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Max-Planck Institute for Immunobiology (W.H., C.S., M.H.), 79108 Freiburg, Germany; Exelixis Germany (B.W., J.O.), 72076 Tuebingen, Germany; and Max-Planck Institute for Developmental Biology (H.-M.M.), 72076 Tuebingen, Germany
Address all correspondence and requests for reprints to: Matthias Hammerschmidt, Max-Planck Institute for Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany. E-mail: hammerschmid{at}immunbio.mpg.de.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In contrast to the adenohypophysis, the neurohypophysis itself does not contain endocrine cells. Rather, it secretes hormones from axonal termini of hypothalamatic neuroendocrine cells that have innervated the neurohypophysis. These hormones are oxytocin (isotocin in fish) and vasopressin (vasotocin in fish), generated in the supraoptic and paraventricular nuclei of the hypothalamus. In addition, neuroendocrine cells from these and other hypothalamic nuclei elaborate peptide hormones (so called releasing or release inhibiting hormones), which control the activity of endocrine cells of the adenohypophysis. In contrast to the neurohypophyseal hormones, these hormones do not reach the pituitary via nerve fibers. Rather, they travel in the blood via the hypothalamic-hypophyseal portal system (1).
The development of the adenohypophysis and the formation of its different cell types from a common primordium is controlled both by intrinsic and extrinsic factors. The adenohypophysis is derived from placodal ectoderm at the anterior neural ridge (reviewed in Ref.3), that becomes committed toward an adenohypophyseal fate via inductive signals from the ventral diencephalon, a subdivision of the forebrain that later gives rise to hypothalamus, infundibulum and neurohypophysis (4, 5). During further development, the adenohypophyseal anlage remains under the control of signals from the hypothalamus. These signals, together with intrinsic signals of the adenohypophysis itself, regulate the maintenance, proliferation and differential specification of adenohypophyseal cells by sequential activation of different transcription factors, such as the Lim class homeodomain proteins Lhx3 (6) or Lhx4 (7), the paired-like homeodomain protein Prophet-of-Pit1 (Prop1; Ref.8), the Pou domain protein Pit1 (9), the T-box factor Tpit/Tbx19 (10, 11, 12), the zinc finger protein Gata2 (13), and several others (reviewed in Refs.14, 15, 16, 17, 18, 19, 20).
Final evidence for the requirement of such factors for pituitary development requires genetic analyses via loss-of-function mutants. In mouse, such mutants have been generated using gene knockout technology, as in the case of Lhx3, that thereby was shown to be required for all adenohypophyseal cell types except the corticotropes (6). More recently, conditional knockout techniques allow one to investigate the pituitary-specific role of genes also involved in other processes, as in the case of the steroidogenic factor Sf1, that is required downstream of Gata2 for the formation of gonadotropes (13, 21). However, such gene targeting approaches are generally biased because they require a priori knowledge of the molecular nature of the genes to be analyzed. As an alternative to this reverse genetics approach, genes essential for mouse pituitary development were isolated via positional cloning of spontaneous, viable mutations, such as Prop1, that is mutated in Ames dwarf mutants and required for somatotropes, lactotropes, thyrotropes, and gonadotropes (8), and Pit1, which is mutated in Snell and Jackson dwarf mutants and required for somatotropes, lactotropes and thyrotropes, defining the so-called Pit1 lineage of the adenohypophysis (9) (also see Ref.20 for recent review). Tpit/Tbx19 requirement for the Pomc lineage (corticotropes and melanotropes) was revealed via the analysis of human patients with isolated Acth deficiencies caused by recessive Tbx19 point mutations (11). However, as in other known human pituitary disorders (see Refs.16 and 22 for reviews), there are too few families and patients to allow a direct positional cloning of the affected human gene. Furthermore, the spontaneous mutation rates in humans and mice are too low to allow a saturating identification of all genes required for adenohypophysis development via forward genetics.
In light of these limitations of reverse and forward genetics in mammalian systems, we carried out a forward genetic analysis of pituitary mutants in a nonmammalian vertebrate, the zebrafish. Due to its extracorporal and rapid development, the transparency of its embryos and larvae, its high fecundity, its relatively small size and the ease of high-density maintenance, the zebrafish is highly suitable for mutant screens. In the past, three independent large-scale screens have been carried out, two of which used the chemical N-ethyl-N-nitrosourea (ENU) to introduce random point mutations over the entire genome (23, 24). Whereas in these screens mutant analyses were largely restricted to examining embryonic and larval morphology at different developmental stages, the more recent Tuebingen 2000 large-scale ENU screen was set up to allow mutant screening with molecular tools (see for cloned mutations isolated in this screen, Refs.25 and 26). As part of the Tuebingen 2000 screen, we searched for genes required for zebrafish adenohypophysis formation and patterning, carrying out large-scale whole mount in situ hybridizations with a probe detecting Gh encoding transcripts, a marker for somatotropes. The staining was necessary because the adenohypophysis of zebrafish larvae is morphologically too indistinct to be analyzed in large numbers without molecular tools.
Recent work has revealed both crucial similarities and differences in the development of the adenohypophysis between fish and mammals. The zebrafish pituitary contains the same cell types (lactotropes, corticotropes, melanotropes, thyrotropes, somatotropes, gonadotropes; Refs.27, 28, 29) as the mammalian pituitary, however, there are crucial differences in the morphogenesis and the architecture of the glands in the different vertebrate species. Thus, the zebrafish adenohypophysis maintains its placodal organization and remains in a subepithelial position after oral cavity formation, whereas no invagination of the oral ectoderm equivalent to Rathke’s pouch formation in mammals takes place (27). Furthermore, there are differences in the patterning of the adenohypophyseal anlage: in zebrafish, the different cell types are distributed in three distinct domains along the antero-posterior axis of the pituitary, rather than along the dorsoventral axis as in mammals (27), with the Msh-generating pars intermedia most posteriorly, and the neurohypophysis located dorsal of adenohypophysis (1). In addition, fish and mammals display significant differences in the onset of the specification of the lactotrope lineage, that is the first lineage to specify in fish, but the last in mouse (27). Despite such differences, crucial mechanisms of adenohypophyseal induction and patterning appear to be highly conserved among fish and mammals, as for instance indicated by the conserved role of Sonic Hedgehog signaling from the ventral diencephalon (27, 28). In light of these data, the large-scale ENU mutagenesis screen was expected to uncover genes regulating fish-specific features of the adenohypophysis, as well as genes with shared functions in mouse and fish. This would further illuminate the degree of conservation between the two vertebrate species, and possibly identify novel regulators also involved in mammalian pituitary development.
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The late developmental stage (120 hpf) for mutant screening was chosen for two reasons: 1) to reveal also defects in later steps of pituitary development or patterning that would be missed when mutants were investigated much earlier (see below for lim3 staining of pit1 and aal mutants at 32 hpf), and 2) to avoid pituitary phenotypes that are secondary consequences of early and general defects, such as early brain necrosis, that occurs very frequently, but usually leads to embryonic death between 24 and 72 hpf (30). Secondary consequences on pituitary formation have also been revealed for midline mutants defective in signaling by Nodal and Hedgehog family members (27, 28, 31, 32, 33, 34, 35, 36). In such mutants, eye field separation and hypothalamus formation are compromised, leading to pituitary defects caused by the lack of inductive signaling from the hypothalamus, consistent with the loss of the pituitary in hypothalamus-deficient mouse embryos (4, 37, 38, 39). However, in contrast to pituitary-specific mutants, such zebrafish midline null mutants usually die before 120 hpf (32), although secondary pituitary defects caused by more subtle, viable midline mutations cannot be ruled out.
In total, we identified 13 mutants that were viable at 120 hpf but displayed a severe reduction or complete absence of gh staining in whole mount in situ hybridization. Other gross abnormalities, such as brain degeneration, which could have secondarily affected the pituitary, were not detected in these mutants. Only two of the 13 mutants (allele numbers t24594 and t20626) showed reduced eye distance and a curled-down tail, indicative for midline defects. Although t20626 was not further investigated, genomic mapping placed t24594 31–32 centimorgans from the top of linkage group 6, just below the marker Z6626. This is the same position as reported for the midline mutation iguana (Ref.32 ; and http://zfin.org/cgi-bin/view_mapplet.cgi, Ref.40), and strongly suggests that t24594 is a weak iguana allele that we did not analyze further.
Of the remaining 11 adenohypophysis-specific mutations, eight were recovered. A combination of complementation crossing and genomic mapping revealed that they fall into four complementation groups, defining four genes essential for adenohypophysis development in the zebrafish. The genomic positions of the four genes, the different alleles, and recognized other specific defects of the mutants are listed in Table 1
. All genes appear to be dispensable for hypothalamus development, as revealed by the normal expression of nkx2.1 (41) in mutant embryos at 32 hpf (Fig. 1, B, E, H, K, and N), and the normal number of isotocin-positive cells at 32 hpf (Fig. 1, C, F, I, L, and O). isotocin cDNA was isolated in our laboratory via degenerate RT-PCR, and is identical with the cDNA recently reported by Unger and Glasgow (42). It is expressed in two nuclei of magnocellular neurons in the anterior hypothalamus (Fig. 1C), possibly the presumptive supraoptic or paraventricular nuclei (see Introduction), from where the peptide hormone is supposed to undergo axonal transport into the more ventrally and posteriorly located region of the neurohypophysis adjacent to the adenohypophysis (see Refs.14 and 43 for review).
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, A–D).
lia and pia mutants lack the expression of all four adenohypophysis hormones at 72 hpf (Fig. 1, F and I; and data not shown) and 120 hpf (Fig. 2, E–L). The remaining expression of pomc in two longitudinal stripes (Fig. 2, F and J) is confined to ß-endorphin-synthesizing cells in the ventral base of the diencephalon (27) (for mouse see Refs.2 and 47), whereas melanotropes and corticotropes of the adenohypophysis, as well as lactotropes, thyrotropes, and somatotropes, are absent.
In contrast to their indistinguishable phenotypes at d 3 and 5 after fertilization, lia and pia mutants differ in lim3 expression during earlier stages of development. Although in lia mutants, lim3 expression in the region of the adenohypophysis anlage is completely absent at 32 hpf (Fig. 1D), lim3 expression in pia mutants is reduced, but clearly present (Fig. 1G in comparison to Fig. 1A for wild-type control). However, prl expression at 32 hpf is absent in both mutants (Fig. 1, E and H, in comparison to Fig. 1B for wild-type control). In sum, the expression pattern analyses suggest that both lia and pia are required for the specification of all adenohypophyseal cell types, with lia acting earlier than pia. lia appears to act upstream of lim3 (but not necessarily via lim3; see Discussion), whereas pia is more likely to act at the level, in parallel or downstream of lim3. The phenotype of pia zebrafish mutants is quite similar to that of Lhx3/Lhx4 double mutant mice (7), suggesting that it might be caused by a mutation in a zebrafish lim gene. However, lim3 itself can be ruled out, as it maps to a different linkage group (LG5; http://zfin.org/ cgi-bin/view_mapplet.cgi, Ref.40) than pia (LG18; Table 1).
Mutations Affecting Adenohypophyseal Patterning and Lineage Specification
The different cell types of the adenohypophysis derive from a common primordium, the placode, that initially consists of identical precursor cells. Subsequently, the placode becomes patterned, and cells in different regions of the primordium undergo differential specifications to form the different cell types characterized by the hormones they produce and secrete. Data from mammalian pituitary development suggest that the patterning process is characterized by subdivision of the common pool of precursor cells into certain cell lineages that then continue to branch until single cell types are generated. It is assumed that cells of the Pomc lineage, giving rise to corticotropes and melanotropes, split off first. In contrast to all other pituitary cell types, they express and require the T-box transcription factor Tpit/Tbx19 (10, 11, 12), but are independent of the Lim class homeodomain transcription factor Lhx3 (6). The complementary lineage, consisting of thyrotropes, somatotropes, lactotropes, and gonadotropes, is characterized by its Lhx3 dependence (6) (and reviewed in Refs.14 , 15 , 17 , 18 , and 20). Another lineage, comprising a subset of the Lhx3-dependent cell types, is defined via its dependence on the Pou domain transcription factor Pit1 (48, 49), and therefore called the Pit1 lineage. It consists of thyrotropes, lactotropes, and somatotropes, all of which are absent in Pit1 mutant mice (9).
In our zebrafish screen, we could identify two genes required for the specification of two different adenohypophyseal cell lineages. Mutants in both genes show normal lim3 expression at 32 hpf (Fig. 1, J and M, in comparison to Fig. 1A for wild-type control), indicating that the pituitary anlage is induced normally and of normal size. However, already at this early stage (32 hpf), the subdivision within the anlage is altered, indicated by the loss of prl expression in pit1 mutants (Fig. 1, K and L), whereas prl expression in aal mutants appears normal (Fig. 1, N and O, in comparison to Fig. 1, B and C, as control).
At 120 hpf, pit1 mutant embryos are characterized by loss of lactotropes (prl, Fig. 2M), somatotropes (gh, Fig. 2K) and thyrotropes (tsh, Fig. 2P), whereas pomc-positive adenohypophyseal cells, most likely anterior corticotropes and posterior melanotropes (27, 29), are present at normal or even slightly elevated numbers (Fig. 2N). The zebrafish pit1 mutants resemble Pit1-deficient mice (9), and indeed could be shown to carry mutations in the zebrafish pit1 gene (50).
aal mutant zebrafish show a very different phenotype; they fail to generate pomc-, tsh-, and gh-expressing pituitary cells (Fig. 2, R–T), whereas the prl-expressing lactotropes are still present both at 32 hpf (Fig. 1, N and O) and 120 hpf (Fig. 2Q). However, at the later developmental stages, the lactotropes are dispersed along the antero-posterior axis (Fig. 2Q), rather than being organized in the sharp anterior domain found in wild-type siblings (Fig. 2A). This phenotype seems to define a new lineage of adenohypophyseal cells, consisting of corticotropes, melanotropes, thyrotropes, and somatotropes, all of which depend on the aal gene, whereas the lactotropes appear to develop independently of aal function (Fig. 3). In contrast to pit1, no phenotype similar to that of aal mutants has been described in mouse thus far. Thus, the aal mutation seems to have revealed a thus far unknown lineage of adenohypophyseal cells that partially overlaps with the Pit1 lineage.
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DISCUSSION |
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Genes Required Upstream and Downstream of Lim3 to Drive Development of All Adenohypophyseal Cell Types
Two genes, lia and pia, are required for all adenohypophyseal cell types. In contrast, other placodal derivatives appear to develop normally in mutant embryos, as for instance indicated by the normal expression of marker genes of the olfactory epithelium (Herzog, W., and M. Hammerschmidt, unpublished data), which derives from placodal positions located left and right of the adenohypophyseal placode at the anterior neural ridge (45). This indicates that lia and pia specifically act on early steps of adenohypophyseal placode formation or maintenance, whereas other placodes are regulated differently. During adenohypophyseal development, lia and pia appear to be required for different, maybe subsequent steps, as indicated by the differences in the lim3 expression in the two mutants. Thus, lia appears to act at an earlier step, before lim3 expression in the adenohypophyseal placode is initiated, whereas pia acts after the initiation of lim3 expression, as indicated in Fig. 3. However, this does not necessarily mean that lia fulfills its indispensable function via the activation of lim3, nor does it necessarily mean that lim3 is required for pia activation. lia could as well activate other essential genes that act in parallel or in addition to lim3, and pia could as well act in parallel rather than downstream of lim3. However, it is interesting to note that pia mutants—despite the early presence of lim3 transcripts—fail to express even early adenohypophyseal hormone genes like prl. This indicates that in the absence of pia, lim3 is not sufficient for early lactotrope specification—although it appears to be necessary. The latter is suggested by our preliminary results obtained with antisense morpholino oligonucleotides (51), according to which inactivation of lim3 leads to the loss of all adenohypophyseal cell lineages except some pomc cells (Herzog, W., and M. Hammerschmidt, unpublished data). Final analyses of the epistatic relationships between lia, lim3, and pia will only be possible after the lia and pia genes have been cloned.
To gain further insight into the biological roles of lia and pia during the development of the zebrafish adenohypophysis, future experiments must reveal the fate of the pituitary precursor cells in the mutants. Thus, cell-tracing experiments (44), bromodeoxyuridine incorporation studies, and transferase deoxyuridine triphosphate nick-end labeling or acridine orange stainings will be carried out to investigate whether adenohypophyseal cells are lost because of transfating, failed proliferation, or cell death. In addition, analyses of chimeric embryos generated via cell transplantation will help to specify in which cell types the two genes are required.
Genes Required for Lineage Specification: Evidence for a Conserved and a Novel Lineage of Adenohypophyseal Cell Types
In contrast to lia and pia, pit1 and aal mutants lose only some of the adenohypophyseal cell types, indicating that they are required for adenohypophyseal patterning and lineage specification processes during later stages of pituitary development. pit1 mutants lack gh, prl, and tsh staining, whereas pomc staining is normal or even enlarged, particularly in its posterior domain that accommodates both corticotropes and melanotropes (29). The phenotype looks very similar to that of mouse Pit1 mutants, characterized by the loss of thyrotropes, somatotropes, and lactotropes. Due to the lack of suitable markers, we could not study the effect of zebrafish pit1 on the gonadotropes, that according to the mouse mutant should be present in pit1 mutant fish. One subtle difference in the phenotypes of mouse and zebrafish mutants is that zebrafish mutants lack all tsh expression, whereas in mouse mutants, only the caudo-medial thyrotropes are lost, although the earlier specifying rostral-tip thyrotropes are present (52) (for review, see Ref.17). Tsh expression in this special thyrotrope lineage is independent of Pit1, and most likely regulated by the leucine zipper transcription factor TEF (thyrotrope embryonic factor; Refs.52 and 53). The functional importance of the rostral-tip thyrotrope cells is not clear (17). However, our data obtained for the zebrafish pit1 mutant suggest that they might be a specialty of mammals.
In contrast to pit1, the zebrafish aal mutants appear to define a thus far unknown lineage of adenohypophyseal cell types, lacking somatotropes, thyrotropes, corticotropes, and melanotropes, but not the lactotropes. Such a combination of lost cell types had not been previously described. In addition, the parallel existence of the Pit1 and Aal lineages indicates mechanisms of lineage specification beyond the thus far believed subsequent branching off of lineages from a common precursor pool. Consistent with such a linear mechanism, only two relative patterns of cell lineages had been observed thus far. Lineages were either complementary (such as the Pomc lineage and the Prop1 lineage), or part of each other (such as Pit1, consisting of somatotropes, lactotropes, and thyrotropes, and Prop1, consisting of the Pit lineage plus the gonadotropes). In contrast, the zebrafish Pit1 and Aal lineages show a novel relative pattern, with shared cell types, as well as cell types specific for one or the other lineage (see Fig. 3). Thus, somatotropes and thyrotropes appear to belong both to the Pit1 and the Aal lineage. However, corticotropes and melanotropes only belong to the Aal lineage and are independent of Pit1, whereas the lactotropes belong to the Pit1 lineage only and are independent of Aal. This indicates that factors driving cell lineage specification are not only used in a mutually exclusive or consecutive fashion, but can also be recruited in different combinations to allow differential parallel cell specifications.
In mammals, no Aal lineage has been identified as yet. However, this does not necessarily mean that the mammalian Aal homolog is not required or involved in adenohypophysis development. Rather, the exclusive regulation of prl expression independently of Aal function might be a specialty of fish, consistent with the much earlier onset of prl expression in fish compared with mouse, and with the earlier and additional function of Prl during osmoregulation in water-living larvae (1, 27, 54). Along these lines, it is tempting to speculate that during the evolution of water-living vertebrates, lactotropes became independent of Aal, or that during evolution of land-based vertebrates, lactotropes got under the control of the Aal homolog, similar to the other pituitary cell types. In this case, mutations in the Aal homolog in mouse would affect all adenohypophyseal cell lineages. Final answers have to await the cloning of the zebrafish aal gene, and the identification and analysis of possible mammalian homologues.
The Cloning of the Mutated Pituitary Genes
Because the introduction of point mutations during the ENU mutagenesis is random, the molecular nature of the genes affected in the isolated mutants is not known a priori, but has to be determined in subsequent steps. However, with the ease of mutant embryo collection, together with recent progress in the zebrafish genome projects, positional cloning of such ENU-mutated genes has become relatively easy, and has been successfully applied in multiple cases (55, 56). Using a combination of positional cloning and candidate testing, we have for instance been able to identify the molecular nature of our two zebrafish pit1 alleles (50). Also, future cloning of lia, pia, and aal will hopefully identify novel genes in pituitary development. In addition, we will be able to investigate whether such genes are specifically required for pituitary development in fish, or whether their roles are conserved between fish and mammals.
Possible Reasons for the Low Number of Identified Pituitary Genes and Perspectives
Based on gene targeting and cloning of spontaneous mutations, a total of approximately 20 genes indispensable for pituitary development in the mouse were identified (for reviews, see Refs.15 and 17). These include genes required for the combined formation of hypothalamus, neurohypophysis, and adenohypophysis (such as Nkx2.5 and Shh; Ref.4 , 38 , 39), genes required for hypothalamus and neurohypophysis only (such as Brn2; Refs.57 and 58), and genes required for the adenohypophysis only (such as Lhx3, Lhx4, Prop1, Pit1). Our screen was designed to find adenohypophysis-specific defects only. Pituitary phenotypes caused secondarily due to loss of the hypothalamus (such as in mouse Nkx2.5 or Shh mutants) would have been missed because screening was performed at a late developmental stage, after mutations in such genes should have been lethal.
In addition, the zebrafish mutants were screened for altered gh expression only—the only adenohypophyseal hormone gene that had been cloned at the time the screen was carried out. The gh probe should have allowed us to identify genes required for adenohypophysis induction and development in general, as well as genes specifically required for somatotropes and somatotropes-including lineages, such as Lhx3, Prop1, and Pit1. On one hand, gh was a good choice, because it allowed us to identify genes that would most likely have been missed with other hormone probes (pit1 mutants still express pomc; aal mutants still express prl). On the other hand, we might have missed genes that would have been revealed with other probes, such as Tpit/Tbx19 (affecting the Pomc lineage only), Gata2 (affecting gonatotropes and thyrotropes only), or Sf1 (affecting gonadotropes only). In light of these possible limitations of the gh probe, we are currently preparing another large-scale ENU screen, looking for the expression of pit1, lim3, isotocin, pomc, and prl at early and late stages of zebrafish development.
Another reason for the low number of identified essential zebrafish genes could be the larger size and complexity of the zebrafish genome. Due to the additional genome duplication that has occurred during teleost evolution (59), genes controlling pituitary development in zebrafish might display a higher degree of functional redundancy than in mammals. However, genome analyses suggest that only approximately 25% of the genes gained in the duplication have remained active, with most of them having evolved a different expression pattern than their paralogs (59, 60). Also, parallel searches for zebrafish genes required for other developmental processes, such as angiogenesis (25) and thymus development (Thomas Boehm, Max-Planck Institute for Immunobiology, Freiburg, Germany; personal communication) have yielded many more mutants, suggesting that high functional redundancy with a low number of indispensable genes might be a feature of some, but not all processes of zebrafish development. On the other hand, we cannot rule out general technical problems during our mutagenesis. Comparing our mutagenesis conditions with those of previous screens (23), the screening of over 4200 mutagenized genomes should have yielded 50–80% saturation, with an average allele frequency per gene between three and four. However, in case of our identified pituitary genes, the average allele rate was two, with single alleles for two of the four genes. This suggests that the ENU mutagenesis might have been less efficient than in previous screens. Further complementation testing of other mutant classes has to be carried out for final conclusion about the mutation rates.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To determine whether two mutations causing similar phenotypes reside in the same or in two different genes, complementation analyses were performed, crossing a heterozygous fish of one mutation with a heterozygous fish of the other mutation. If the mutations are alleles of the same gene, they fail to complement each other in trans-heterozygous embryos, which show the mutant phenotype like homozygotes of either allele. If the mutations are in different genes, the double-heterozygous offspring show a wild-type phenotype.
Mapping
Genomic localization of zebrafish mutations was performed as described, using the Tübingen marker set for genome scans (version 4) on F2 Tuebingen x Wik crosses of the mutant carriers (55). Primer sequences are available from the Massachusetts General Hospital web site (http://zebrafish.mgh.harvard.edu). The linkage groups (genetic equivalents to chromosones) determined for the different mutations are given in Table 1.
Cloning of the Zebrafish Isotocin cDNA
Isotocin cDNA was cloned via degenerate RT-PCR, using the following primers: sense 5'TGY TAY ATH CAR AAY TGY CC, antisense 5'CCR CAR CAD ATN BWN GGN CC, with 35 cycles and an annealing temperature of 53 C. To obtain the full-length cDNA sequence, this was followed by a 3'-rapid amplification of cDNA ends (RACE), using the sense primer and the SMART RACE Kit (CLONTECH, Palo Alto, CA) according to the manufacturer’s instructions. PCR fragments were cloned into pCRII (Invitrogen, Carlsbad, CA). For isotocin in situ antisense probe synthesis, the plasmid was digested with KpnI and transcribed using T7 RNA polymerase.
In Situ Hybridizations
For initial screening, F3 clutches were incubated in the presence of 0.25 mM 1-phenyl-2-thiourea (Sigma, St. Louis, MO) to avoid melanin synthesis, and fixed in 4% paraformaldehyde/PBS at 120 hpf. Whole mount in situ hybridization was carried out in specially designed 48-well plates (Aldinger, Nagold, Germany) with digoxygenin-labeled probes for gh (27), and rag1, a marker for thymic T cells (61), after standard protocols (62). Red/blue double in situ hybridizations were carried out as described (27). prl, pomc, tsh, gh (27), lim3 (31), and nkx2.1 (41) probes were synthesized as reported (27).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ENU, N-ethyl-N-nitrosourea; hpf, hours post fertilization; POMC, proopiomelanocortin; PRL, prolactin; Prop1, Prophet-of-Pit1.
1 B.W., J.O., and H.-M.M. represent the Tuebingen 2000 screen consortium. 
Received for publication September 26, 2003. Accepted for publication January 21, 2004.
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