The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis

General and Comparative Endocrinology 177 (2012) 315–321

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Review

The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis Bruce G. Jenks ⇑, Miyuki Kuribara, Adhanet H. Kidane, Bianca M.R. Kramer, Eric W. Roubos, Wim J.J.M. Scheenen Department of Cellular Animal Physiology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands

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Article history: Available online 10 January 2012 Keywords: BDNF POMC a-MSH Autocrine ERK DCLK-short TrkB p75NTR Pars intermedia Melanotrope IRES

a b s t r a c t Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in the mammalian pituitary gland is well documented its functional significance remains obscure. Studies on the pars intermedia of the pituitary of the amphibian Xenopus laevis have shown that BDNF is produced by the neuroendocrine melanotrope cells, its expression is physiologically regulated, and the melanotrope cells themselves express receptors for the neurotrophin. The neurotrophin has been shown to act as an autocrine factor on the melanotrope to promote cell growth and regulate gene expression. In doing so BDNF supports the physiological function of the cell to produce and release a-melanophore-stimulating hormone for the purpose of adjusting the animal’s skin color to that of its background. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction and overview of BDNF in the vertebrate pituitary gland Brain-derived neurotrophic factor (BDNF) is a 14 KDa protein belonging to the neurotrophin family of growth factors. It supports neuron survival and stimulates the growth and differentiation of neurons [8,13]. BDNF also plays an essential role in the regulation of long-term potentiation, where there is a strengthening of synapses between neurons in support of memory processes [42,52,64]. Despite its name, BDNF is not only found in the brain but also in many peripheral organs [32,65,80] including the rat pituitary gland. Here, BDNF and/or its mRNA is found in both the anterior [14,23,25,36,37,58,68] and intermediate lobe [14,26,36,37,53,58] as well as in nerve terminals of the pars nervosa [14]. The above studies on the rat do not report the cell-type expressing BDNF in the anterior lobe, with the exception of Höpker et al. [25] who showed colocalization of BDNF-immunoreactivity with thyroid-stimulating hormone, and Rage et al. [58], reporting colocalization of the neurotrophin in the anterior pituitary lobe with S-100 protein, a marker in the pituitary for folliculo-stellate ⇑ Corresponding author. Address: Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands. Fax: +31 243652714. E-mail address: [email protected] (B.G. Jenks). 0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2012.01.001

cells. The presence of the BDNF-specific tropomyosin-receptorkinase B (TrkB) receptor in the rat pituitary gland [23,36,37,58] suggests that the neurotrophin could have a local autocrine/paracrine function. Immobilization stress increases anterior lobe BDNF mRNA [23,68], suggesting involvement of BDNF in the regulation of the hypothalamo–pituitary–adrenal axis. This idea is supported by the observation that adrenalectomy decreases the amount of BDNF mRNA in the anterior pituitary [37]. In the rat pars intermedia BDNF-immunoreactivity is found throughout the lobe, reflecting BDNF’s presence in melanotrope cells [58]. In vitro addition of BDNF to intermediate lobe fragments from young (but not middle-aged or old) rats weakly stimulates secretion of a-melanocyte-stimulating hormone (a-MSH), an observation favoring the idea of an autocrine/paracrine function for the neurotrophin [58]. Höpker et al. [26] noted a depletion of immunoreactive BDNF from incubated intermediate lobe cells, which could be prevented by adding the dopamine receptor agonist apomorphine during the dissociation and cultivation of the cells. This result indicates that melanotrope cells sequester BDNF in the regulated secretory pathway, because secretory activity of rat melanotropes is known to be under inhibitory dopaminergic control [4]. From the above it is evident that BDNF and its receptor are present in the rat pituitary gland, but the functional significance of these observations is elusive. To our knowledge, the only other

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vertebrate species where pituitary BDNF has been studied is the amphibian, Xenopus laevis. Here, BDNF-immunoreactivity is found in melanotropes throughout the intermediate lobe [40] and in neurohemal nerve terminals ending in the pars nervosa [5,73]. Because the function of the Xenopus melanotrope cell is well established (see below) this is an ideal cell-type to investigate the functional significance of endocrine BDNF. This review introduces the Xenopus melanotrope cell as a model to study neuroendocrine integration, and then gives an overview of our studies on the role of BDNF in this integrative process. 2. The Xenopus melanotrope cell as a model for neuroendocrine integration Amphibian intermediate lobe melanotrope cells are responsible for regulating skin color in a process known as ‘‘background adaptation’’. They produce and release a-MSH when an animal is on a black background and, through the action of circulating a-MSH on dermal melanophores, the black pigment melanin in the melanophores disperses and consequently the skin darkens. When the animal is placed on a white background release of the hormone is inhibited, the melanin pigment becomes punctuate in a perinuclear position, and the skin blanches [2,77]. The neuroendocrine reflex regulating this background adaptation process involves the eyes, with the optic information concerning color of background being processed by various brain centers that regulate the secretory activity of the melanotrope cell via neuronal projections to the pituitary gland (for reviews see: [41,63,71]). This regulation involves the action of a number of inhibitory and stimulatory neurotransmitters and neuropeptides acting directly on the endocrine cell. Details of this regulation have been extensively studied in two amphibian species, Rana ridibunda and Xenopus laevis; these studies reveal that the two species have developed similar strategies for the regulation of the secretory activity of the melanotrope cell (for reviews see: [21,31,50,62,63,74]). For the Xenopus melanotrope, many of the regulatory transmitters, acting through their specific receptors, converge on the adenylyl cyclase system to regulate the production of cyclic-AMP (see Fig. 1). This second messenger activates protein kinase A (PKA), which in turn regulates membrane ion channel complexes to stimulate the influx of Ca2+ through voltage-operated Ca2+ channels (VOCC). This Ca2+ signal induces exocytosis of a-MSH and, through the process of Ca2+-induced Ca2+ release, it mobilizes Ca2+ from intracellular Ca2+ stores, thereby creating a self-propagating Ca2+ wave that travels through the cytoplasm and enters the nucleus. Activation of the melanotrope cell not only stimulates release of a-MSH but also increases the transcription and translation of proopiomelanocortin (POMC), the precursor protein of a-MSH. The intracellular signaling cascades regulating Xenopus melanotrope cell function have been extensively reviewed [30,29]. While cyclic-AMP and Ca2+ are the two major intracellular second messengers generated by the signal transduction machinery of Xenopus melanotropes, surprisingly, the promoter of POMC lacks cyclic-AMP responsive elements and Ca2+ responsive elements [16,28]. POMC expression in these cells is regulated, at least in part, through indirect mechanisms involving the immediate early genes c-Fos [35] and Nur77 [45]. 3. The Xenopus melanotrope cell expresses BDNF In situ hybridization showed the presence of BDNF mRNA in the Xenopus melanotropes and immunohistochemistry extended this finding to the protein level [40]. Western blot analysis revealed the presence of both the precursor protein (proBDNF) and mature BDNF. Analysis of the subcellular distribution of BDNF, using a combination of high-pressure freezing, cryosubstitution and

immunoelectron microscopy, demonstrated it to be sequestered within secretory granules of Xenopus melanotrope cells [76]. These same studies, using triple immunogold-labeling, established BDNF coexistence with POMC and a-MSH within these granules. This intragranular location and coexistence implies that BDNF follows the same regulated secretory pathway as POMC and its end-products, including a-MSH. Presumably, BDNF is released together with a-MSH from the actively secreting melanotropes of black-adapted animals. BDNF is an extremely potent protein (inducing biological responses at the sub-nanomolar level e.g. [18,56] and we have found that the amount released from the melanotrope cell remains below the detection limit of our BDNF assays (Jenks et al., unpublished).

4. Expression of BDNF in Xenopus melanotrope cells is physiologically regulated Quantitative reverse-transcriptase polymerase chain reaction (Q-RT-PCR) revealed a 25-fold increase in BDNF mRNA in melanotropes of black compared to white background-adapted Xenopus [40]. In these studies the forward and reverse primers for the PCR were within the BDNF coding sequence, and therefore total BDNF mRNA was measured. The BDNF gene, however, possesses multiple promoters, each capable of producing a specific transcript [1,24,57]. To determine if there is promoter-specific expression of BDNF in Xenopus, we first characterized the structure of the Xenopus gene [35]. For this purpose BDNF transcripts of the X. laevis brain were sequenced and then mapped to the Xenopus tropicalis genome to determine exon–intron structure. This showed that Xenopus BDNF contains seven exons, giving rise to seven exon-specific transcripts, with each transcript containing the protein encoding exon VII (Fig. 2A). Q-RT-PCR analysis revealed transcriptspecific expression in melanotropes during background adaptation [35]. The most highly upregulated transcript was transcript IV which displayed 130-fold increase in expression in animals on black background; in contrast transcript VII (a 50 extension of exon VII), showed no difference between white- and black-adapted animals (Fig. 2B). Clearly the expression pattern of BDNF transcripts is highly promoter-specific. Analysis of the nucleotide sequence in the promoter region upstream of Xenopus exon IV revealed two potential Ca2+ responsive elements (CaRE1 and CaRE2) and a potential cyclic-AMP responsive element (CRE). This promoter region shows high sequence homology with that of rat and human BDNF [35]. In rat cortical neurons these elements are involved in the regulation of expression of BDNF transcript IV [10,69,70]. The high expression of transcript IV displayed by melanotropes of animals on black background fits well with the importance of Ca2+ and cyclic-AMP signaling in such cells. The Xenopus promoter region also has a potential binding site for a repressor transcription factor, namely a down-stream responsive element (DRE) that partially overlaps with the CRE element [35]. In mammals DRE functions as a binding site for the Ca2+ binding protein DRE antagonist modulator (DREAM), which in its Ca2+-bound form, lifts itself from the DRE site to promote gene expression [7]. DREAM is involved in Ca2+-dependent regulation of the expression of mammalian BDNF [51,60] and the same is likely true for BDNF expression in the Xenopus melanotrope. In the latter, Ca2+ waves generated at the membrane and entering the nucleus [38,67] might not only act on CaRE1 and CaRE2 but also on DREAM to induce BDNF expression. Many of the BDNF transcripts of Xenopus possess long upstream untranslated regions (uUTRs), a phenomenon associated with inefficient mRNA translation [72]. Lengthy uUTRs are also found in BDNF transcripts of zebrafish [24], human [57] and rat and mouse [1]. Moreover, all BDNF transcripts have AUG start codons upstream to the translation initiation codon for pre–proBDNF. In

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Fig. 1. Schematic overview of the regulation of the melanotrope cell of Xenopus laevis. The secretion of a-MSH is regulated by multiple neurotransmitters, some of which are indicated in the figure, together with their receptors that activate (cyan) or inhibit (red) adenylyl cyclase. Cyclic-AMP binds to the regulatory subunit (R) of PKA leading to the dissociation of the catalytic subunit (C) which then activates components of Ca2+ signaling machinery on the plasma membrane, resulting in opening of voltage-operated Ca2+ channels (VOCC) and influx of Ca2+. This Ca2+ signal stimulates exocytosis and mobilizes intracellular Ca2+ stores to initiate a self-propagating wave of Ca2+ that ultimately enters the nucleus. In the nucleus Ca2+ and the catalytic subunit of PKA are responsible for activating transcription factors (e.g. CREB, USF1/2, CaRF1) that act on responsive elements (grey boxes) within the promoters of various genes. Among the first genes to be activated are the immediate early genes c-Fos and Nur77, along with BDNF, POMC and proBDNF mRNA are translated at the rough endoplasmic reticulum (RER) and co-packaged into secretory granules. Mature BDNF acts as an autocrine factor, with TrkB signaling activating ERK that subsequently phosphorylates DCLK-short to translocate into the nucleus. In the nucleus DCLK-short stimulates, through unknown pathways, the expression of POMC; it is likely also involved in the regulation of other genes, such as those governing cell growth. ERK may act via Nur77 on POMC expression, possibly in a compartment regulated by PKA associated with AKAP. Abbreviations: AKAP, A kinase-associated protein; AP1, activator protein 1 promoter site; BDNF, brain-derived neurotrophic factor; b, b-adrenergic receptor; b/c, b/c subunit of G proteins; CaRE, calcium-responsive element; CaRF, calcium-responsive transcription factor; CRE, cyclicAMP-responsive element; CRH, corticotropin-releasing hormone; D2, dopamine D2 receptor; DCLK, doublecortin-like kinase; DRE, down-stream responsive element; DREAM, DRE antagonist modulator; ERK, extracellular-signal-regulated kinase; Gb, GABAb receptor; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase-activating polypeptide; POMC, proopiomelanocortin; PLCc, phospholipase Cc; PKA, protein kinase A; R1, CRH receptor 1; USF, upstream stimulatory factor; V1, VPAC1 receptor; VOCC, voltageoperated Ca2+ channel; Y1, NPY Y1 receptor. (Adapted from Kidane [33] and Kuribara [43].

Xenopus the number of upstream AUGs varies from one, in transcript II, to eleven in transcript VII (Fig. 2A). These AUGs represent initiation sites for upstream open reading frames (uORFs) many of which have been conserved among mammals, Xenopus and zebrafish [31,35]. The presence of uORFs is often associated with poor translation efficiency [72], possibly reflecting a protective mechanism whereby potent proteins are inefficiently produced to avoid harmful overproduction (cf. [22]). In case of Xenopus BDNF transcripts I and IV, the presence of the uUTR drastically reduces translation efficiency [35], probably reflecting the presence of the many uORFs. We suggest that the reduced translational efficiency imposed by uORFs could represent a protective mechanism [35]. Possibly these uORFs could be bypassed in the translation process in e.g. stressed cells through the presence of a so-called internal ribosome entry site (IRES) within the BDNF transcripts [31]. While it remains to be determined if Xenopus BDNF transcripts possess IRESes,

interestingly, the presence of IRESes has been reported for some human BDNF transcripts [79]. 5. Xenopus melanotrope cells express receptors for BDNF RT-PCR established the presence of mRNA of the full length TrkB receptor (TrkB.FL) and the truncated form of this receptor (TrkB.T) in the Xenopus neurointermediate lobe [34]. This same study also showed that there is expression of the p75 neurotrophin receptor (p75NTR), a receptor with low affinity for neurotrophins but can form a complex with Trk receptors, such as TrkB, to generate high-affinity binding sites for neurotrophins [11,19]. In situ hybridization shows that TrkB.FL and p75NTR mRNAs are present in the melanotrope cells [34], and the same is probably true for TrkB.T mRNA (due to the small size of the unique sequence in this receptor it is not possible to produce specific in situ probes). Q-RT-PCR

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Fig. 2. (A) Schematic representation of the Xenopus BDNF gene showing its promoter (P) specific transcripts (T1–T7). Each transcript possesses the pre–proBDNF coding exon VII (dark blue indicates BDNF coding region within the exon). Transcript VII is a 50 extension of exon VII (VII 50 ext). AUG codons upstream of the AUG start codon for pre– proBDNF are indicated by vertical red lines within the exons. (B) Effect of transfer of white-background adapted Xenopus to a black background on the relative level of BDNF transcripts IV and VII. For both, the level of mRNA in white background control animals (WA) was set at 1. Black-adapted (BA) control animals stayed minimally 3 weeks on black background. Data are ±SEM (n = 5) and asterisks indicate significant difference from the WA-group. ⁄P < 0.005; ⁄⁄P < 0.0005 (data from Kidane et al. [35]).

reveals that the concentrations of TrkB.T and p75NTR mRNA are about 3-fold higher in neurointermediate lobes of black- than of white-background adapted Xenopus [34]. We suggest that the amount of p75NTR might set the sensitivity of the melanotrope cell to BDNF. In this scenario animals on a black background produce more p75NTR, which drives the formation of high-affinity TrkB.FL–p75NTR complexes, thus increasing the sensitivity of the cell to BDNF. The physiological significance of the higher expression of TrkB.T in animals on black background is unknown. While this receptor, lacking the kinase domain, has been thought to act as negative effector of the full-length receptor [3,6,17], there is evidence that it may have unique signaling properties [12,55,61]. 6. BDNF promotes growth of Xenopus melanotrope cells in an autocrine way Xenopus melanotrope cells are primarily under inhibitory control, and ‘‘spontaneously’’ produce and release a-MSH when removed from hypothalamic control and cultured in vitro [66,75]. Because BDNF is co-sequestered with a-MSH in secretory granules [76] such disinhibited cells would presumably release BDNF. Therefore, in order to demonstrate autocrine BDNF action, a series of experiments were designed in which different strategies were used to block endogenous BDNF (see Fig. 3). As read-out parameter in these studies, reported by Kuribara et al. [46], the cell diameter was measured as criterion for cell growth (because of the association of BDNF with growth processes). In all cases, during in vitro culture control cells increased in cell diameter (Fig. 3), indicating that in vivo the melanotropes in the intermediate lobe are under (probably hypothalamic) inhibition, which is lifted when the cells

are isolated and placed in vitro. To block potential signaling by release of endogenous BDNF we sequestered the neurotrophin by adding specific BDNF antiserum to the incubation medium or, alternatively, excess soluble chimeric TrkB receptor fragment (containing the BDNF binding domain of the receptor). Both treatments blocked the increase in cell size, indicating involvement of endogenous BDNF in melanotrope cell growth. To demonstrate involvement of the TrkB receptor in this growth process we used cyclotraxin-B, a cyclic peptide that selectively blocks BDNF binding to its TrkB receptor [9]. Again, cell growth was blocked, thus establishing involvement of the TrkB receptor. The attenuation of cell growth by the various treatments could, in all cases, be overcome by adding excess BDNF [46]. Clearly, endogenous BDNF is acting in an autocrine, stimulatory way on the Xenopus melanotrope cells. In stimulating cell growth, one of the most common pathways used by BDNF involves the activation of the mitogen-activated protein kinase (MAPK) cascade [27,59]. One component of this cascade is extracellular signal-regulated kinase (ERK), for which there is a well known inhibitor, U0126 [20]. In our in vitro analysis of melanotrope cell growth we found that addition of U0126 to the incubation medium blocks melanotrope cell growth [46] (Fig. 3), indicating the likely involvement of the MAPK cascade in melanotrope BDNF-signaling. 7. ERK regulates DCLK-short to act on POMC gene expression Further evidence for the participation of ERK in the regulation of Xenopus melanotrope functioning comes from experiments showing that this enzyme is activated (phosphorylated) in melanotropes when Xenopus is placed on a black background [48]. Treatment of

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Fig. 3. Scheme of strategies used to block (yellow bolt) the action of endogenous BDNF released from the Xenopus melanotrope cell, together with effects of each treatment on the in vitro growth of the melanotropes, as assessed by determining cell diameter. Cells from black-adapted Xenopus were incubated for 3 days in complete medium or in medium containing either antiserum to BDNF or TrkB fragment (each to sequester released BDNF), cyclotraxin-B (to block BDNF binding to TrkB) or U0126 (to block BDNF signaling through MAPK). Asterisks indicate significant difference with the control group at 0 days (P < 0.05; n = 3) (data from Kuribara et al. [46]).

melanotropes in vitro with U0126 markedly reduced this ERK phosphorylation and lowered transcription as well as translation of POMC. In further elucidating the signaling cascades leading to the regulation of POMC expression the possible involvement of another kinase, double cortin-like kinase short (DCLK-short), was studied. In mammals BDNF-induced synaptic consolidation is associated with an up-regulation of DCLK-short suggesting a role for this kinase in the regulation of cellular plasticity [49]. The observation that DCLK-short contains a target sequence for its phosphorylation by ERK prompted us to determine if this kinase plays a role in regulating events in Xenopus melanotropes. DCLK-short mRNA is indeed expressed by the melanotropes and is up-regulated in black-adapted animals [47]. Moreover, the activation of these cells is accompanied by an increase in phosphorylated DCLK-short (pDCLK-short). In these cells pDCLK-short is translocated from the cytoplasm into the nucleus, a process that is prevented by incubation with the ERK blocker U0126 [47]. Studies with transfected Xenopus melanotrope cells show that introducing a mutation in the ERK-phosphorylation site of DCLK-short dramatically reduced translocation of DCLK-short whereas expression of POMC mRNA in melanotropes transfected with this mutated DCLK construct was 2 times lower than in melanotropes transfected with the wild-type construct [47]. Altogether it can be concluded that DCLK-short in the Xenopus melanotrope cell is controlled at both the level of its gene expression and ERK-mediated phosphorylation, and that the kinase is involved in regulating POMC gene expression. The pathway(s) DCLK-short uses inside the nucleus to act on the POMC gene remain(s) to be determined. Another potential signaling pathway of melanotrope ERK is via the transcription factor Nur77, which in mammals is one of the most important transcription factors regulating POMC expression in corticotrope cells (for review, [28]). In corticotropes, cyclicAMP sets in motion a signaling cascade that activates ERK, with ERK then phosphorylating and thus activating Nur77 to promote POMC expression [39]. It has already been shown that there is an upregulation in Nur77 expression in melanotropes of black adapted Xenopus [45]. In view of the importance of cyclic-AMP in the regulation of intracellular events in Xenopus melanotropes, it is conceivable that in this cell the cyclic-AMP- and ERK-dependent

mechanisms are operating on Nur77 to regulate POMC expression. Interestingly, there seem to be at least two compartments of PKA signaling in Xenopus melanotropes, one associated with the regulation of Ca2+ signaling and the other, compartmentalized through interaction with A kinase-anchoring protein (AKAP), involved in the regulation of other cellular functions [15]. Possibly, PKA associated with ERK signaling could be part of an AKAP/PKA signaling domain. 8. BDNF mobilizes intracellular Ca2+ to act on its own expression Besides signaling through the MAP-kinase cascade, the TrkB receptor is known to signal through activation of phospholipase Cc (PLCc) with the generation of inositol triphosphate (IP3) [54]. In melanotropes of black-adapted Xenopus Ca2+ is mobilized through a Ca2+-induced Ca2+ mechanism [67]. As this mechanism is thought to work through the activation of IP3 receptors on intracellular Ca2+ stores, possible effects of BDNF on Ca2+ dynamics in the melanotrope were investigated. BDNF caused a dose-dependent increase in Ca2+ oscillation frequency and induced a Ca2+ transient in Ca2+-free medium [44]. This transient was absent in melanotropes treated in vitro with thapsigargin (which depletes intracellular Ca2+ stores) or with 2-aminoethoxydiphenyl borate (a blocker of IP3 receptors), thus indicating that BDNF stimulates release of Ca2+ from IP3-sensitive intracellular Ca2+ stores. Thapsigargin treatment also inhibited the expression of BDNF transcript IV [44], the highly upregulated transcript in melanotropes of black-adapted animals [35]. This suggests that BDNF stimulates its own expression through a Ca2+ mobilization mechanism. Interestingly, it has been reported that BDNF stimulates its own expression in rat cortical neurons and this stimulation also concerns transcript IV [78]. 9. Concluding remarks While the significance of the presence of BDNF outside the CNS is rather enigmatic, it is clear that the protein has an important physiological function in a pituitary endocrine cell. In Xenopus

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melanotropes the neurotrophin acts as an autocrine factor to weakly stimulate aMSH secretion [40], strongly stimulate the translation of POMC [40], promote its own expression [44], and stimulate cell growth [46]. Several challenges remain in defining the actions of melanotrope BDNF. Among these will be determining which genes fall under BDNF regulation in promoting cell growth and identifying the intracellular and intranuclear pathways involved in this process. In this endeavor the melanotrope cell may make a good model system to further establish the function of DCLK-short in the regulation of gene expression. Finally, at the level of translation, the melanotrope cell may someday play a role in establishing the importance of IRESes to BDNF signaling. Acknowledgments We would like to thank our many Bachelor and Master students who participated in these studies. We also gratefully acknowledge the contributions of our technical staff, Peter Cruijsen, Debby Tilburg-Ouwens, Frouwke Kuijpers-Kwant and Tony Coenen and thank Ron Engels for the excellent animal care and management of our aquatic facility. The contribution of Liangchun Wang to the immuno-EM studies is also acknowledged. This work was supported by grants from the Space Research Organization Netherlands (SRON), The Netherlands Organization for Scientific Research (NWO) and by a Fulbright Fellowship to A.H.K. References [1] T. Aid, A. Kazantseva, M. Piirsoo, K. Palm, T. Timmusk, Mouse and rat BDNF gene structure and expression revisited, J. Neurosci. Res. 85 (2007) 525–535. [2] J.T. Bagnara, M.E. Hadley, Chromatophores and Color Change: the Comparative Physiology of Animal Pigmentation, Prentice Hall, Eaglewood Cliff, NJ, USA, 1973. [3] S. Biffo, N. Offenhäuser, B.D. Carter, Y.A. Barde, Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development, Development 121 (1995) 2461–2470. [4] M. Boschetti, F. Gatto, M. Arvigo, D. Esposito, A. Rebora, M. Talco, M. Albertelli, E. Nazzari, U. Goglia, F. Minuto, D. Ferone, Role of dopamine receptors in normal and tumoral pituitary corticotropic cells and adrenal cells, Neuroendocrinology 92 (2010) 17–22. [5] M. Calle, L. Wang, F.J. Kuijpers, P.M. Cruijsen, L. Arckens, E.W. Roubos, Brainderived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin, J. Neuroendocrinol. 18 (2006) 454– 465. [6] L. Carim-Todd, K.G. Bath, G. Fulgenzi, S. Yanpallewar, D. Jing, C.A. Barrick, J. Becker, H. Buckley, S.G. Dorsey, F.S. Lee, L. Tessarollo, Endogenous truncated TrkB. T1 receptor regulates neuronal complexity TrkB kinase receptor function in vivo, J. Neurosci. 29 (2009) 678–685. [7] A.M. Carrión, W.A. Link, F. Ledo, B. Mellström, J.R. Naranjo, DREAM is a Ca2+ regulated transcriptional repressor, Nature 398 (1999) 80–84. [8] B.J. Casey, C.E. Glatt, M. Tottenham, F. Soliman, K. Bath, D. Amso, M. Altemus, S. Pattwell, R. Jones, L. Levita, B. McEwen, A.M. Magariños, M. Gunnar, K.M. Thomas, J. Mezey, A.G. Clark, B.L. Hempstead, F.S. Lee, Brain-derived neurotrophic factor as a model system for examining gene by environment interactions across development, Neuroscience 164 (2009) 108–120. [9] M. Cazorla, A. Jouvenceau, C. Rose, J.P. Guilloux, C. Pilon, A. Dranovsky, J. Prémont, Cyclotraxin-B the first highly potent selective TrkB inhibitor has anxiolytic properties in mice, PLoS One 19 (2010) e9777. [10] W.G. Chen, A.E. West, X. Tao, G. Corfas, M.N. Szentirmay, M. Sawadogo, C. Vinson, M.E. Greenberg, Upstream stimulatory factors are mediators of Ca2+ responsive transcription in neurons, J. Neurosci. 23 (2003) 2572–2581. [11] Y. Chen, J. Zeng, L. Cen, Y. Chen, X. Wang, G. Yao, W. Wang, W. Qi, K. Kong, Multiple roles of the p75 neurotrophin receptor in the nervous system, J. Int. Med. Res. 37 (2009) 281–288. [12] A. Cheng, T. Coksaygan, H. Tang, R. Khatri, R.J. Balice-Gordon, M.S. Rao, M.P. Mattson, Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism, J. Neurochem. 100 (2007) 1515–1530. [13] S. Cohen-Cory, A.H. Kidane, N.J. Shirkey, S. Marshak, Brain-derived neurotrophic factor and the development of structural neuronal connectivity, Dev. Neurobiol. 70 (2010) 271–288. [14] J.M. Conner, Q. Yan, S. Varon, Distribution of brain-derived neurotrophic factor in the rat pituitary gland, Neuroreport 7 (1996) 1937–1940. [15] G.J. Corstens, R. van Boxtel, M.J. van den Hurk, E.W. Roubos, B.G. Jenks, The effects of disruption of A kinase anchoring protein-protein kinase A association on protein kinase A signalling in neuroendocrine melanotroph cells of Xenopus laevis, J. Neuroendocrinol. 18 (2006) 477–483.

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