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.
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ABSTRACT
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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 1820 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
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.
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INTRODUCTION
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PITUITARY PROOPIOMELANOCORTIN (POMC) lineage cells include ACTH-producing corticotrophs and
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 Rathkes 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 1020% of the cell population. The majority of human ACTH-dependent Cushings disease arises from tumors of the anterior pituitary (2, 3, 5). Cushings 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 Cushings 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
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.
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RESULTS
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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,
MSH, and ß-lipotropin. Regions encoding ACTH,
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. 1A
). 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.

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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 13 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.
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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. 1B
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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. 1A
). 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 1820 h post fertilization (hpf; Fig. 2
, 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. 2C
). 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. 2D
). 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. 2
, E and F). The anterior and posterior expression pattern of POMC-GFP transgene continues and becomes more apparent in 10-d-old larvae (Fig. 2G
).

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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. AG, Left, anterior; right, posterior. A and G, Top, dorsal; bottom, ventral.
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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
MSH on sagittal sections of POMC-GFP transgenic zebrafish with anti-ACTH and anti-
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-
MSH antiserum, endogenous pituitary
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
MSH immunoreactivity (Fig. 3
, 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. 3
, 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. 4
, AC). At this stage,
glycoprotein subunit (
GSU)-expressing cells are apparent and distributed ventrally to POMC-expressing cells (Fig. 4D
). 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
GSU expression.

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Figure 3. Pituitary Expression of the POMC-GFP Transgene, Endogenous POMC, and MSH in 13-d-Old Transgenic Zebrafish Larvae
AD, 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- 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 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.
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Figure 4. Pituitary Expression of the POMC-GFP Transgene, Endogenous POMC, and GSU Gene in 1-Month-Old Transgenic Zebrafish Larvae
AD, 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 GSU antibody. Image was then superimposed with that of GFP expression. Green, GFP expression; red, rhodamine-conjugated antirabbit IgG.
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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 34 hpf. Overall pituitary GFP expression was suppressed by dexamethasone at 2 d post fertilization (dpf; Fig. 5
, A and B). By 5 dpf, there was a significant reduction of GFP expression in the anterior group of cells (Fig. 5
, 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. 5E
, 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. 5
, 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.

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Figure 5. Anterior Pituitary POMC-GFP Expression is Down-Regulated by Dexamethasone
AD, Live transgenic zebrafish embryos were continuously treated with or without dexamethasone starting from 34 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 34 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 = 410). **, P = 0.0007; ***, P < 0.0001, compared with 0 µM control.
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DISCUSSION
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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 1820 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, Rathkes 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 Rathkes 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 Rathkes 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 1820 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 Cushings 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
MSH antiserum showed that the posterior group of GFP-positive cells is posterior to, but not significantly colocalized with,
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.
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MATERIALS AND METHODS
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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-
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.
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ACKNOWLEDGMENTS
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We thank H. Pan for assistance in identifying and maintaining the transgenic zebrafish and L. Zhang for technical support on immunohistochemistry.
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FOOTNOTES
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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;
GSU,
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.
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