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Molecular Endocrinology, doi:10.1210/me.2003-0127
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Molecular Endocrinology 18 (4): 995-1003
Copyright © 2004 by The Endocrine Society

Tpit-Independent Function of NeuroD1(BETA2) in Pituitary Corticotroph Differentiation

Bruno Lamolet, Gino Poulin, Khoi Chu, François Guillemot, Ming-Jer Tsai and Jacques Drouin

Laboratoire de Génétique Moléculaire (B.L., G.P., J.D.), Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada H2W 1R7; Department of Molecular and Cellular Biology (K.C., M.-J.T.), Baylor College of Medicine, Houston, Texas 77030-3498; and Division of Molecular Neurobiology (F.G.), National Institute for Medical Research, London NW7 1AA, United Kingdom

Address all correspondence and requests for reprints to: Dr. Jacques Drouin, 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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NeuroD1(BETA2) and Tpit are cell-specific activators of pituitary proopiomelanocortin (POMC) gene transcription. Expression of both factors slightly precedes that of POMC at embryonic d 12.5 of mouse pituitary development. We now report that NeuroD1(BETA2) is required for early corticotroph differentiation. In agreement with the transcriptional synergism observed between Tpit and basic helix-loop-helix dimers containing NeuroD1(BETA2), POMC expression is delayed in NeuroD1-deficient mice. However, this differentiation defect does not reflect a change of corticotroph commitment as revealed by Tpit expression. The delay of corticotroph terminal differentiation is transient and coincides with the developmental window of NeuroD1 expression in corticotrophs. In contrast to their requirement in other NeuroD1-expressing cells, the neurogenin genes do not appear to be necessary for corticotroph differentiation. Taken together with a similar requirement of Tpit for corticotroph differentiation but not for commitment, the present data indicate that the POMC promoter is a point of convergence for independent corticotroph differentiating signals.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DURING MAMMALIAN PITUITARY gland development, the first functional hormone-producing cells to differentiate are the corticotrophs that appear at embryonic day (e) 12.5 in the ventral part of the mouse pituitary (1). The molecular mechanisms that regulate this differentiation are being unraveled, particularly by identification of transcription factors controlling various aspects of organogenesis and/or cell differentiation. Homeobox transcription factors Pitx1 and 2 are both expressed early in the oral ectoderm and its derivative, Rathke’s pouch, which develops into the pituitary gland (2, 3). Inactivation of the Pitx2 gene blocks cell proliferation and differentiation of all lineages in the pituitary primordium, except corticotrophs (4, 5, 6). A similar phenotype was observed after knockout of the Lhx3 gene (7), which appears to be under control of Pitx genes (8, 9). This suggests that determination of corticotroph progenitors occurs early and/or in a significantly different way by comparison to those of other pituitary lineages.

Recent work identified early signals that may influence corticotroph differentiation. Ex vivo experiments performed with tissues obtained at early stages of pituitary development indicated that fibroblast growth factor 8 and bone morphogenetic proteins 2/4 block corticotroph differentiation (10). However, ectopic expression of these factors in transgenic mice suggested otherwise (11). Another approach to identify mechanisms of corticotroph differentiation has been through analysis of tissue-specific mechanisms for POMC gene regulation. This led to the identification of two corticotroph-specific regulators, the basic helix-loop-helix (bHLH) transcription factor NeuroD1(BETA2) (12, 13) and the T-box factor Tpit (14, 15). NeuroD1(BETA2) is a class B bHLH expressed in pancreas, intestine (16, 17), various parts of the nervous system (18), and in the pituitary corticotroph lineage (12, 13). Other class B bHLH factors were shown to be required for differentiation of muscle cells (19), the hematopoietic system (20), and neurons (21). In corticotrophs, the NeuroD1 protein was only detected between e12.0 and e15.5, although mRNA is still detectable in the adult gland (13). For transcriptional activation, Neuro-D1 heterodimerizes with ubiquitous class A bHLH, such as E47(Pan1), and these heterodimers bind to a specific E-box, the E-boxneuro, within the POMC promoter to activate transcription. This transcriptional effect is enhanced synergistically by physical and functional interaction with Pitx1 (12, 13).

The other corticotroph marker Tpit (Tbx19) was identified as an obligate partner of Pitx1 for activation of POMC transcription (14). Tpit binds to a T element situated next to the Pitx1 binding site. T-box factors have been involved in differentiation in various tissues such as embryonic mesoderm (22), neural cortex (23), and T helper cells (24). Tpit is only expressed in the two POMC-expressing lineages of the pituitary gland, the corticotrophs and the melanotrophs (14). As for NeuroD1, the onset of Tpit expression in corticotrophs is around e12, just before POMC itself.

This strikingly restricted expression pattern suggested that Tpit may have a role in corticotroph differentiation. This is indeed the case because Tpit-deficient mice fail to terminally differentiate corticotrophs and melanotrophs, although early corticotroph commitment appeared to be intact as assessed by NeuroD1 expression (15). In addition, Tpit was shown to have a negative role for gonadotroph differentiation through antagonism with the gonadotroph-restricted orphan nuclear receptor steroidogenic factor 1 (SF1) (15). The loss of Tpit function also results in ACTH deficiency in mice and human TPIT gene mutations are the most frequent cause for early onset isolated ACTH deficiency (14, 25).

Gain-of-function experiments in transgenic mice, using the {alpha}GSU (glycoprotein hormone subunit) promoter to drive Tpit expression in undifferentiated cells of the rostral tip of the developing pituitary gland, showed that this factor could induce ectopic expression of POMC (14). However, these cells do not express NeuroD1 and do not lead to long-term establishment of corticotrophs. Therefore, Tpit appears to be required but not sufficient for complete corticotroph differentiation. NeuroD1 is an obvious candidate for involvement in corticotroph differentiation.

In this paper, we used NeuroD1-deficient mice to show that NeuroD1 is required for proper onset of corticotroph differentiation. This process appears independent of Tpit expression. Thus, independent signals appear to control Tpit and NeuroD1 expression and appear to converge at the transcriptional level for activation of POMC expression and terminal corticotroph differentiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NeuroD1/Pan1 and Tpit are both corticotroph-specific transcriptional activators of the POMC gene and they interact individually with Pitx1. Indeed, the NeuroD1/Pan1 heterodimers synergistically enhance transcription with Pitx1, and this appears to result from direct physical interactions between the Pitx1 homeodomain and the bHLH domain of Pan1, but not that of NeuroD1 (13). NeuroD1 is nonetheless required because it is essential for specific DNA sequence recognition of the E-boxneuro. Similarly, Tpit enhances transcription synergistically with Pitx1, and this results from cooperative DNA binding of the two proteins to their contiguous sites on the POMC promoter (14).

Synergism and Physical Interactions between bHLH Factors and Tpit
To assess whether both corticotroph-specific factors act in synergism on POMC transcription, we used a reconstitution system by cotransfection in CV1 cells of a POMC promoter luciferase reporter, together with relevant transcription factors (Fig. 1AGo). As shown in Fig. 1AGo, Tpit and NeuroD1/Pan1 exert Pitx1-dependent synergism on the POMC reporter. This synergism was also observed on an artificial promoter containing two copies of the E-boxneuro and three of the Pitx1-Tpit binding site in GH3 cells that express endogenous Pitx1 (Fig. 1BGo). Because previous work had shown physical interaction between Pitx1 and Pan1 (13), and between Tpit and Pitx1 (Ref.14 ; and Lamolet, B., and J. Drouin, unpublished data), we tested the possibility of a physical interaction between Tpit and HLH factors. In vitro pull-down assays showed that Tpit could only interact with Pan1 (Fig. 1CGo, lane 4) but not NeuroD1 (lane 8). Interestingly, Pan1 did not interact with the T-box of Tpit (lane 2), suggesting that the interaction domain of Tpit with HLH factors is located outside the T-box. The transcriptional synergism between bHLH factors and Tpit/Pitx therefore relies on protein:protein interactions that focus on Pan1; the contribution of NeuroD1 to cell-specific transcription thus depends entirely on its DNA binding specificity for the Eboxneuro (Fig. 1DGo).



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  		Fig. 1. TPIT and Pan1/NeuroD1 Heterodimers Interact to Activate POMC Transcription

A, Tpit and Pan1/NeuroD1 heterodimers (bHLH) activate POMC transcription synergistically in CV1 cells using the POMC promoter (-480 to +63 bp) reporter. Fold activation is shown relative to the POMC promoter construct (CTL) in presence of Pitx1. The homeobox factor Pitx1 was required for synergism between Tpit and bHLH. B, Synergism was also observed in GH3 cells that express endogenous Pitx1. C, Tpit interacts with Pan1 in vitro. Pull-down assays were performed with in vitro-synthesized Pan1 or NeuroD1 and purified MBP-ßGal, MBP-Tpit or MBP-T-box of Tpit. Input was 10% of amount used. D, Protein interactions of the bHLH heterodimers NeuroD1/Pan 1 with Tpit and Pitx. NeuroD1 provides DNA binding specificity to the bHLH dimers for recognition of the Eboxneuro (12 ), and these heterodimers interact with Pitx (13 ) and Tpit (this paper) through Pan1.

 
Requirement for NeuroD1 in Corticotroph Differentiation
We already showed that ectopic expression of Tpit could induce POMC expression but not that of NeuroD1, suggesting that Tpit is not upstream of NeuroD1. Also, inactivation of these genes has different consequences. Tpit-null mice have very few corticotroph cells (15), but NeuroD1-null pituitaries contain a normal number of corticotrophs at e17.5 (26). Tpit thus seems essential for corticotroph differentiation, but not NeuroD1. However, NeuroD1 protein is not detectable in the late developing pituitary (13).

To address a putative role of NeuroD1 on Tpit and/or corticotroph differentiation during early pituitary development, we assessed the onset of corticotroph differentiation in mouse mutants for NeuroD1 (27). Because NeuroD1 is only detectable in corticotrophs between e12.5 and about e15.5, we first assessed the number of corticotrophs in the anterior lobe of e13.5 pituitaries from wild-type and NeuroD1 mutant mice. Using ACTH immunohistochemistry to assess POMC expression and terminal differentiation of corticotrophs, a clear reduction in number of ACTH(POMC)-positive cells was observed in mice heterozygous (+/-) for the NeuroD1 null allele (Fig. 2Go, B compared with A), and this reduction was even greater in NeuroD1-/- mice (Fig. 2CGo). Not only was the number of positive cells decreased but also ACTH immunoreactivity was lower in the remaining cells. The distribution of POMC cells is also affected by NeuroD1 dosage. In wild-type pituitaries, corticotrophs are present throughout the anterior lobe (Fig. 2AGo), whereas in heterozygous and null mice, the remaining POMC-positive cells were mostly in the ventral part of the gland (Fig. 2Go, B and C), as observed in normal early development (14). Thus, NeuroD1 is required for the appropriate timing of corticotroph differentiation.



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  		Fig. 2. Delayed Corticotroph Differentiation in e13.5 NeuroD1(BETA2)-Null Mice

In wild-type mice (+/+), POMC (A) and Tpit (D) expression patterns as assessed by immunohistochemistry are similar and {alpha}GSU (G) is mostly restricted to the rostral tip of the pituitary. In NeuroD1 heterozygous mice (+/-), the number of corticotrophs was markedly decreased (B), but Tpit (E), {alpha}GSU (H), and Pitx1 (K) expression patterns are similar to those of wild-type animals. The remaining cells are located ventrally. In null mice (-/-), only a few corticotroph cells are detected in the ventral region of the pituitary (C). In contrast, Tpit (F), {alpha}GSU (I), and Pitx1 (L) expression patterns are unchanged. Sections A–L are sagittal with the embryo facing to the right.

 
Whereas the reduced POMC expression indicates a delay in corticotroph differentiation, it may not reflect the loss of corticotroph cells. To assess this possibility, we investigated expression of another corticotroph-specific marker, Tpit. Strikingly, the expression of Tpit in heterozygous (Fig. 2EGo) and null (Fig. 2FGo) pituitaries is similar to its expression in wild-type pituitary (Fig. 2DGo). Expression of another hormone marker {alpha}GSU (Fig. 2Go, G–I) and of the pan-pituitary transcription factor Pitx1 (Fig. 2Go, J–L) were not affected by NeuroD1 dosage. These results indicate that corticotroph commitment is not affected in absence of NeuroD1, but that only the last step in corticotroph differentiation (as revealed by POMC expression) is delayed, but not completely abolished in NeuroD1-/- mice.

Corticotrophs are the first anterior pituitary hormone-producing cells to fully differentiate. The role of corticotrophs on differentiation of other pituitary lineages is not known, although their number is not required to maintain the distribution of other cell types (28). To assess whether the delay in terminal corticotroph differentiation may affect other lineages, we investigated expression of other hormones that appear later. At e14.5, the delay of corticotrophs terminal differentiation is still observed in NeuroD1-deficient mice (Fig. 3Go, B and C) relative to normal pituitaries (Fig. 3AGo), although Tpit expression is similar in all pituitaries (Fig. 3Go, D–F). This delay in corticotroph differentiation does not seem to affect expression of other pituitary markers. {alpha}GSU expression is restricted in the rostral and ventral parts of pituitaries of the three genotypes (Fig. 3Go, G–I). Pit1-independent expression of ßTSH in the rostral tip is also unaffected as is the appearance of Pit1-dependent ßTHS cells in the most ventral part of the anterior lobes (Fig. 3Go, J–L). Finally, expression of Pitx1 is not affected by the delay of corticotroph differentiation (Fig. 3Go, M–O). The NeuroD1 differentiation delay is thus specific to corticotroph cells.



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  		Fig. 3. Corticotroph Deficiency Is Restricted to this Lineage

As in e13.5 pituitaries (Fig. 2Go), the distribution and number of POMC cells relative to those of wild-type animals (A) is affected by NeuroD1 dosage at e14.5. The lower the dosage, the less there are corticotrophs and the more ventrally they are located (B to C). Tpit expression is unaffected (D–F). Expression of other pituitary markers {alpha}GSU (G–I), ßTSH (J–L), and Pitx1 (M–O) is unchanged by lower dosage of NeuroD1. Sections A–O are sagittal with the embryo facing to the right.

 
The NeuroD1-Dependent Differentiation Delay Is Transient
The expression of NeuroD1 is transient during pituitary development. At e15.5, NeuroD1 protein is not detected anymore (13). We therefore analyzed e16.5 pituitaries to evaluate the putative consequences of NeuroD1 deficiency beyond the time of detectable NeuroD1 expression. At that age, POMC-positive cells were in normal number and distribution (Fig. 4Go, A–C), and the distribution of those cells is similar to that of Tpit-positive cells (Fig. 4Go, D–F). It therefore appears that the delay in corticotroph differentiation has fully recovered by e16.5. The delay of corticotroph differentiation had no secondary effect on the expression of other pituitary markers. {alpha}GSU, ßTSH, and GH were normally expressed in the anterior lobe (Fig. 4Go, G–O). Similarly, transcription factors Pitx1, Pit1 (a factor restricted to GH, prolactin, and ßTSH cells), and SF1 (a gonadotroph-restricted factor) were expressed normally in NeuroD1 +/- and -/- pituitaries (Fig. 4Go, P–X). The dosage effect of NeuroD1 on corticotroph differentiation (as revealed by POMC expression) is therefore restricted to the time period when NeuroD1 is detectable in the developing corticotrophs, and this delay appears to be fully recovered beyond that point.



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  		Fig. 4. Recovery of Corticotroph Differentiation at e16.5 in NeuroD1(BETA2)-Deficient Mice

In contrast to early stages of pituitary development, the distribution of POMC cells was similar in wild-type (A), heterozygous (B), and NeuroD1-null mice (C). Tpit expression patterns were also similar (D–F), as were those of {alpha}GSU (G–I), ßTSH (J–L), GH (M–O), which starts to be detectable at e16.5 and those of transcription factors Pit1 (P–R), Pitx1 (S–U), and SF1 (V–X). Sections A–X are sagittal with the embryo facing to the right.

 
Neurogenins (Ngn) 1–3 Are Not Required for POMC Expression
In other systems that require NeuroD1 for their development, NeuroD1 expression depends on prior action of transcription factors of the Ngn family. For example, Ngn1 and/or Ngn2 expression precedes that of NeuroD1 in the nervous system (21, 29, 30) and Ngn3 precedes NeuroD1 in the hypothalamus and pancreas (31, 32). We therefore tested the possibility that such a cascade of neurogenic bHLH transcription factors could also exist in the pituitary for corticotroph differentiation.

To address this question, we first investigated Ngn factors expression in the developing pituitary. Immunohistochemical analysis of mouse pituitaries from e10.5 to e15.5 using anti-Ngn1 and anti-Ngn3 antibodies did not reveal any expression of Ngn1 or Ngn3 (data not shown). However, we detected Ngn2 expression in a small number of nuclei of the anterior part of the pituitary at e12.5 and e13.5 (Fig. 5Go, A and E). Whereas the Ngn2 antibody revealed Ngn2 protein in tissues known to express this gene by in situ hybridization (33, 34) such as in dorsal root ganglia (Fig. 5BGo) or in cerebral cortex (Fig. 5CGo) but not in ventral thalamus for example (Fig. 5DGo), it also suggested the presence of nonnuclear Ngn2 in the periphery of the developing pituitary (Fig. 5AGo). This labeling is likely artifactual because only the nuclear staining was lost in Ngn2-/- pituitaries (Fig. 5FGo).



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  		Fig. 5. Ngn2 Is Expressed during Early Pituitary Development

Ngn2 was detected by immunohistochemistry in a few nuclei (arrowheads) of the pituitary at e12.5 (A) and e13.5 (E). As expected, from published in situ hybridization data (33 34 ), Ngn2 was detected in (B) dorsal root ganglia (drg) and (C) cerebral cortex (cc) but not in (D) ventral thalamus. Only the nuclear signal (arrowheads in A and E) is lost in e13.5 Ngn2-/- pituitary (F); other immunoreactivity is still present in mutant tissues and therefore is artifactual. Sections are sagittal with the embryo facing to the right and ventral at bottom.

 
Because Ngn2 appears to be transiently expressed in the developing pituitary, we investigated its role during pituitary development. Mouse mutants for Ngn1 and Ngn2 genes were investigated for putative delays in corticotroph differentiation as shown in NeuroD1-/- mice. The absence of these Ngn genes did not affect the timing of corticotroph differentiation at e13.5 as assessed by staining for POMC (Fig. 6Go, A and B) or for Tpit (Fig. 6Go, C and D), and it did not alter expression of NeuroD1 (Fig. 6Go, E and F). Similar results were obtained in mice mutant for either Ng1 or Ngn2 (data not shown). The Ngn1 and 2 genes are therefore not upstream regulators of NeuroD1 as they are in neural tissues.



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  		Fig. 6. Ngn1 and 2 Genes Are Not Required for Differentiation of Pituitary Corticotrophs

POMC (A, B, G, H), Tpit (C, D, I, J) and NeuroD1 (E, F) expression patterns are similar in wild-type (A, C, E, G, I) and animal mutants for Ngn1 and Ngn2 (B, D, F, H, J) at e13.5 (A–F) and e15.5 (G–J). Sections are sagittal with the embryo facing to the right.

 
The Ngn genes could have played a role, directly or indirectly, in maintenance of POMC expression beyond the window of NeuroD1 expression and to exclude this possibility, we also assessed POMC expression and corticotroph differentiation at e15.5. Both POMC (Fig. 6Go, G and H) and Tpit (Fig. 6Go, I and J) expression were not affected in Ngn mutant mice. The Ngn1 and 2 genes are therefore not required for definitive corticotroph differentiation. Further work will be required to determine whether other cell-restricted bHLH factors are essential either before or after NeuroD1 for corticotroph differentiation and for POMC transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous work identified two cell-specific regulators of POMC transcription, Tpit and NeuroD1, that constitute early markers of corticotroph differentiation. They are expressed in the corticotroph progenitors, or precorticotrophs, of the ventral part of the anterior lobe just before POMC at e12.5 (Fig. 7AGo). Precorticotrophs express very little, if any, POMC and they clearly form in absence of NeuroD1 (Figs. 2Go, 3Go, and 7BGo) or of Tpit [Fig. 7CGo and (15)]. The present work has defined fetal corticotrophs (e12.5-e15.5) and shown their dependence on NeuroD1. After e15.5 and cessation of NeuroD1 protein expression, definitive or adult corticotrophs are no longer dependent on NeuroD1. Future work will determine whether and which factor(s) take over the role of NeuroD1 in adult corticotrophs.



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  		Fig. 7. NeuroD1 and Tpit Are Both Required for Corticotroph Differentiation, But Not for Commitment

A, Tpit and NeuroD1 expression in precorticotrophs starts at around e12 and they are the first markers of corticotrophs. Terminal corticotroph differentiation occurs at e12.5 when POMC is detected. NeuroD1 is expressed until e15 and it is a marker of early fetal corticotrophs. Another factor may replace it in definitive corticotrophs. B, In NeuroD1-null animals, expression of Tpit in precorticotrophs indicates that commitment has occurred but early corticotrophs do not fully differentiate to express POMC. However, definitive corticotrophs do not express NeuroD1 and do not require it to reach terminal differentiation. C, Mutagenesis of Tpit does not prevent NeuroD1 expression and precorticotroph commitment but Tpit is essential for maintenance/proliferation of adult corticotroph (15 ).

 
The overall effect of NeuroD1 deficiency in the pituitary is a delay in the appearance of corticotrophs that is recovered by e16.5. Therefore, only NeuroD1-expressing fetal corticotrophs are dependent on this factor for POMC expression and differentiation (Fig. 7BGo). Others have suggested that NeuroD1(BETA2) has no effect on corticotroph differentiation (26), but these authors missed the importance of NeuroD1 because they did not investigate its role in early development.

The commitment of pituitary corticotrophs does not require NeuroD1 (Figs. 2Go, 3Go, and 7BGo), nor does it require Tpit (Fig. 7CGo and Ref.15). However, maintenance and/or expansion of adult corticotroph does require Tpit (15) but not NeuroD1. Does NeuroD1 only play a transient role for early onset of POMC expression or is it replaced by another factor around e15? Other tissues that require neurogenic bHLH factors for their development or differentiation sequentially express different members of this transcription factor family (21). Our data indicate that the Ngn1, Ngn2, and Ngn3 genes are not essential for pituitary corticotroph differentiation (Fig. 6Go), despite limited expression of Ngn2 in the developing pituitary (Fig. 5Go). The control of corticotroph NeuroD1 expression thus appears to be different from the pancreas and brain where Ngn genes precede and control NeuroD1 expression (21, 31, 32). It is possible that other neurogenic bHLH or other types of transcription factors take over after e15 the early role played by NeuroD1: indeed, it is at about e15 that the developing pituitary starts to receive portal blood and signals from the hypothalamus (35, 36). Other classes of transcription factors may thus be induced in response to signals and responsible for maintenance of POMC transcription.

The requirement for NeuroD1 during early differentiation is not absolute, and we could detect a few POMC-expressing cells in the ventral part of the anterior lobe in NeuroD1-/- pituitaries (Figs. 2Go and 3Go). This indicates that ventral and dorsal precorticotrophs of the anterior pituitary are different, possibly because of ventral signals that are stimulatory for corticotroph differentiation or because of dorsal signals that may be inhibitory. Various signals such as bone morphogenetic proteins and fibroblast growth factor 8 have been proposed to influence corticotroph differentiation, but divergent conclusions have resulted on their putative involvement depending on experimental paradigm (10, 11). Irrespective of those possibilities, it appears that NeuroD1 may set the sensitivity of precorticotrophs to these signals.

The present work clearly indicates that NeuroD1 does not contribute to control of Tpit expression (Figs. 2Go and 3Go), and conversely, the opposite was also shown for NeuroD1expression in Tpit-/- mice (15). Thus, Tpit and NeuroD1 appear to be regulated completely independently of each other, both being required as positive regulators of POMC transcription and corticotroph differentiation. Interestingly, commitment of precorticotrophs is not controlled by either of these factors individually, and future work will determine whether they may do so jointly. The independent action of Tpit and NeuroD1 on corticotrophs is suggestive of separate extracellular signals for control of NeuroD1 and Tpit expression. We currently do not know what these signals may be, but their identification will be essential to understand early events of cell fate decisions during pituitary differentiation.

How do the developmental roles of NeuroD1 and Tpit relate to their transcriptional activity on the POMC promoter? We have shown that these two factors can functionally interact on the POMC promoter for transcriptional activation. The synergistic activation by bHLH factors and Tpit indicated that these two factors are not redundant for POMC transcription because they are not redundant for differentiation. Although bHLH factors and Tpit can interact physically, the presence of Pitx1 is required for their synergistic activation of the POMC promoter. This is similar to the ßLH promoter where Egr and SF1 interact with Pitx1 for transcriptional activation (37, 38). Because Tpit and bHLH factors interact individually with Pitx1, it suggests that tissue-specific regulation of POMC transcription depends on a complex containing Pitx1, Tpit, and NeuroD1/Pan1. This complex may be the point of convergence of different early differentiation signals and/or pathways acting through Tpit and NeuroD1 for transcriptional activation of POMC and onset of corticotroph differentiation at e12.5. Analysis of the control of Tpit and NeuroD1 expression should lead to identification of earlier upstream regulators of corticotroph differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice Antibodies and Immunohistochemistry
Knockout mice for NeuroD1(BETA2) and for Ngn1 and 2 were described in (27, 30), respectively. Production of NeuroD1 (13), Tpit (14), and Pitx1 (39) antibodies was described elsewhere. The Ngn1 and Ngn2 antibodies were obtained from Chemicon (Temecula, CA). The Ngn3 antibody provided by Dr. M. German was described (40). Pituitary hormone antibodies were obtained through the National Institutes of Health National Hormone and Pituitary Program, except for the ACTH antibody (Cortex Biochem, San Leandro, CA). Immunohistochemistry was performed as described (14).

Plasmids and Transfection Assays
Plasmids were described previously (12, 14). CV1 and GH3 cells were grown and transfected as described (14).

Pulldown Assay
MBP-LacZ, MBP-Tpit, and MBP-Tpit deletion mutant were purified from Escherichia coli BL21 after the manufacturer’s recommendations (New England Biolabs, Beverly, MA), and 500 ng of each fusion protein coupled to amylose beads were used in all assays. Pan1 and NeuroD1 were synthesized in vitro using [35S]methionine and the TnT-coupled transcription-translation rabbit reticulocyte lysate system (Promega, Madison, WI). Pull-downs were performed as described (41).


   ACKNOWLEDGMENTS
 
We are thankful to Dr. A. F. Parlow of the NIH Pituitary Hormone Program for providing antibodies against various pituitary hormones. We thank Dr. David Anderson, Caltech, for Ngn1 mutant mice and Dr. Michael S. German (University of California-San Francisco, San Francisco, California), for the anti-Ngn3 antibody. We also thank Dr. Ken Morohashi (Okazaki, Japan) for the SF-1 antibody and Dr. Carol Schuurmans (University of Calgary, Alberta) for help with Ngn1-/-, Ngn2-/- embryos. We are indebted to Annie Vallée for preparation of tissue sections and to Lise Laroche for her expert secretarial assistance.


   FOOTNOTES
 
B.L. was suported by a fellowship from the Canadian Institutes for Health Research (CIHR). M.J.T. is supported by NIH grants. This work was supported by the National Cancer Institute of Canada with funds provided by the Canadian Cancer Society.

Abbreviations: bHLH, Basic helix-loop-helix; e, embryonic day; GSU, glycoprotein hormone subunit; Ngn, neurogenin; POMC, proopiomelanocortin; SF1, steroidogenic factor 1.

Received for publication April 9, 2003. Accepted for publication January 8, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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