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.2002-0392
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, N.-A.
Right arrow Articles by Melmed, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, N.-A.
Right arrow Articles by Melmed, S.
Molecular Endocrinology 17 (5): 959-966
Copyright © 2003 by The Endocrine Society

Pituitary Corticotroph Ontogeny and Regulation in Transgenic Zebrafish

Ning-Ai Liu, Haigen Huang, Zhongan Yang, Wiebke Herzog, Matthias Hammerschmidt, Shuo Lin and Shlomo Melmed

Department of Medicine, Cedars-Sinai Research Institute (N.-A.L., S.M.), University of California Los Angeles School of Medicine, Los Angeles, California 90048; Department of Molecular, Cell and Developmental Biology (H.H., Z.Y., S.L.), University of California Los Angeles, Los Angeles, California 90095; and Hans-Spemann-Laboratorium (W.H., M.H.), Max-Planck-Institut fuer Immunbiologie Stuebeweg 51, D-79108 Freiburg, Germany

Address all correspondence and requests for reprints to: Shlomo Melmed, Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048. E-mail: Melmed{at}CSMC.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and {alpha}MSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10-5 M) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PITUITARY PROOPIOMELANOCORTIN (POMC) lineage cells include ACTH-producing corticotrophs and {alpha}MSH-producing melanotrophs. Anterior lobe corticotrophs are the first hormone-producing cells to differentiate in the embryonic adenohypophysis. Human fetal ACTH-expressing cells are first evident at 5 wk gestation within Rathke’s pouch and then appear within the rudimentary pars intermedia (PI) during later gestation. In the rat fetus, corticotrophs arise from anterior pituitary primordia between embryonic day (e)13 and e14 and from PI on e16 (1, 2). The human postnatal hypophysis lacks a distinct PI, but corticotrophs scattered in the zona intermedia, the junction zone between the anterior and posterior pituitary, are considered a functional equivalent of those derived from the PI. Starting from young adulthood, some corticotrophs in the zona intermedia proliferate into the posterior pituitary lobe and become more prominent with aging, a phenomenon referred to as basophilic invasion (2, 3, 4).

In the adult human and rodent, corticotrophs principally reside in the anterior lobe, where they comprise 10–20% of the cell population. The majority of human ACTH-dependent Cushing’s disease arises from tumors of the anterior pituitary (2, 3, 5). Cushing’s disease in the horse, however, usually results from tumors of the intermediate lobe, and only rarely results from those of the anterior lobe (6, 7). Approximately 30% of canine Cushing’s disease exhibits tumors of the PI. In addition to typical melanotrophs, the canine PI contains a substantial percentage of a second cell type that stains intensely for ACTH but not for {alpha}MSH. The concentration of bio- and immunoreactive ACTH in the dog PI is 50% of that in the pars distalis (8).

CRH, adrenal glucocorticoids, and cytokines are the major physiological regulators of adult corticotroph secretion (9, 10, 11, 12, 13). At e15.5, soon after the initiation of their differentiation, fetal rat corticotrophs already respond to physiological regulators in an adult-like manner, indicating that embryonic corticotrophs possess functional regulatory mechanisms before structural maturation of the hypothalamic-hypophyseal portal system (14).

Studies on the developmental effects of signaling molecules and transcription factors suggest that corticotroph ontogeny involves molecular pathways distinct from those of other pituitary cell lineages (15, 16). However, developmentally essential, extrinsic signaling and/or intrinsic lineage-restricted factors have thus far not been confirmed for corticotroph specification and differentiation. Transcription factors such as Neuro D1, Nur77, Pitx1/2, and T-pit/Tbx19 activate specific cis-elements on the POMC promoter and target POMC expression to corticotrophs (17, 18, 19, 20, 21, 22, 23). Mutations of the human T-pit/Tbx19 gene are related to defective ACTH production, suggesting that T-pit/Tbx19 plays an important role in corticotroph development (17). Cytokines such as leukemia-inhibitory factor, acting through the Janus kinase-signal transducers and activators of transcription (STAT) pathway, stimulate POMC expression, expand cells of the POMC lineage, and decrease Lhx3 expression and the number of pituitary gonadotrophs, somatotrophs, and lactotrophs (24, 25, 26, 27, 28).

Zebrafish, Danio rerio, are an ideal vertebrate model for studying lineage-specific pathways of endocrine organ development (29, 30, 31, 32, 33, 34). However, zebrafish pituitary embryonic development has yet to be documented. To monitor the dynamic ontogeny of the pituitary POMC lineage during zebrafish development, we generated germline transgenic zebrafish expressing green fluorescent protein (GFP) under control of regulatory sequences of the zebrafish POMC gene. Pituitary GFP expression in each transgenic line recapitulated the pattern of endogenous pituitary POMC protein and was specifically targeted to the corticotroph lineage, as shown by immunohistochemical studies. The results indicate that zebrafish POMC progenitor cells arise within the most anterior structure bilateral to the neural ridge midline before formation of the nascent pituitary. Anterior pituitary POMC-GFP expression is selectively suppressed by treatment of transgenic embryos with dexamethasone. Despite some temporal and spatial differences, zebrafish corticotroph development shares a conserved basic mechanism with higher vertebrates, providing useful insights into vertebrate pituitary development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Zebrafish POMC Promoter Contains Conserved Regulatory Elements Similar to Higher Vertebrates
The zebrafish POMC gene contains a 5' regulatory sequence followed by three exons and two introns. Exon 1 is 85 bp long and is not translated. Exon 2 contains 143 bp corresponding to the entire hydrophobic signal peptide and the first eight amino acids of POMC N-terminal glycopeptide. Most POMC coding sequences are located in exon 3, which encodes N-terminal glycopeptide, joining peptide, ACTH, {alpha}MSH, and ß-lipotropin. Regions encoding ACTH, {alpha}MSH, ß-lipotropin, and N-terminal glycopeptide are 75, 100, 40, and 35% homologous to those of mouse and human, respectively (data not shown). The 5' promoter sequence from -451 to +61 contains putative response elements important for POMC expression, as identified in the rat POMC gene. However, unlike rat POMC promoter, which has a high affinity and a low affinity binding site for STAT3 (26), zebrafish POMC promoter only contains one conserved STAT3 binding site, TT(5N)AA, in positions -439 to -431. Interestingly, the negative glucocorticoid responsive element (nGRE) is located in the cDNA strand of zebrafish POMC gene promoter (Fig. 1AGo). The complete zebrafish POMC genomic sequence was submitted to GenBank (accession no. BankIt510482 AY212967) and was searched against a zebrafish genomic DNA sequence database, all of which suggests the presence of a single POMC gene.



View larger version (54K):
[in this window]
[in a new window]
  		Figure 1. Zebrafish POMC Gene Structure

A, Schematic representation of genomic structure of zebrafish POMC gene and GFP reporter construct used for generating transgenic zebrafish. Exons 1–3 of zebrafish POMC gene are shown with open boxes. The 5', 3' noncoding region and introns are shown as a straight line. GFP reporter construct contains a 1006-bp 5' fragment including the entire exon 1 and the first 22 bp of exon 2 of zebrafish POMC gene as a promoter. Promoter sequence -451 to + 61 contains putative regulatory elements as previously identified in the rat. Position +1 corresponds to the transcription initiation site. Underlined sequences represent known cis elements for transcription factors. Bold sequences are those conserved between zebrafish and rat. NurRE, Nur responsive element; E Box, binding site for heterodimers containing NeuroD; Pitx1, binding site for bicoid-related homeodomain transcription factors; T Box, binding site for T Box factor, T-pit/Tbx19; AP1, binding site for CRH-inducible transcription factors. A putative STAT3 binding site within the nGRE is indicated with a line above the sequence. B, Hematoxylin-eosin staining of a cross-section of adult zebrafish brain. The pituitary gland is located ventral to the hypothalamus. p, Pituitary; h, hypothalamus.

 
In parallel to the genetic conservation of POMC, a pituitary prehormone, the zebrafish pituitary gland shares anatomical and histological similarities with that of other vertebrates. However, in adult zebrafish, the pituitary is located ventral to the hypothalamus, connected closely without a distinct stalk structure (Fig. 1BGo).

Zebrafish POMC Lineage Starts to Differentiate in the Anterior Neural Ridge before Pituitary Formation
Germline transgenic zebrafish were generated with a GFP reporter construct containing a 1006-bp 5' fragment including entire exon 1 and the first 22 bp of exon 2 of zebrafish POMC gene as promoter (Fig. 1AGo). The transparent transgenic embryos expressing GFP targeted to pituitary POMC-expressing cells facilitated monitoring of the dynamic POMC lineage ontogeny in live zebrafish.

In POMC-GFP transgenic zebrafish, GFP-positive cells start to become evident at the most anterior portion of the neural ridge, lateral to both sides of the neural tube, as early as 18–20 h post fertilization (hpf; Fig. 2Go, A and B). At this stage, the appearance of GFP-positive cells is asymmetric, showing a single-cell mass on the left side and two smaller groups on the right side. This asymmetric pattern is reproducible from three independent transgenic lines. GFP fluorescence was also observed in the olfactory and otic anlage, which completely disappeared after 24 hpf. Between 24 and 48 hpf, GFP-positive cells form a single-cell cluster and subsequently organize as distinct anterior and posterior domains (Fig. 2CGo). At this stage, the pattern of POMC-driven GFP expression in pituitary primordial faithfully recapitulated that observed for pituitary POMC mRNA expression in wild-type zebrafish embryos, as shown by in situ hybridization (Fig. 2DGo). The transgene expression is clearly restricted to the embryonic pituitary, whereas wild-type embryos also show additional POMC expression in hypothalamic regions. This is consistent with findings in the mouse that distal 5' sequences of the POMC gene up to 13 kb are required for hypothalamic expression of POMC protein (35). After 48 hpf, a second group of GFP-expressing cells appears in the pituitary anlage along the anterior-posterior axis. In 5-d-old larvae, expression patterns of POMC-GFP transgene and endogenous POMC appear similar, except that anterior and posterior expression domains of the POMC transcripts are of similar size, whereas, for the transgene, the posterior domain is smaller (Fig. 2Go, E and F). The anterior and posterior expression pattern of POMC-GFP transgene continues and becomes more apparent in 10-d-old larvae (Fig. 2GGo).



View larger version (69K):
[in this window]
[in a new window]
  		Figure 2. POMC Expression in Germline Transgenic and Wild-Type Zebrafish

In transgenic zebrafish, POMC-driven GFP expression first appears within bilateral anterior portions of the neural ridge at 20 hpf. At this stage, GFP is also expressed in the olfactory and otic anlage, which later disappears (A, lateral view; B, ventral view). Pituitary GFP-positive cells fuse into a single-cell mass between 24 and 48 hpf (C, dorsal view). At this stage, GFP expression recapitulates endogenous pituitary POMC expression as shown by in situ hybridization with POMC mRNA in wild-type zebrafish (D, ventral view). In 5-d-old larvae, pituitary expression patterns of POMC-GFP transgene (E, ventral view) and endogenous POMC mRNA (F, ventral view) are similar. The two longitudinal arcs of positive cells anterior of the two pituitary domains in F correspond to endorphin-generating cells in the hypothalamus. This pattern continues and becomes more apparent in 10-d-old larvae (G, sagittal cryosection of transgenic zebrafish pituitary; high magnification). di, Diencephalon; h, hypothalamus; of, olfactory; op, otic primordia; p, pituitary. A–G, Left, anterior; right, posterior. A and G, Top, dorsal; bottom, ventral.

 
POMC-GFP Expression in Germline Transgenic Zebrafish Is Specifically Targeted to Both Anterior and Posterior Corticotroph Groups But Not to Melanotrophs
POMC-expressing cells of the adenohypophysis give rise to ACTH-generating corticotrophs and MSH-generating melanotrophs. To study whether the observed reduced size of the posterior GFP expression domain might be due to differential transgene expression in corticotrophs vs. melanotrophs, we performed immunohistochemistry to localize endogenous POMC and {alpha}MSH on sagittal sections of POMC-GFP transgenic zebrafish with anti-ACTH and anti-{alpha}MSH antisera. Immunohistochemistry with monoclonal mouse anti-GFP antibody was also performed, which showed the same expression pattern of GFP as that observed by fluorescent microscopy (data not shown). Using rabbit anti-{alpha}MSH antiserum, endogenous pituitary {alpha}MSH expression was shown to be selectively present in a region posterior to the anterior group of GFP-positive cells in 13-d-old transgenic zebrafish. In this region, the majority of GFP signal is posterior to, without significant overlap with, the area of {alpha}MSH immunoreactivity (Fig. 3Go, A, C, E, and G). Within the anterior pituitary, GFP expression recapitulated that of endogenous POMC protein, shown by immunohistochemistry with a polyclonal rabbit antihuman ACTH antibody, which also cross-reacts with POMC protein. In contrast, the posterior pituitary GFP signal only partially colocalizes with the posterior portion of ACTH/POMC immunoreactivity (Fig. 3Go, B, D, F, and H), whereas the anterior POMC- and MSH-positive portion is devoid of transgene expression. This expression pattern was confirmed on multiple sections derived from three different transgenic lines, suggesting that transgene expression is specifically lacking in the melanotroph lineage. By 1 month, the distribution of GFP-positive cells becomes more diffuse within the pituitary. Endogenous ACTH expression continues to colocalize with GFP expression in the anterior pituitary and only partially in the posterior portion of the gland (Fig. 4Go, A–C). At this stage, {alpha} glycoprotein subunit ({alpha}GSU)-expressing cells are apparent and distributed ventrally to POMC-expressing cells (Fig. 4DGo). Taken together, these results indicate that the transgenic POMC promoter fragment is only active within corticotrophs, and GFP expression is specifically targeted to the corticotroph lineage in the transgenic zebrafish, whereas melanotroph POMC expression is regulated by distinct elements not present in the transgenic fragment. During embryonic development, zebrafish pituitary corticotrophs are evident as both an anterior and posterior group, reflecting a general anterior-posterior polarity of the early zebrafish pituitary anlage, rather than a dorsoventral polarity, as described for mammals. By 1 month, this anterior-posterior polarity is less apparent, and POMC-GFP-positive cells become more diffusely distributed along the entire anterior-posterior length of the gland, whereas they are excluded from ventral regions, characterized by {alpha}GSU expression.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Pituitary Expression of the POMC-GFP Transgene, Endogenous POMC, and {alpha}MSH in 13-d-Old Transgenic Zebrafish Larvae

A–D, Saggital cryosection of zebrafish larvae. A and B, Pituitary GFP expression under fluorescent microscopy. C, Imminohistochemistry of the same cryosection from A using rabbit anti-{alpha}MSH antiserum. D, Immunohistochemistry of the same cryosection from B using rabbit antihuman ACTH polyclonal antibody. E, A and C superimposed. F, B and D superimposed. G and H, Enlargement of pituitary area from E and F. Within the posterior pituitary region, GFP expression is posterior to endogenous {alpha}MSH. GFP expression colocalizes with endogenous ACTH/POMC expression in the anterior pituitary and partially overlaps with the posterior group of endogenous ACTH/POMC-expressing cells. Green, GFP expression; red, rhodamine-conjugated antirabbit IgG.

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. Pituitary Expression of the POMC-GFP Transgene, Endogenous POMC, and {alpha}GSU Gene in 1-Month-Old Transgenic Zebrafish Larvae

A–D, Sagittal cryosection of zebrafish pituitary (left, anterior; right, posterior; top, dorsal; bottom, ventral). A, GFP expression under fluorescent microscopy. B, Immunohistochemistry of the same cryosection from A using rabbit antihuman ACTH polyclonal antibody. C, A and B superimposed. D, Immunohistochemistry of a different cryosection from the same pituitary as in A and B, using rabbit antimouse {alpha}GSU antibody. Image was then superimposed with that of GFP expression. Green, GFP expression; red, rhodamine-conjugated antirabbit IgG.

 
POMC-GFP Expression in the Anterior Group of Corticotrophs Is Down-Regulated by Dexamethasone
To examine whether zebrafish coticotrophs respond to glucocorticoid during early development, live POMC-GFP transgenic embryos were treated continuously with 50 µM dexamethasone starting from 3–4 hpf. Overall pituitary GFP expression was suppressed by dexamethasone at 2 d post fertilization (dpf; Fig. 5Go, A and B). By 5 dpf, there was a significant reduction of GFP expression in the anterior group of cells (Fig. 5Go, C and D). Green fluorescence intensity within anterior and posterior pituitary clusters was measured for semiquantitative analysis to determine the dose response of GFP expression to dexamethasone treatment. As show in Fig. 5EGo, the ratio of GFP intensity in anterior and posterior pituitary clusters decreased by more than 60% after embryos were treated with 25 µM dexamethasone for 5 d. At higher doses, dexamethasone causes abnormal embryonic development, including pericardial sac enlargement, and death before 72 hpf (data not shown). Immunohistochemistry using anti-ACTH antibody on embryos treated with 25 µM dexamethasone for 5 d also showed selective suppression of endogenous ACTH expression in the anterior group of corticotrophs (Fig. 5Go, C and D, inset). These results suggest that embryonic corticotrophs respond to glucocorticoid regulation soon after their initial differentiation. Dexamethasone selectively down-regulates POMC-GFP expression in corticotrophs clustered in the anterior domain, while having less or no effect on posterior corticotrophs.



View larger version (56K):
[in this window]
[in a new window]
  		Figure 5. Anterior Pituitary POMC-GFP Expression is Down-Regulated by Dexamethasone

A–D, Live transgenic zebrafish embryos were continuously treated with or without dexamethasone starting from 3–4 hpf and observed by fluorescent microscopy at different developmental stages. By 2 dpf, pituitary POMC-GFP transgene expression is down-regulated by dexamethasone (A, without; B, with 50 µM dexamethasone). At 5 dpf, anterior pituitary GFP expression is suppressed in treated vs. nontreated groups (C, without; D, with 50 µM dexamethasone). Inset in C and D, Immunohistochemistry using ACTH antibody. E, Live transgenic zebrafish embryos were treated continuously with increasing dexamethasone concentrations starting from 3–4 hpf. By 5 dpf, the intensity of GFP expression in the anterior and posterior pituitary clusters was obtained separately using Openlab software for measurement of the area of interest. The ratio of anterior vs. posterior GFP intensity in each individual embryo was calculated and analyzed using GraphPad (San Diego, CA) Prism. GFP intensity ratios are mean ± SEM (n = 4–10). **, P = 0.0007; ***, P < 0.0001, compared with 0 µM control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This transgenic analysis of zebrafish POMC lineage ontogeny indicates that zebrafish POMC-GFP transgene is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 hpf. POMC-GFP-positive cells then form a single-cell mass within the pituitary anlage after 24 hpf, and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Dexamethasone down-regulates corticotroph POMC expression soon after the onset of their differentiation (2 dpf) and selectively suppresses POMC expression within the anterior group of corticotrophs by 5 dpf.

The chick and amphibian adenohypophysis arises from the anterior ventral neural ridge during early development (36, 37). In human and rodents, the anterior pituitary lobe arises from oral ectoderm, which makes contact with the embryonic midline ventral diencephalon (22). No experimental data on localization of the presumptive territory of the mammalian adenohypophysis at the open neural stage are available. However, it has been suggested that mammalian pituitary cell commitment may occur very early, before formation of the adenohypophyseal pituitary anatomic anlage, Rathke’s pouch (38). In zebrafish, pituitary ontogeny has not been well documented. Herzog et al. (39) have recently reported studies on whole-mount in situ hybridizations detecting endogenous POMC transcripts. Like the transgene, endogenous POMC expression in wild-type zebrafish is initiated in a bilateral fashion in the anterior neural ridge, moves medially and caudally, and splits into an anterior and a posterior part within the pituitary anlage. POMC also displays specific additional expression in endorphin-generating neurons of the hypothalamus. Studies by Glasgow et al. (40) on zebrafish pituitary expression of Lim3, a LIM homeodomain transcription factor essential for development of Rathke’s pouch and determination of pituitary cell lineages other than corticotrophs, provide a valuable marker for the early stages of zebrafish pituitary development. In the zebrafish pituitary anlage, Lim3-positive cells appear at the 21-somite stage, lateral to the midline and adjacent to the anterior ventral diencephalons in an asymmetric pattern. At 28 hpf, the two lateral Lim3-positive regions fuse into a single pituitary cluster that then translocates caudally through the second day of development. The pattern of Lim3 expression suggests that zebrafish pituitary specification occurs in cells lateral to the midline, rather than within the Rathke’s pouch-like structure (40). In the POMC-GFP transgenic zebrafish described here, expression of POMC-GFP transgene within pituitary primordia is similar to that of endogenous POMC and Lim3 in wild-type zebrafish. GFP-positive cells were first observed within the most anterior structure bilateral to the midline at 18–20 hpf; distribution of GFP-expressing cells is asymmetric at this stage. Between 24 and 48 hpf, GFP-positive cells form a single cluster. Subsequently, they organize as distinct anterior and posterior domains. The pattern of endogenous POMC and POMC-GFP transgene expression supports the notion, derived from studies on Lim3 expression, that zebrafish pituitary determination occurs asymmetrically and laterally within a region closely related to the anterior neural ridge, when the pituitary is still organized in a placodal-like fashion and before posterior migration into the head. The temporal parallel of Lim3 and POMC expression indicates that zebrafish pituitary POMC lineage development is Lim3 independent, similar to the pattern observed in higher vertebrates.

Embryonic and adult corticotroph distributions within the pituitary vary among species (2, 3, 5, 6, 7, 8). Unlike the human and rodent, most dog and horse Cushing’s disease arises from ACTH hypersecretion of the PI. Human adult pituitary contains no distinct intermediate lobe. However, ACTH-expressing cells still exist in the junctional zone between the anterior and posterior pituitary lobes. It has been suggested that human ACTH-secreting pituitary adenomas originate from corticotrophs within the anterior lobe or the region equivalent to the intermediate lobe, which respond differently to regulators of hormone secretion such as glucocorticoids (41). Human corticotroph basophilic invasion has also been implicated as the possible origin of the extremely rare pituitary ACTH-secreting adenomas arising from within the posterior lobe (4).

In POMC-GFP transgenic zebrafish, pituitary GFP-positive cells appear as anterior and posterior groups after 48 hpf. GFP expression exactly colocalized with POMC immunoreactivity within the anterior group of cells. However, the posterior group of GFP-positive cells only constituted a partial ACTH/POMC immunoreactivity. Further immunohistochemical study using a {alpha}MSH antiserum showed that the posterior group of GFP-positive cells is posterior to, but not significantly colocalized with, {alpha}MSH immunoreactivity. Therefore, it seems that the zebrafish posterior pituitary contains two major cell types, corresponding to distinct melanotrophs and corticotrophs. POMC-GFP germline transgenic zebrafish contain GFP specifically targeted to the corticotroph lineage. This selectivity may be due to absence of an endogenous melanotroph-specific activating sequence within the promoter of the reporter construct used. In the mouse, a transgene containing rat 5' flanking sequences of less than 300 bp, corresponding to the region of zebrafish POMC promoter used in this study, targets reporter protein expression in both corticotrophs and melanotrophs (42). However, a 529-bp sequence in the similar region of Xenopus POMC promoter is primarily activated in intermediate-lobe melanotrophs (43). In keeping with this study on zebrafish POMC expression, there is an apparent species difference in activation of the pituitary POMC promoter. Due to the lack of specific markers for zebrafish melanotrophs, we cannot completely exclude the possibility that the posterior group of GFP-positive cells may contain some populations of melanotrophs. Interestingly, the distinction of an anterior and a posterior group of embryonic corticotrophs becomes less obvious in 1-month-old zebrafish. This observation is similar to the observed involution of the human embryonic PI, leading to the zona intermedia, that is continuous with and morphologically indistinguishable from the posterior lobe (3).

The temporal relation between anterior and posterior pituitary corticotrophs during zebrafish development is still unclear. It is also unknown whether one group of corticotrophs is derived from the other or whether each arises independently. Further time-lapse studies on the germline transgenic zebrafish described here should provide information to answer such questions.

In summary, this study of corticotroph development in live transgenic zebrafish indicates that the zebrafish pituitary POMC lineage shares conserved molecular, genetic, and developmental mechanisms with higher vertebrates. However, the precise temporal and spatial patterns may be divergent among different species. POMC regulatory sequence can target GFP reporter specifically to corticotroph cells within the pituitary and allow a dynamic monitoring of their ontogeny in a live system. This transgenic model therefore offers a unique system for in vivo study of molecular and genetic mechanisms underlying corticotroph development and function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Promoter Isolation and Generation of POMC-GFP Reporter Construct
A zebrafish genomic Pi-derived artificial chromosome (PAC) library was screened by PCR using the following pair of primers: POMC3, 5'-CGAGGAAGGATGAGTGAGTG-3'; and POMC4, 5'-GTGCTATTGTTAACATTGCC-3'. Five positive clones were identified. Restriction digestion of genomic DNA from one of the positive clones (clone 591) yielded a 3574-bp HindIII fragment identified by Southern blot and subcloned into pBluescript KS- (Stratagene, La Jolla, CA). Sequence analysis revealed that the HindIII fragment contained the entire coding region, a 586-bp 5' flanking region and a 377-bp 3' flanking region of POMC. A 1006-bp 5' fragment was isolated by PCR amplification using the T3 vector primer and a primer corresponding to the first 22 bp of POMC gene exon 2 (5'-ATTGGATCCTCACTCCCCTCACCAT-3'). The PCR fragment was digested with BamHI and ligated to a modified GFP reporter gene (44) to generate the POMC-GFP reporter construct.

Generation of Germline POMC-GFP Transgenic Zebrafish
Germline transgenic zebrafish were generated with the POMC-GFP reporter construct as previously described (45). Briefly, PCR products of the POMC-GFP reporter construct (without vector DNA) were purified using a GENECLEAN III kit (Bio 101, Vista, CA) and resuspended in 5 mM Tris, 0.5 mM EDTA, 0.1 M KCl at a final concentration of 100 µg/ml. Fertilized embryos from wild-type zebrafish were injected at the one-cell stage. Microinjections were carried out five times to generate approximately 300 surviving embryos. Injected founder fish were mated to wild-type fish and their progeny observed for GFP expression under a Carl Zeiss (Thornwood, NY) fluorescent microscope. Founder fish that produced GFP-positive eggs were considered transgenic and bred to generate F1 heterozygotes and F2 homozygotes.

Whole-Mount RNA in Situ Hybridization
Sense and antisense digoxigenin-labeled RNA probes were generated from a cDNA clone of the zebrafish POMC gene using a DIG/Genius 4 RNA Labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). RNA in situ hybridizations and plastic sections of zebrafish whole-mount embryos were performed as previously described (46).

Immunohistochemistry
Cryosection and immunohistochemistry of zebrafish adults and embryos were performed as previously described (46). ACTH immunoreactivity was detected with a rabbit polyclonal antibody against human ACTH (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA; 1:1000), and secondary rhodamine-conjugated antirabbit IgG was from Sigma (St. Louis, MO). Rabbit anti-{alpha}MSH serum was obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA; 1:500).

Fluorescent Microscopy and Imaging
Wild-type and transgenic embryos were examined at various developmental stages under a fluorescein isothiocyanate filter on a Carl Zeiss microscope (Axioplan-2). Live embryo and tissue section images were generated with an Axiocam video system (Carl Zeiss). Fluorescence intensity of POMC-GFP positive cells was measured by the area of interest function in Openlab software (Improvision, Lexington, MA).

Maintenance of Zebrafish and Drug Treatment
Zebrafish embryos were maintained and raised as previously described (46). Embryo staging was carried out according to Kimmel et al. (47). Dexamethasone (Sigma, D2915) was dissolved in distilled water at a stock concentration of 1 mM, diluted in fish medium, and added to live embryos at the stages indicated.


   ACKNOWLEDGMENTS
 
We thank H. Pan for assistance in identifying and maintaining the transgenic zebrafish and L. Zhang for technical support on immunohistochemistry.


   FOOTNOTES
 
This work was supported by NIH Grants RO1-CA-75979 (to S.M.), RO1-RR-13227 (to S.L.), and the Doris Factor Molecular Endocrinology Laboratory.

GenBank accession number for POMC genomic sequence: bankit510482 AY212967.

Abbreviations: dpf, Days post fertilization; e, embryonic day; GFP, green fluorescent protein; {alpha}GSU, {alpha} glycoprotein subunit; hpf, hours post fertilization; nGRE, negative glucocorticoid responsive element; PI, pars intermedia; POMC, proopiomelanocortin; STAT, signal transducers and activators of transcription.

Received for publication November 21, 2002. Accepted for publication January 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Watanabe YG, Daikoku S 1979 An immunohistochemical study on the cytogenesis of adenohypophysial cells in fetal rats. Dev Biol 68:557–567[CrossRef][Medline]
  2. Ikeda H, Suzuki J, Sasano N, Niizuma H 1988 The development and morphogenesis of the human pituitary gland. Anat Embryol 178:327–336[CrossRef][Medline]
  3. Doniach I 1985 Histopathology of the pituitary. Clin Endocrinol Metab 14:765–789
  4. Kuebber S, Ropte S, Hori A 1990 Proliferation of adenohypophyseal cells into posterior lobe. Their normal anatomical condition and possible neoplastic potentiality. Acta Neurochir (Wien) 104:21–26
  5. Leroux P, Pelletier G 1984 Radioautographic study of binding and internalization of corticotropin-releasing factor by rat anterior pituitary corticotrophs. Endocrinology 114:14–21[Abstract/Free Full Text]
  6. Orth DN, Holscher MA, Wilson MG, Nicholson WE, Plue RE, Mount CD 1982 Equine Cushing’s disease: plasma immunoreactive proopiolipomelanocortin peptide and cortisol levels basally and in response to diagnostic tests. Endocrinology 110:1430–1441[Abstract/Free Full Text]
  7. Wilson MG, Nicholson WE, Holscher MA, Sherrell BJ, Mount CD, Orth DN 1982 Proopiolipomelanocortin peptides in normal pituitary, pituitary tumor, and plasma of normal and Cushing’s horses. Endocrinology 110:941–954[Abstract/Free Full Text]
  8. Halmi NS, Peterson ME, Colurso GJ, Liotta AS, Krieger DT 1981 Pituitary intermediate lobe in dog: two cell types and high bioactive adrenocorticotropin content. Science 211:72–74[Abstract/Free Full Text]
  9. Melmed S 2001 Series introduction. The immuno-neuroendocrine interface. J Clin Invest 108:1563–1566[CrossRef][Medline]
  10. Muglia LJ, Jacobson L, Weninger SC, Karalis KP, Jeong K, Majzoub JA 2001 The physiology of corticotropin-releasing hormone deficiency in mice. Peptides 22:725–731[CrossRef][Medline]
  11. Rivier C, Brownstein M, Spiess J, Rivier J, Vale W 1982 In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin, ß-endorphin, and corticosterone. Endocrinology 110:272–278[Abstract/Free Full Text]
  12. Vale W, Vaughan J, Smith M, Yamamoto G, Rivier J, Rivier C 1983 Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells. Endocrinology 113:1121–1131[Abstract/Free Full Text]
  13. Venihaki M, Dikkes P, Carrigan A, Karalis KP 2001 Corticotropin-releasing hormone regulates IL-6 expression during inflammation. J Clin Invest 108:1159–1166[CrossRef][Medline]
  14. 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]
  15. Ericson J, Norlin S, Jessell TM, Edlund T 1998 Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary. Development 125:1005–1015[Abstract]
  16. Treier M, Gleiberman AS, O’Connell SM, Szeto DP, McMahon JA, McMahon AP, Rosenfeld MG 1998 Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 12:1691–1704[Abstract/Free Full Text]
  17. 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]
  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. Poulin G, Turgeon B, Drouin J 1997 NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 17:6673–6682[Abstract]
  20. Philips A, Lesage S, Gingras R, Maira MH, Gauthier Y, Hugo P, Drouin J 1997 Novel dimeric Nur77 signaling mechanism in endocrine and lymphoid cells. Mol Cell Biol 17:5946–5951[Abstract]
  21. Lamonerie T, Tremblay JJ, Lanctot C, Therrien M, Gauthier Y, Drouin J 1996 Ptx1, a bicoid-related homeo box transcription factor involved in transcription of the pro-opiomelanocortin gene. Genes Dev 10:1284–1295[Abstract/Free Full Text]
  22. Scully KM, Rosenfeld MG 2002 Pituitary development: regulatory codes in mammalian organogenesis. Science 295:2231–2235[Abstract/Free Full Text]
  23. Liu J, Lin C, Gleiberman A, Ohgi KA, Herman T, Huang HP, Tsai MJ, Rosenfeld MG 2001 Tbx19, a tissue-selective regulator of POMC gene expression. Proc Natl Acad Sci USA 98:8674–8679[Abstract/Free Full Text]
  24. Akita S, Conn PM, Melmed S 1996 Leukemia inhibitory factor (LIF) induces acute adrenocorticotrophic hormone (ACTH) secretion in fetal rhesus macaque primates: a novel dynamic test of pituitary function. J Clin Endocrinol Metab 81:4170–4178[Abstract/Free Full Text]
  25. Bousquet C, Melmed S 1999 Critical role for STAT3 in murine pituitary adrenocorticotropin hormone leukemia inhibitory factor signaling. J Biol Chem 274:10723–10730[Abstract/Free Full Text]
  26. Bousquet C, Zatelli MC, Melmed S 2000 Direct regulation of pituitary proopiomelanocortin by STAT3 provides a novel mechanism for immuno-neuroendocrine interfacing. J Clin Invest 106:1417–1425[Medline]
  27. Yano H, Readhead C, Nakashima M, Ren SG, Melmed S 1998 Pituitary-directed leukemia inhibitory factor transgene causes Cushing’s syndrome: neuro-immune-endocrine modulation of pituitary development. Mol Endocrinol 12:1708–1720[Abstract/Free Full Text]
  28. Auernhammer CJ, Melmed S 2001 The central role of SOCS-3 in integrating the neuro-immunoendocrine interface. J Clin Invest 108:1735–1740[CrossRef][Medline]
  29. Maures TJ, Duan C 2002 Structure, developmental expression, and physiological regulation of zebrafish IGF binding protein-1. Endocrinology 143:2722–2731[Abstract/Free Full Text]
  30. Maures T, Chan SJ, Xu B, Sun H, Ding J, Duan C 2002 Structural, biochemical, and expression analysis of two distinct insulin-like growth factor I receptors and their ligands in zebrafish. Endocrinology 143:1858–1871[Abstract/Free Full Text]
  31. Yee NS, Yusuff S, Pack M 2001 Zebrafish pdx1 morphant displays defects in pancreas development and digestive organ chirality, and potentially identifies a multipotent pancreas progenitor cell. Genesis 30:137–140[CrossRef][Medline]
  32. Perz-Edwards A, Hardison NL, Linney E 2001 Retinoic acid-mediated gene expression in transgenic reporter zebrafish. Dev Biol 229:89–101[CrossRef][Medline]
  33. Huang H, Vogel SS, Liu N, Melton DA, Lin S 2001 Analysis of pancreatic development in living transgenic zebrafish embryos. Mol Cell Endocrinol 177:117–124[CrossRef][Medline]
  34. Zeng S, Gong Z 2002 Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary. Gene 294:45[CrossRef][Medline]
  35. Young JI, Otero V, Cerdan MG, Falzone TL, Chan EC, Low MJ, Rubinstein M 1998 Authentic cell-specific and developmentally regulated expression of pro-opiomelanocortin genomic fragments in hypothalamic and hindbrain neurons of transgenic mice. J Neurosci 18:6631–6640[Abstract/Free Full Text]
  36. Couly GF, Le Douarin NM 1985 Mapping of the early neural primordium in quail-chick chimeras. I. Developmental relationships between placodes, facial ectoderm, and prosencephalon. Dev Biol 110:422–439[CrossRef][Medline]
  37. Eagleson GW, Jenks BG, Van Overbeeke AP 1986 The pituitary adrenocorticotropes originate from neural ridge tissue in Xenopus laevis. J Embryol Exp Morphol 95:1–14[Medline]
  38. Dubois PM, ElAmraoui A 1995 Embryology of the pituitary gland. Trends Endocrinol Metab 6:1–7
  39. Herzog W, Zeng X, Lele Z, Sonntag C, Ting J, Chang C, Hammerschmidt M 2003 Adenohypophysis formation in the zebrafish and its dependence on Sonic Hedgehog. Dev Biol 254:36–49[CrossRef][Medline]
  40. Glasgow E, Karavanov AA, Dawid IB 1997 Neuronal and neuroendocrine expression of lim3, a LIM class homeobox gene, is altered in mutant zebrafish with axial signaling defects. Dev Biol 192:405–419[CrossRef][Medline]
  41. Lamberts SW, de Lange SA, Stefanko SZ 1982 Adrenocorticotropin-secreting pituitary adenomas originate from the anterior or the intermediate lobe in Cushing’s disease: differences in the regulation of hormone secretion. J Clin Endocrinol Metab 54:286–291[Abstract/Free Full Text]
  42. Liu B, Hammer GD, Rubinstein M, Mortrud M, Low MJ 1992 Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice. Mol Cell Biol 12:3978–3990[Abstract/Free Full Text]
  43. Jansen EJR, Holling TM, van Herp F, Martens GJM 2002 Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis. FEBS Lett 516:201–207[CrossRef][Medline]
  44. Long Q, Meng A, Wang H, Jessen JR, Farrell MJ, Lin S 1997 GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124:4105–4111[Abstract]
  45. Lin S 2000 Transgenic zebrafish. Methods Mol Biol 136:375–383[Medline]
  46. Westerfield M 1995 The zebrafish book. Eugene, OR: University of Oregon Press
  47. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF 1995 Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310[Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
B. Guner, A. T. Ozacar, J. E. Thomas, and R. O. Karlstrom
Graded Hedgehog and Fibroblast Growth Factor Signaling Independently Regulate Pituitary Cell Fates and Help Establish the Pars Distalis and Pars Intermedia of the Zebrafish Adenohypophysis
Endocrinology, September 1, 2008; 149(9): 4435 - 4451.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Qi, J. A. Ranish, X. Zhu, A. Krones, J. Zhang, R. Aebersold, D. W. Rose, M. G. Rosenfeld, and C. Carriere
Atbf1 is required for the Pit1 gene early activation
PNAS, February 19, 2008; 105(7): 2481 - 2486.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
V. F. Bumaschny, F. S. J. de Souza, R. A. Lopez Leal, A. M. Santangelo, M. Baetscher, D. H. Levi, M. J. Low, and M. Rubinstein
Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence
Mol. Endocrinol., November 1, 2007; 21(11): 2738 - 2749.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
X. Zhu, A. S. Gleiberman, and M. G. Rosenfeld
Molecular Physiology of Pituitary Development: Signaling and Transcriptional Networks
Physiol Rev, July 1, 2007; 87(3): 933 - 963.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. T. To, S. Hahner, G. Nica, K. B. Rohr, M. Hammerschmidt, C. Winkler, and B. Allolio
Pituitary-Interrenal Interaction in Zebrafish Interrenal Organ Development
Mol. Endocrinol., February 1, 2007; 21(2): 472 - 485.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
R. J. Weil, A. O. Vortmeyer, L. K. Nieman, H. L. DeVroom, J. Wanebo, and E. H. Oldfield
Surgical Remission of Pituitary Adenomas Confined to the Neurohypophysis in Cushing's Disease
J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2656 - 2664.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
I M McGonnell and R C Fowkes
Fishing for gene function - endocrine modelling in the zebrafish.
J. Endocrinol., June 1, 2006; 189(3): 425 - 439.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
N.-A. Liu, Q. Liu, K. Wawrowsky, Z. Yang, S. Lin, and S. Melmed
Prolactin Receptor Signaling Mediates the Osmotic Response of Embryonic Zebrafish Lactotrophs
Mol. Endocrinol., April 1, 2006; 20(4): 871 - 880.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
H.-M. Pogoda, S. von der Hardt, W. Herzog, C. Kramer, H. Schwarz, and M. Hammerschmidt
The proneural gene ascl1a is required for endocrine differentiation and cell survival in the zebrafish adenohypophysis
Development, March 15, 2006; 133(6): 1079 - 1089.
[Abstract] [Full Text] [PDF]

Home page
 Mol Biol EvolHome page
F. S. J. de Souza, V. F. Bumaschny, M. J. Low, and M. Rubinstein
Subfunctionalization of Expression and Peptide Domains Following the Ancient Duplication of the Proopiomelanocortin Gene in Teleost Fishes
Mol. Biol. Evol., December 1, 2005; 22(12): 2417 - 2427.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
F. S. J. de Souza, A. M. Santangelo, V. Bumaschny, M. E. Avale, J. L. Smart, M. J. Low, and M. Rubinstein
Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting
Mol. Cell. Biol., April 15, 2005; 25(8): 3076 - 3086.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
W. Herzog, C. Sonntag, B. Walderich, J. Odenthal, H.-M. Maischein, and M. Hammerschmidt
Genetic Analysis of Adenohypophysis Formation in Zebrafish
Mol. Endocrinol., May 1, 2004; 18(5): 1185 - 1195.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
G. Nica, W. Herzog, C. Sonntag, and M. Hammerschmidt
Zebrafish pit1 Mutants Lack Three Pituitary Cell Types and Develop Severe Dwarfism
Mol. Endocrinol., May 1, 2004; 18(5): 1196 - 1209.
[Abstract] [Full Text] [PDF]

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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, N.-A.
Right arrow Articles by Melmed, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, N.-A.
Right arrow Articles by Melmed, S.

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