.5 (Fig. 5Go). Whereas the e14.5 pituitary has a limited number of POMC-expressing cells, the e17.5 pituitary exhibits a large number of POMC-expressing corticotrophs in the anterior lobe, and by this time, most melanotrophs of the intermediate lobe express detectable POMC. In a way, it is also surprising that between these two time points, the concentration of POMC mRNA does not increase more than a few fold; however, this time period also sees expansion of the pituitary precursor pool and differentiation of other lineages (1). In the interval between e17.5 and adult, the total weight of the pituitary increases by about 10-fold. During this same time interval, the proportion of the POMC-positive cells in the anterior lobe decreases to about 10% of all cells in the adult anterior lobe because during this same time period, the number of somatotroph and lactotroph cells becomes predominant. The 200-fold increase of POMC mRNA relative to 40S rRNA during this period is therefore a clear indication that POMC mRNA increases markedly during this period. The mechanism for this is unknown: it may involve enhanced transcription rates and/or stabilization of POMC mRNA. Hypothalamic signaling, including CRH and vasopressin, may be implicated in increasing POMC mRNA concentration. Alternatively, paracrine signals within the pituitary may be implicated in this process.

The assessment of POMC-EGFP transgene expression in the developing pituitary by qRT-PCR and scoring of double positive cells for EGFP and ACTH did not yield comparable results at all times examined (Fig. 5Go and 6Go). The percentage of double positive cells is a more reliable assessment of transgene activity in relevant POMC-expressing cells. However, this measurement is likely subject to a minimal threshold of detection, such that cells expressing the transgene below this threshold will go undetected. Be that as it may, this value is likely a reliable assessment of transgene activity because at all developmental times, the percentage of EGFP over ACTH-positive cells is about 80% in the anterior lobe for the reference transgene (Fig. 6Go, B, D, and F). Thus, the intact promoter transgene is expressed above the detection limit at all developmental times despite the fact that total level of POMC-EGFP mRNA (as assessed by PCR) is about 100-fold higher in the adult compared with e14.5 or e17.5. Consequently, the much lower percentage of EGFP over ACTH-positive cells in transgenic pituitaries carrying the mutant reporters likely reflects the decrease in promoter activity resulting from the mutation. It is also possible that the mutated promoters are not only much weaker in activity compared with the wild-type POMC promoter but also that they may lead to nonspecific expression in other pituitary cells at levels that are below the detection threshold. In such a situation, a significant amount of POMC-EGFP mRNA could be present in other cells but would go undetected by immunofluorescence. It is currently not possible to discriminate between these two possibilities.

Sustained Dependence on EboxNeuro for POMC Expression in Development
The EboxNeuro was defined through mutagenesis of the POMC promoter and assessment of its activity in AtT-20 cells, a model taken to represent pituitary corticotrophs because these cells are responsive to CRH and subject to Gc repression. In this context, the EboxNeuro is an essential component of the distal region of the promoter that mediates its effect on transcription through interaction with the Tpit/PitxRE and its cognate TFs, Pitx1 and Tpit (5, 18). NeuroD1 was identified in this same cell line as the neurogenic bHLH that specifically interacts with the EboxNeuro as heterodimers with A class bHLH such as E47, E12, and ITF2, which is the most abundant in the AtT-20 cells. The POMC promoter also contains an active Ebox in its central domain, but this Ebox is only bound in vitro and activated in transfection by a class A bHLH factor. NeuroD1 is thus essential for protein-DNA interactions at the EboxNeuro (18). Given that NeuroD1 expression decreases significantly after e16 in the mouse pituitary, the AtT-20 cells may be considered as a model of the early corticotrophs because of the prevalence of NeuroD1 in these cells; thus, the AtT-20 cells may not be a suitable model by which to identify bHLH factors of the neurogenic class that may take over the role of NeuroD1 after its down-regulation in mouse development. Alternatively, the EboxNeuro and NeuroD1 might only have a transient role in development and their function be taken over by TFs of a different structural class. It is for this reason that we chose to mutate the EboxNeuro by specific nucleotide changes that destroy the Ebox motif (Fig. 2AGo) but hopefully do not perturb other protein-DNA interactions. We do not know of any other proteins that interact with overlapping sequences of the POMC promoter, but it is not possible to formally exclude such a possibility. Accepting this caveat, the loss of function observed with the POMC promoter mutated at the EboxNeuro is a strong indication that bHLH factors of a neurogenic class continue to be important throughout later fetal development and in the adult pituitary.

We currently do not know what the(se) factors may be. We have previously assessed two candidate neurogenic bHLH factors on the basis of their involvement in the NeuroD1 cascade in pancreas, namely, neurogenin 1 and neurogenin 2. Neither of these factors appeared to take over the role of NeuroD1 in corticotrophs, nor to be required for activation of NeuroD1 expression before e12 of development (19).

The NurRE and Nur Factors in Development
The NurRE is situated next to the EboxNeuro in the distal region of the POMC promoter, and the activity of these two elements is interdependent (5). It was expected that mutagenesis of this element should hamper POMC promoter activity in the adult pituitary because this target has been proposed as a major site of action for effectors of the CRH signaling pathway (20, 22). This pathway ultimately leads to activation of Nur factors, in particular NGFI-B, through dephosphorylation of the DNA-binding domain (permissive for DNA binding) and through phosphorylation of the N-terminal AF1 domain (21). Because CRH may not reach the pituitary efficiently before establishment of the hyophothalamo-pituitary portal system at e16, this element could have much less importance for POMC expression in early fetal development. In contrast, we found that mutagenesis of the NurRE has a significant importance in the POMC promoter activity in corticotrophs (Fig. 6Go, B and D) at e14.5 of development. It is noteworthy, however, that the relative importance of the NurRE increases with time, and its mutation is most detrimental to promoter activity in the adult (Fig. 6FGo). This increase on NurRE dependence for promoter activity later in development is consistent with an increase important in CRH signaling in the adult. Promoter activation through the NurRE at e14.5 may rely on the constitutive activity of Nur factors; alternatively, the MAPK pathway may be activated through signals other than CRH in the fetal pituitary and thus Nur factor activation may take place independently of CRH. However, we currently do not know any candidate signals for such a role.

Collectively, the present work supports a model of promoter activation that relies on occupancy of a constant set of regulatory elements during development despite the fact that during the same time frame, some of the cognate TFs may change (such as the bHLH factors on the EboxNeuro) or that the ligands responsible for signaling pathway activation may also differ. Ultimately, the analysis of chromatin organization and the association of specific TFs with the POMC promoter will be required to directly resolve the questions that derive from the present work. Although such studies could be conducted in adult tissues with presently available technologies, their application on cell-heterogeneous fetal tissues remains a challenge.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
Human embryonic kidney (HEK)293 cells were grown in DMEM with 10% fetal bovine serum and penicillin/streptomycin and maintained at 37 C and 5% CO2. Cells were transfected by the calcium phosphate coprecipitation method on 250,000 cells plated in 12-well plates. A total of 6 µg of DNA was used for each transfection, performed in duplicate. Luciferase activity was measured 24 h after transfection as described previously (23). In each transfection experiment, cytomegalovirus (CMV)-β-galactosidase was used as internal standard.

Plasmids
The different reporter plasmids were constructed in the vector pXP1-luciferase by insertion of different lengths of the rat POMC promoter or oligonucleotides of this promoter and have been previously described (6, 20). The mutated EboxNeuro and half-site NurRE plasmids were generated as previously reported (18). The double mutation of the NurRE (boldface letters indicate mutant nucleotides) was also generated with the following oligonucleotide: 5'-ATGCTCATCGATGAGACCTCACCCGTCCAGGAAGGCAGATGGACGCAC-3'. The TF expression vectors used in this study were described previously, Tpit, NGFI-B, NeuroD1 (11, 18, 20).

Transgenics
Wild-type or mutant POMC promoters (–480/+63) were introduced in front of the EGFP cDNA by replacing the CMV promoter (AseI/HindIII) of the pEGFP-N1 plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA). The purified BglII/AflII 1.6-kb fragment containing the POMC promoter, the EGFP cDNA, and the simian virus 40 polyA was then microinjected, and founder lines were derived as already described (18).

EMSA
The gel-shift assays were performed as previously described (18, 20) with in vitro-translated proteins (TNT from Promega Corp., Madison, WI). We used the following oligonucleotides: wild-type EboxNeuro 5'-GATCCGGAAGGCAGATGGACGCA-3', mutant Eboxneuro 5'-GATCCGGAAGGCTACCGGACGCA-3', wild-type NurRE 5'-ATGCAGTGATATTTACCTCCAAATGCCA-3', half-site mutant NurRE 5'-ATGCCATCGATGAGACCTCCAAATGCCA-3', and double mutated NurRE 5'-ATGCCATCGATGAGACCTCACCCGTCCA-3'.

qRT-PCR
Total RNA from embryonic or adult pituitary was prepared with the RNAeasy columns (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. Reverse transcription was performed with all this RNA using oligo dT12–18 (Invitrogen, Carlsbad, CA) and AMV-RT (Promega). The cDNA was then used for quantitative real-time PCR (MX 3005, Stratagene, La Jolla, CA) with the SYBR Green kit (QIAGEN). The primers used are the following: EGFP, 5'-TGAAGGGCATCGACTTCAAGGA-3' and 5'-TGGCGGATCTTGAAGTTCACCT-3'; POMC, 5'-TGGAAGATGCCGAGATTCTGCTACAGT-3' and 5'-G ATGCAAGCCAGCAGGTTGCTCTC-3'; and 40S, 5'-TCTGGGCAAGGAGAGATTTG-3' and 5'-CCGCCAAACTTCTTGGATTC-3'.

Immunohistochemistry and Immunofluorescence
Immunohistochemistry was performed on embedded paraffin tissue sections for horseradish peroxidase revelation using antibodies against NeuroD1 (19) and Nurr1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The present work used a new affinity-purified rabbit polyclonal antibody raised against the same NeuroD1 epitope [amino acids 120–170 of mouse NeuroD1 fused to glutathione-S-transferase (GST)] as were used previously (18, 19). For purification, the antiserum was passed first on a GST column, and the flow-through from this column was further purified by affinity on a GST-NeuroD1 column. This purified antibody does not detect any signal in tissue sections from NeuroD1–/– mice (data not shown). Immunohistochemistry was performed as previously described (32). For immunofluorescence, EGFP fluorescence was first directly observed after rehydration, after which the slides were treated as for immunohistochemistry with an antibody against POMC (Cortex Biochem, Concord, MA).


   ACKNOWLEDGMENTS
 
We thank our many colleagues from the laboratory for their comments and we thank Lise Laroche for expert secretarial assistance.


   FOOTNOTES
 
This work was supported by fellowship from Fonds des chercheurs et aide à la recherche-Fonds de la recherche en santé du Québec and by research grants (to J.D.) from the Canadian Institutes of Health Research and National Cancer Institute of Canada.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2008

Abbreviations: bHLH, Basic helix-loop-helix; CMV, cytomegalovirus; e12.5, embryonic d 12.5; EGFP, enhanced green fluorescent protein; Gc, glucocorticoid; GR, glucocorticoid receptor; GST, glutathione-S-transferase; HEK, human embryonic kidney; NGFI-B, nerve growth factor-induced-B; POMC, proopiomelanocortin; qRT-PCR, quantitative RT-PCR; SRC2, steroid receptor coactivator 2; TF, transcription factor.

Received for publication December 19, 2007. Accepted for publication March 25, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Drouin J 2006 Molecular mechanisms of pituitary differentiation and regulation: implications for hormone deficiencies and for hormone resistance syndromes. In: Arzt E, Bronstein M, Guitelman M, eds. Frontiers of hormone research: pituitary today. Basel: S. Karger AG; 74–87
  2. Therrien M, Drouin J 1991 Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements. Mol Cell Biol 11:3492–3503[Abstract/Free Full Text]
  3. Tremblay Y, Tretjakoff I, Peterson A, Antakly T, Zhang CX, Drouin J 1988 Pituitary-specific expression and glucocorticoid regulation of proopiomelanocortin fusion gene in transgenic mice. Proc Natl Acad Sci USA 85:8890–8894[Abstract/Free Full Text]
  4. Liu B, Mortrud M, Low MJ 1995 DNA elements with AT-rich core sequences direct pituitary cell-specific expression of the pro-opiomelanocortin gene in transgenic mice. Biochem J 312:827–832[Medline]
  5. Therrien M, Drouin J 1993 Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene. Mol Cell Biol 13:2342–2353[Abstract/Free Full Text]
  6. Poulin G, Turgeon B, Drouin J 1997 NeuroD1/BETA2 contributes to cell-specific transcription of the POMC gene. Mol Cell Biol 17:6673–6682[Abstract]
  7. Lamonerie T, Tremblay JJ, Lanctôt C, Therrien M, Gauthier Y, Drouin J 1996 PTX1, a bicoid-related homeobox transcription factor involved in transcription of pro-opiomelanocortin (POMC) gene. Genes Dev 10:1284–1295[Abstract/Free Full Text]
  8. Suh H, Gage PJ, Drouin J, Camper SA 2002 Pitx2 is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification. Development 129:329–337[Medline]
  9. Tremblay JJ, Lanctôt C, Drouin J 1998 The pan-pituitary activator of transcription, Ptx-1 (pituitary homeobox1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 12:428–441[Abstract/Free Full Text]
  10. Tremblay JJ, Marcil A, Gauthier Y, Drouin J 1999 Ptx1 regulates SF-1 activity by an interaction that mimics the role of the ligand-binding domain. EMBO J 18:3431–3441[CrossRef][Medline]
  11. Lamolet B, Pulichino AM, Lamonerie T, Gauthier Y, Brue T, Enjalbert A, Drouin J 2001 A pituitary cell-restricted T-box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104:849–859[CrossRef][Medline]
  12. Szeto DP, Ryan AK, O'Connell SM, Rosenfeld MG 1996 P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development. Proc Natl Acad Sci USA 93:7706–7710[Abstract/Free Full Text]
  13. Pulichino AM, Vallette-Kasic S, Tsai JPY, Couture C, Gauthier Y, Drouin J 2003 Tpit determines alternate fates during pituitary cell differentiation. Genes Dev 17:738–747[Abstract/Free Full Text]
  14. Drouin J, Bilodeau S, Vallette-Kasic S 2007 Of old and new diseases: genetics of pituitary ACTH excess (Cushing) and deficiency. Clin Genet 72:175–182[CrossRef][Medline]
  15. Pulichino AM, Vallette-Kasic S, Couture C, Gauthier Y, Brue T, David M, Malpuech G, Deal C, Van Vliet G, De Vroede M, Riepe FG, Partsch CJ, Sippell WG, Berberoglu M, Atasay B, Drouin J 2003 Human and mouse Tpit gene mutations cause early onset pituitary ACTH deficiency. Genes Dev 17:711–716[Abstract/Free Full Text]
  16. Vallette-Kasic S, Brue T, Pulichino AM, Gueydan M, Barlier A, David M, Nicolino M, Malpuech G, Déchelotte P, Deal C, Van Vliet G, De Vroede M, Riepe FG, Partsch CJ, Sippell WG, Berberoglu M, Atasay B, de Zegher F, Beckers D, Kyllo J, Donohoue P, Fassnacht M, Hahner S, Allolio B, Noordam C, et al. 2005 Congenital isolated adrenocorticotropin deficiency: an underestimated cause of neonatal death, explained by Tpit gene mutations. J Clin Endocrinol Metab 90:1323–1331[Abstract/Free Full Text]
  17. Vallette-Kasic S, Couture C, Balsalobre A, Gauthier Y, Metherell LA, Dattani M, Drouin J 2007 The Tpit gene mutation M86R associated with isolated ACTH deficiency interferes in protein:protein interactions. J Clin Endocrinol Metab 92:3991–3999[Abstract/Free Full Text]
  18. Poulin G, Lebel M, Chamberland M, Paradis FW, Drouin J 2000 Specific protein:protein interaction between basic helix-loop-helix transcription factors and homeoproteins of the Pitx family. Mol Cell Biol 20:4826–4837[Abstract/Free Full Text]
  19. Lamolet B, Poulin G, Chu K, Guillemot F, Tsai MJ, Drouin J 2004 Tpit-independent function of NeuroD1(β2) in pituitary corticotroph differentiation. Mol Endocrinol 18:995–1003[Abstract/Free Full Text]
  20. Philips A, Lesage S, Gingras R, Maira MH, Gauthier Y, Hugo P, Drouin J 1997 Novel dimeric Nur77 signaling mechanisms in endocrine and lymphoid cells. Mol Cell Biol 17:5946–5951[Abstract]
  21. Maira MH, Martens C, Batsche E, Gauthier Y, Drouin J 2003 Dimer-specific potentiation of NGFI-B (Nur77) transcriptional activity by the protein kinase A pathway and AF-1-dependent coactivator recruitment. Mol Cell Biol 23:763–776[Abstract/Free Full Text]
  22. Maira MH, Martens C, Philips A, Drouin J 1999 Heterodimerization between members of the Nur subfamily of orphan nuclear receptors as a novel mechanism for gene activation. Mol Cell Biol 19:7549–7557[Abstract/Free Full Text]
  23. Batsche E, Desroches J, Bilodeau S, Gauthier Y, Drouin J 2005 Rb enhances p160/SRC coactivator-dependent activity of nuclear receptors and hormone responsiveness. J Biol Chem 280:19746–19756[Abstract/Free Full Text]
  24. Batsche E, Moschopoulos P, Desroches J, Bilodeau S, Drouin J 2005 Retinoblastoma and the related pocket protein p107 act as coactivators of NeuroD1 to enhance gene transcription. J Biol Chem 280:16088–16095[Abstract/Free Full Text]
  25. Maira MH, Couture C, Le Martelot G, Pulichino AM, Bilodeau S, Drouin J 2003 The T-box factor Tpit recruits SRC/p160 coactivators and mediates hormone action. J Biol Chem 278:46523–46532[Abstract/Free Full Text]
  26. Philips A, Maira MH, Mullick A, Chamberland M, Lesage S, Hugo P, Drouin J 1997 Antagonism between Nur77 and glucocorticoid receptor for control of transcription. Mol Cell Biol 17:5952–5959[Abstract]
  27. Martens C, Bilodeau S, Maira M, Gauthier Y, Drouin J 2005 Protein:protein interactions and transcriptional antagonism between the subfamily of NGFI-B/Nur77 orphan nuclear receptors and glucocorticoid receptor. Mol Endocrinol 19:885–897[Abstract/Free Full Text]
  28. Bilodeau S, Vallette-Kasic S, Gauthier Y, Figarella-Branger D, Brue T, Berthelet F, Lacroix A, Batista D, Stratakis S, Hanson J, Meij B, Drouin J 2006 Role of Brg1 and HDAC2 in GR trans-repression of pituitary POMC gene and misexpression in Cushing disease. Genes Dev 20:2871–2886[Abstract/Free Full Text]
  29. Lugo DI, Pintar JE 1996 Ontogeny of basal and regulated secretion from POMC cells of the developing anterior lobe of the rat pituitary gland. Dev Biol 173:95–109[CrossRef][Medline]
  30. Lugo DI, Pintar JE 1996 Ontogeny of basal and regulated proopiomelanocortin-derived peptide secretion from fetal and neonatal pituitary intermediate lobe cells: melanotrophs exhibit transient glucocorticoid responses during development. Dev Biol 173:110–118[CrossRef][Medline]
  31. Liu B, Hammer GD, Rubinstein M, Mortrud M, Low MJ 1992 Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary gland of transgenic mice. Mol Cell Biol 12:3978–3990[Abstract/Free Full Text]
  32. Lanctôt C, Gauthier Y, Drouin J 1999 Pituitary homeobox 1 (Ptx1) is differentially expressed during pituitary development. Endocrinology 140:1416–1422[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   NGFIB  |  NURR1  |  NOR1




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lavoie, P.-L.
Right arrow Articles by Drouin, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lavoie, P.-L.
Right arrow Articles by Drouin, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals