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Department of Human Genetics (I.J.), Mount Sinai School of Medicine, New York, New York 10029; and National Institutes of Health (L.N., H.L., D.F.), National Institute of Diabetes and Digestive and Kidney Diseases, Clinical Endocrinology Branch, Bethesda, Maryland 20892-1772
Address all correspondence and requests for reprints to: Douglas Forrest, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Clinical Endocrinology Branch, 10 Center Drive, Bethesda, Maryland 20892-1772. E-mail: forrestd{at}niddk.nih.gov.
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ABSTRACT |
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INTRODUCTION |
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). Although several physiological functions have been ascribed to these isoforms, little is known of the underlying mechanisms that direct their expression in vivo.
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To elucidate the regulation of Thrb that underlies its tissue-specific roles, we have investigated the cis-acting elements that mediate induction of TRß2 in vivo. Previous studies defined a basal promoter region in pituitary cell lines in culture (31) but not its function or regulation in vivo. By detailed mapping with reporter transgenes in mice, we find that the promoter alone is active only in cochlea, whereas a complex intron region confers both pituitary and retinal expression. Specific domains within this intron region cooperate with the common but distant promoter to direct pituitary and retinal expression. The specialized expression and functions of TRß2 therefore depend upon an unusual multifunctional control region in an intron of the Thrb gene.
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RESULTS |
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, A–C). LacZ activity was analyzed at optimal ages for TRß2 expression in the outer neuroblastic layer of the retina that contains immature photoreceptors at embryonic day (E)17.5, the anterior pituitary at postnatal day (P)25, and the interior sensory tissues of the cochlea at E17.5. A transgene carrying the TRß2 promoter with 570 bp of upstream sequence (transgene A, TgA) was sufficient for expression in the cochlea but not retina or pituitary (Fig. 1D). Further extension to 6.5 kb upstream of the exon failed to give retinal or pituitary expression while cochlear expression was retained (TgB). Comparison of mouse and human genomic DNA sequences revealed significant homology in the TRß2 promoter and also in a 0.6-kb intron region located approximately 2 kb downstream from the exon. A transgene carrying the promoter and a 2.5-kb fragment encompassing this intron region (TgC) gave expression in both pituitary and retina. Expression in the neural retina was localized to cells at the periphery, recapitulating the distribution of endogenous TRß2. Unlike endogenous TRß2, the transgene was also expressed in the inner zone of the retina that forms the ganglion cell layer; this expression was weak and appeared as punctate staining. The expression in two populations of retinal cells suggests that both ganglion cell and cone precursors respond to shared signals, consistent with both of these cell types being among the earliest to be generated in the retina (30). The endogenous Thrb gene presumably restricts expression to photoreceptors by additional suppressive sequences not present in the transgene.
Distinct Elements Direct TRß2 Expression in Retina and Pituitary
Finer analysis revealed that distinct but overlapping intron elements activate the TRß2 promoter in the retina and pituitary (Fig. 2). Intron fragments of 550 bp (TgD) or longer (data not shown) gave expression in retina and pituitary (Fig. 2B). However, deletion of 170 bp at the 3' end of the 550-bp fragment abolished retinal expression, indicating that the remaining 380-bp fragment (TgE) is sufficient for expression in pituitary but not retina.
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). This direct analysis of founders bypassed the prolonged times necessary for establishing stable carrier lines. The preceding analyses on established founder lines allowed testing at optimal ages for both retina (E17.5) and pituitary (P25), whereas transient analysis of embryos was informative only for retina. Although retinal expression was given by the downstream 170-bp region in conjunction with the 380-bp pituitary-determining fragment (TgD above), the 170-bp fragment alone gave expression only in rare sporadic cells in the outer neuroblastic layer at E17.5 (TgF). Extension upstream by 110 bp but not 35 bp restored a full expression pattern (280-bp fragment, TgH). Thus, the 170-bp downstream sequence specified expression but only in sporadic cells, whereas a distinct upstream sequence amplified expression in a wider cell population, recapitulating the pattern of endogenous TRß2. The upstream region alone (TgJ or TgE) failed to give expression, supporting its role as an amplifier that cooperates with the specifier element. From the above-described analyses, it was inferred that the amplifier and specifier elements were separated by nonessential sequences. Therefore the putative amplifier (85 bp) and specifier (115 bp) elements were combined in the absence of the 80-bp intervening sequence (TgK). This transgene gave expression in a wider cell population, similar to the pattern of endogenous TRß2. Thus, the extended retinal-determining region consists of at least two distinct domains that specify and amplify expression, respectively.
Developmental Expression of the TRß2-lacZ Transgene
The intron control region directed transgene expression in the retina beginning around E12.5 and rising to a peak in the late embryo at E17.5, followed by a neonatal decrease. Little or no detectable signal remained by P8 (Fig. 3A). This time course resembled that of endogenous TRß2 mRNA and indicated that the profile is determined at the transcriptional level. Previous studies by northern blot or RNAse protection analysis have shown that low residual levels of endogenous mRNA remain detectable at older postnatal ages in the mouse and chick (16, 25), suggesting that detection of lacZ activity is somewhat less sensitive.
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). This lack of zonal restriction is consistent with the proposal that other factors interact with the TRß2 product to give spatially divergent expression of M and S opsins (25).
In contrast to the embryonic peak of expression in the retina, expression in the anterior pituitary followed a later time course, with steadily increasing levels at postnatal stages (Fig. 3C). This developmental pattern was consistent with that described for endogenous TRß2 mRNA in the rat pituitary, where levels were low in the late embryo and increased at postnatal stages (14). In the cochlea, transgene expression was detected at both embryonic and postnatal stages (Fig. 3, D and E). Expression was strongest in the region of the greater and lesser epithelial ridges, which contain the formative inner sulcus, immature sensory hair cells, and other supporting cells. Weaker expression was also detected (as punctate staining) in other regions of the epithelium around the cochlear duct that form the lateral wall and stria vascularis. The pattern was largely in accord with that described for endogenous TRß2 mRNA based on in situ hybridization in the rat cochlea (23).
TRß2 Expression in Immature Postmitotic Cones
Retinal neurogenesis is thought to involve an arrest of cell division as multipotent progenitor cells adopt a particular differentiation fate (30). The determinative events in cone differentiation are unknown, but TRß2 provides a unique marker for studying early events in this pathway. Given that peak TRß2 expression occurs in the late embryo and neonate when large numbers of mitotic progenitors exist, we used a lacZ reporter line to rule out that TRß2-positive cells might represent an unusual population of proliferating progenitors rather than committed postmitotic cones. 5-Bromo-2'-deoxyuridine (BrdU) was administered to mark dividing cells in transgenic neonates or embryos. At P0, BrdU labeling was found in many cells in internal areas of the neural retina but not in the lacZ-positive cells, which were mainly located at the periphery (Fig. 4A). Similar results were found by examining expression of lacZ targeted into the endogenous Thrb gene in TRß2-deficient mice (Thrbtm2/tm2) (Fig. 4B). Thus, loss of TRß2 did not alter the generation of postmitotic cones, consistent with the normal cone numbers found in adult TRß2-deficient mice (25). At earlier stages (E15.5), the lacZ-positive cell population in the transgenic or TRß2-deficient lines had similar characteristics. A denser population of BrdU-positive cells was detected at E15.5 than at P0, but most if not all identifiable lacZ-positive cells were negative for BrdU. Thus, TRß2 is detected primarily in postmitotic cells.
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). These cells were identified as cones based on their relatively small numbers, time of generation in the late embryo (29), and their immunoreactivity for S opsin (Fig. 4D). Low levels of S opsin begin to be detectable in late embryonic cones, before the neonatal decline of TRß2 levels, and these cells were positive for both S opsin and TRß2.
The Intron Control Region Reprograms Expression of a Heterologous Promoter
To determine whether the Thrb intron control region is constrained to be active only with its natural TRß2 promoter or whether it can regulate a heterologous promoter, a 990-bp intron fragment was incorporated into a transgene driven by the promoter of the mouse S opsin gene (Opn1sw) (33), which bears no obvious sequence resemblance to the TRß2 promoter. The Thrb intron was inserted downstream from the S opsin promoter, resembling its natural context relative to the TRß2 promoter. The S opsin/lacZ transgene without the intron gave little or no detectable expression at P0 but was readily detectable by P14 and at older ages. Expression followed a similar distribution gradient as endogenous S opsin, with strongest signals in the inferior retina (Fig. 5A).
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). This hybrid transgene (S opsin/lacZ+Thrb intron) was active at P0 around the entire periphery of the retina. At P14, expression persisted in both superior and inferior areas of the retina, although expression was relatively stronger in the inferior, indicating that a gradient was in the process of being imposed. By P28, expression of the hybrid transgene had reverted to an S opsin-like pattern, with little or no signal remaining in the most superior part of the retina. At P14, the hybrid transgene was expressed in similar cell numbers in all superior and inferior areas of the retina, contrasting with the gradient shown by the parental S opsin/lacZ transgene (Fig. 5C). The Thrb intron fragment used contained elements for both retinal and pituitary expression in conjunction with the TRß2 promoter; however, this fragment gave only retinal expression with the S opsin promoter. Thus, the pituitary-determining elements in the intron operate only in conjunction with a promoter that is predisposed for activity in the pituitary as well as in cone photoreceptors.
To rule out the possibility that the artificial combination of S opsin promoter and Thrb intron gave expression in an undefined cell population, the cone identity was confirmed by costaining with peanut agglutinin (PNA), a lectin that reacts with cone inner and outer segments (34). In both inferior and superior retina, cells were identified as positive for both PNA and lacZ staining (Fig. 5D).
Activity of the Intron Control Region in Vitro
The location and complexity of the intron control region suggested that it regulates the promoter by long-range mechanisms. Biochemical approaches to isolating the interacting factors or elucidating the chromatin structure of the intron region in vivo present difficulties because of the limited source material, particularly the immature cones in utero: the cone population in the mouse represents only 3% of retinal photoreceptors with the remaining 97% being rods (29). As an alternative indicator of a potential role for chromatin, the TRß2 promoter with or without the intron control region was introduced into an episomal reporter vector, pREP4, that can replicate extrachromosomally and adopt a chromatin conformation (35, 36). Figure 6 shows that a construct with the promoter and intron gave minimal activity in 293T embryonic kidney cells. However, in Weri retinoblastoma cells or GH3 pituitary cells, both of which are permissive for expression of endogenous TRß2 (11, 37), the intron conferred a significant induction of luciferase activity (
6-fold and >60-fold, respectively) compared with a construct with the promoter alone. The TRß2 promoter itself had a very low basal activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 isoforms, encoded by the Thrb and Thra genes, respectively, are governed to a considerable extent by their individual expression patterns. The restricted expression of TRß2 contrasts with the wider distribution of TRß1 and the near-ubiquitous expression of TR1 from early stages. The present study shows that the specialized expression of TRß2 is determined by a versatile control region in an intron of the Thrb gene. The intron control region contains overlapping but distinct regions for retinal and pituitary expression, suggesting that separate inducing mechanisms have been independently acquired by the Thrb gene. Despite the presumably independent origins of these regions, they reside in a shared region of the intron, suggesting a beneficial arrangement for the regulation of the gene. Although the pituitary and retinal regions are likely to respond to different trans-acting signals, it is possible that the consequent activation of the distant promoter involves related long-range changes in chromatin configuration in both cases. In accord with this idea, it has been reported that, in vitro in pituitary GH3 cells, endogenous TRß2 mRNA is suppressed by sodium butyrate, which was suggested to act by altering histone acetylation (41).
The transcriptional mechanisms are unknown but may involve cell-specific complexes that bind to both the promoter and intron control regions in a cooperative manner to activate transcription through the TATA box-binding complex. In immature cones, a particular combination of inducing factors would be present and may decline postnatally to give the characteristic rise and fall of TRß2 expression. In pituitary, TRß2 would be induced later, postnatally, by a different set of transcription factors acting on distinct sites within the same control regions. In both cases, bridging contacts may form between the intron and promoter complexes, perhaps involving looping of the intervening sequences (42). One may speculate whether the pituitary-determining elements in the intron also control the far upstream TRß1 promoter, given the overlap of TRß2 and TRß1 expression in this tissue (14). This could not occur in retina, given the lack of TRß1 in immature cones.
The promoter is trifunctional, being active itself in cochlea as well as being inducible in retina or pituitary in conjunction with the necessary intron elements. Previously, the TRß2 promoter alone was shown to respond to the Pit-1 transcription factor in GH3 somatotrope and TtT97 thyrotrope pituitary cell lines in culture (31). In vivo, however, our results indicate that the promoter is insufficient for pituitary expression, suggesting the importance of in vivo studies to reveal the full array of sequences needed for biological function. It is possible that, in transfected cells, overexpression of Pit-1 is able to mimic functionally the pituitary-specific stimulus that is normally mediated by the intron in vivo. A speculation arising from our finding that the promoter itself is active in the cochlea and not other tissues is that the cochlea may be a primordial site of TRß2 action. The promoter region shows greater conservation across mammalian and nonmammalian species than does the intron control region.
The intron control region in mammals is conserved over several hundred base pairs, whereas homology degenerates in nonmammalian species, suggesting that the multi-tissue control region is a mammalian adaptation (Fig. 7). Limited stretches of possible homology within the retinal-determining region were observed in the chick, but little or no homology was evident in zebrafish or pufferfish. However, the conserved expression of TRß isoforms in retina in mammals and nonmammalian species (16, 43) suggests that, in more distant species, photoreceptor-determining elements are present but are more scattered in the intron or reside elsewhere in the gene. TRß2 probably mediates a conserved role in cone differentiation, at least in mammals, as it is also detected in human fetal cones (44). In rats, polymorphic changes in TRß2 have been associated with differences in S opsin expression (45).
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Thyroid hormone receptors have many actions in the nervous system (48), some of which involve the decision of undifferentiated progenitors to continue to proliferate (38) or to differentiate into a particular cell type (49, 50). For example, TR1 is implicated in neuronal differentiation of embryonic stem cells (50), differentiation of oligodendrocytes (51), and proliferation of neural stem cells in adult mouse brain (52). However, in the retina, TRß2 expression is largely in postmitotic cells, as indicated by BrdU labeling, consistent with TRß2 acting in cells that are already specializing as cones rather than in the initial adoption of a cone fate. This suggests a distinction between TR1 acting in relatively uncommitted progenitors in some areas of the nervous system and TRß2 in more specialized cell types at later stages of differentiation.
Human resistance to thyroid hormone is characterized by elevated levels of thyroid hormones and TSH due to defective feedback control of the pituitary-thyroid axis, as well as a variety of other symptoms (9). The syndrome is typically associated with mutations in exons encoding the C-terminal domain of TRß, although up to 15% of cases lack mutations in THRB coding exons (53). Conceivably, some such cases could involve mutations in cis-acting control sites that impair expression of TRß2 in pituitary and/or retina. This could provide one explanation for some of the cases that have been described as exhibiting a pituitary-selective resistance to thyroid hormone (9, 18). Mentions of color visual defects in this syndrome are scarce (54, 55). However, a systematic study has not been performed such that the incidence and severity of such deficiencies remain to be determined for this syndrome or other congenital thyroid disorders.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Immunostaining.
Sections were rinsed in PBS, blocked for 1 h in 10% (vol/vol) normal goat serum, and incubated overnight with a polyclonal antiserum raised against the N-terminal 108 residues of mouse TRß2 (rabbit, 1:10,000) (Ng, L., and D. Forrest, manuscript in preparation) and 1% (vol/vol) normal goat serum in PBS. Slides were washed three times for 5 min each in PBS and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated antirabbit IgG (1:2,000) in 1% (vol/vol) normal goat serum in PBS, then washed three times for 5 min each in PBS and developed using diaminobenzidine (DAB) peroxidase reagents (Vector Laboratories, Burlingame, CA). For fluorescent double-labeling, slides were incubated TRß2 antiserum (1:10,000) then with fluorescein-labeled antirabbit second antibody (1:200; Vector Laboratories). Slides were incubated with anti-S opsin antibody (1:3000; Chemicon, Temecula, CA) overnight, then washed, incubated with antirabbit Texas Red-labeled second antibody, and coverslipped with Vectashield (Vector Laboratories). To colocalize endogenous TRß2 and transgenic ß-galactosidase, sections were incubated with monoclonal anti-ß-galactosidase antibody (1:200; Promega, Madison, WI) using M.O.M. reagent and detected with fluorescein-labeled antimouse second antibody (1:200; Vector Laboratories). Slides were incubated with anti-S opsin antibody (1:3000) overnight and then detected with Texas Red-conjugated antirabbit second antibody. Cryosections were incubated with biotinylated PNA (Vector Laboratories) for 2 h at room temperature. Sections were washed in PBS and then processed using the Elite ABC kit and DAB reagents (Vector Laboratories).
BrdU Labeling.
Mice were injected with BrdU dissolved in 0.9% (wt/vol) NaCl at a final concentration of 200 µg/g body weight. Pregnant females were injected ip 24 h before dissecting embryonic tissues; neonatal pups were injected sc and dissected 5 h later. Retinal sections were stained for ß-galactosidase, then dehydrated and rehydrated through a graded series of ethanol washes [30, 50, 80% (vol/vol)] for 5 min each and rinsed in PBS and treated with 2 M HCl in PBS for 30 min and 0.1 M B4Na2O7 in PBS for 15 min. Sections were rinsed in PBS, blocked for 1 h in 10% (vol/vol) normal horse serum dissolved in PBS, then incubated overnight with antibody against BrdU (1:1000) in 1% (vol/vol) normal horse serum in PBS. Slides were washed three times for 5 min each in PBS, incubated for 1 h at room temperature with horseradish peroxidase-conjugated antimouse IgG antibody (1:5000) in 1% (vol/vol) normal goat serum in PBS, washed three times for 5 min each in PBS, and then developed using DAB peroxidase reagents.
Transgenic Analyses
A LacZ reporter cassette with a simian virus 40 polyadenylation signal was fused in-frame into the TRß2 exon at the StuI site and cloned into pNEB193. Transgenic constructs were based on this TRß2-LacZ fusion construct using extensions of upstream or downstream (intron) genomic fragments. Transgene fragments were released from the vector by restriction digestion and microinjected into one-cell stage B6CBAF1/J embryos at the Mount Sinai Transgenic Core Facility. Founder mice were identified and transgene copy numbers determined by Southern blot analysis using a lacZ probe. Estimated copy numbers typically ranged from 2 to 67; copy numbers did not correlate with whether or not a given construct was expressed. Founders were crossed with B6CBAF1/J mice to generate stable lines or founder embryos that were analyzed directly as transients. Transgene carrier progeny were identified by a PCR genotyping assay (25). The targeted Thrbtm2 allele deletes the TRß2 product and substitutes a lacZ reporter into the TRß2-specific exon (25) and was maintained on a mixed 129/Sv x C57BL/6J x B6DBAF1/J background. The 562-bp S opsin promoter/lacZ transgene has been described (56) and was derived from a previously reported longer fragment of the mouse Opn1sw gene (33). The S opsin/lacZ + Thrb intron transgene was made by ligating a 990-bp SphI-PstI fragment of the intron downstream of the lacZ cassette. Animal experiments followed approved protocols at Mount Sinai School of Medicine and at the National Institutes of Health.
Sequence Analyses
The exons of the mouse Thrb gene were mapped on a 510-kb genomic sequence downloaded from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) (mouse chromosome 14, NCBIM36 partial sequence, coordinates 16350000 : 16860000). The mouse genomic sequence around the TRß2 exon was used in a basic local alignment search tool (BLAST) search to identify corresponding sequences from the human and other species genomes. Human and mouse sequences were aligned using the Clustal W algorithm (version 1.7; http://www.ebi.ac.uk/clustalW/) using default settings (Gap Opening Penalty 10.0, Gap Extension Penalty 0.2, Gap Separation Distance 4.0). Initial alignments of human and mouse genes were performed using Celera genomic sequences (accession numbers: human, NT_005564; mouse, GA_X5J8B7W68UK). Putative transcription factor binding sites were investigated using Consite (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite?rm=t_input_single) and TRANSFAC 4.0 (http://www.gene-regulation.com/pub/programs/alibaba2/index.html) programs.
Transfection and Reporter Assays
The TRß2 promoter (–570 to first ATG in the exon) was excised from transgenic construct TgA and was cloned into pREP4, a promoterless luciferase reporter, based on an episomal vector carrying an Epstein-Barr virus replication origin and a gene encoding the EBNA1 nuclear antigen (35). The Thrb intron control region (730 bp, extending 40 bp upstream and 60 bp downstream of the fragment shown in Fig. 7B) was amplified by PCR using Fusion polymerase (New England Biolabs, Beverly, MA), cloned downstream of the luciferase cassette in pREP4 and subjected to confirmation by sequence analysis. The promoter and intron fragments were also cloned in the same relative locations in the pGL3 luciferase reporter (Promega). Weri cells were grown in RPMI 1640 with 15% fetal bovine serum; GH3 and 293T cells were grown in DMEM with 10% fetal bovine serum. All media contained 2 mM glutamine and 1% penicillin/streptomycin. Cells grown in 12-well plates were transfected with 200 ng of luciferase reporter plasmid DNA and 10 ng of pSV-RL Renilla internal control using Superfect reagent (QIAGEN, Valencia, CA) for 293T cells and Fugene HD (Roche, Indianapolis, IN) for Weri and GH3 cells. Cells were harvested for preparation of cell lysates after 48 h (293T, GH3) or 60 h (Weri). Luciferase activity was assayed using the dual-luciferase system (Promega). Three wells were used per experiment, and each experiment was performed in triplicate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Disclosure Statement: The authors have nothing to declare.
First Published Online March 6, 2007
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; DAB, diaminobenzidine; E, embryonic day; P, postnatal day; PNA, peanut agglutinin; Tg, transgene; TRß, thyroid hormone receptor ß; X-gal, 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside;
Received for publication January 19, 2007. Accepted for publication February 27, 2007.
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and TRß in the control of thyroid hormone production and post-natal development. EMBO J 18:623–631[CrossRef][Medline] and ß thyroid hormone receptors from thyrotrope cells: the mouse pituitary-specific ß2 isoform differs at the amino terminus from the corresponding species from rat pituitary tumor cells. Mol Endocrinol 5:1049–1061[Abstract]- and ß-thyroid hormone receptor mRNAs, including the ß 2-subtype, in the developing mammalian nervous system. J Neurosci 12:2288–2302[Abstract] and N-terminal variant ß receptors. Development 114:39–47[Abstract] and ß thyroid hormone receptor (TR) gene expression during auditory neurogenesis: evidence for TR isoform-specific transcriptional regulation in vivo. Proc Natl Acad Sci USA 91:439–443
in the Xenopus embryo. Differentiation 72:239–249[CrossRef][Medline] gene dominant-negative mutation (P398H) selectively impairs gene expression in differentiated embryonic stem cells. Endocrinology 143:2664–2672 1. EMBO J 21:6452–6460[CrossRef][Medline] receptor. FASEB J 19:863–865NURSA Molecule Pages Link:
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, insertion site of lacZ fusion in the targeted allele and transgene constructs shown in panels C and D (53 codons after the first ATG initiator codon of TRß2). HIII, HindIII; BII, BglII restriction enzyme sites. C, Activity of the endogenous TRß2 promoter in retinal photoreceptors, anterior pituitary, and cochlea within the inner ear. Expression is shown using a targeted knock-in of lacZ in the TRß2 exon of the Thrb gene for sections of retina and whole mounts of pituitary and inner ear. RPE, Retinal pigmented epithelium; ONBL, outer neuroblastic layer; c, cochlea; v, vestibular parts of the inner ear; AP, anterior pituitary. D, Transgenes carrying the promoter give cochlear expression, whereas intron sequences between the HindIII and BglII restriction sites were necessary for pituitary and retinal expression. In the retina, expression is detected in immature photoreceptors and, unlike the endogenous gene, in the inner ganglion cell layer (weaker, punctate staining); open arrowheads. In the cochlea, open arrowheads indicate signal in the basal turn, which continues round the spiral to the apex. For a given transgene (Tg), the fraction of Tg-carrying founder lines that gave expression in any of the three tissues is indicated next to the identifier number (e.g. TgA, 3/5 denotes expression in three of five lines that carried TgA). TgA and TgB gave similar results; pictures are shown for TgA. Scale bars in C and D: retina, 100 µm; pituitary, 1000 µm; cochlea, 500 µm.
