Development 113, 1093-1104 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

1093

The transcription factor TTF-1 is expressed at the onset of thyroid and lung

morphogenesis and in restricted regions of the foetal brain

DOMENICO LAZZARO, MELANIE PRICE, MARIO DE FELICE and ROBERTO DI LAURO

European Molecular Biology Laboratory (EMBL), Meyerhofstrasse, 1, 6900 Heidelberg (FRG)

Summary

TTF-1, a homeodomain-containing transcription factor,which is required for the specific expression of thethyroglobulin and thyroperoxidase gene promoters indifferentiated thyroid cell lines, is expressed at the verybeginning of rat thyroid differentiation. TTF-1 mRNA isdetected in the endodermal cells of the thyroid rudimentin the rat embryo and precedes the expression of the twoknown target genes by 5 days. No delay is observedbetween the appearance of TTF-1 mRNA and protein,which shows a clear nuclear localization. In the adultthyroid, TTF-1 is present only in the endoderm-derivedfollicular cells.

Two additional domains of expression of TTF-1 havebeen observed, the lung and restricted areas of the

brain. In the lung, TTF-1 mRNA and protein are alsopresent at the earliest stages of differentiation and arelater confined to the bronchial epithelium. In the brain,TTF-1 appears to be restricted to structures ofdiencephalic origin, including the developing neurohy-pophysis. The early detection of TTF-1 in the endoder-mal cells of the thyroid and lung anlage and in restrictedneuroblast populations indicates that TTF-1 may have arole in cell determination in these three systems and thatadditional mechanisms may be involved in the activationof thyroid-specific gene expression.

Key words: transcription factor, TTF-1, thyroid, lung,brain, rat.

Introduction

Homeodomain-containing proteins play a role in theregulation of morphogenesis in Drosophila and havebeen shown to bind to specific DNA sequences and toexert their effects through transcriptional regulation(Hoey and Levine, 1988; Desplan etal. 1988; Biggin andTijan, 1989; Dearolf etal. 1989). Counterparts of thesegenes have been isolated in mammals, most beingrelated to the Drosophila Antennapedia gene (the Class1 Hox genes) and their patterns of expression duringembryogenesis would suggest that homeodomain pro-teins also play a role in mammalian development(Holland and Hogan, 1988; Gaunt et al. 1988; Kesseland Gruss, 1990). Recently, a number of tissue-specifictranscription factors have been isolated in mammals,for example Oct2 (Clerc et al. 1988; Staudt et al. 1988;Scheidereit et al. 1988), GFHl/Pitl (Bodneref al. 1988;Ingraham et al. 1988), and LFB1 (Frain et al. 1990)which have homeodomains highly divergent from thatof the prototype Antennapedia. In several cases, it hasbeen established that these factors are required for thetranscriptional activation of genes that define a cell type(e.g. Mullerera/. 1988; Frain et al. 1990), indicating thatthey are involved in at least one stage in thedevelopment of a tissue-specific phenotype. In the caseof one tissue specific transcription factor, GHFl/Pitl,which has a role in the activation of growth hormone

(GH) gene expression in differentiated cell lines, it hasbeen demonstrated that synthesis of GHFl/Pitl corre-lates temporally with the activation of GH geneexpression (Dolle" et al. 1990; Simmons et al. 1990).

We have recently isolated the cDNA for a thyroidtranscription factor, TTF-1 (Guazzi etal. 1990). TTF-1contains a novel homeodomain that is necessary andsufficient for its DNA-binding activity. The homeo-domain of TTF-1 is most closely related to that of NK2,a member of a family of homeobox-containing genes inDrosophila (Kim and Niremberg, 1989). In the differ-entiated thyroid cell line FRTL-5, the binding of TTF-1to the thyroglobulin (Tg) promoter is essential for thecell-type-specific expression of this gene (Civitareale etal. 1988) and binding to the promoter of a secondthyroid specific gene, thyroperoxidase, (TPO), isrequired for the full activity of this promoter in thyroidcells (Francis-Lang et al. 1990). Co-transfection of thecloned TTF-1 cDNA and a Tg reporter construct intoseveral non-thyroid cell lines which do not express TTF-1 has demonstrated a specific transactivation of the Tgand TPO promoters as a result of the production ofTTF-1 protein, therefore establishing TTF-1 as atranscriptional activator (M. De Felice, H. Francis-Lang and R. D. L., unpublished observations).

Preliminary analysis demonstrated that TTF-1mRNA is found only in mRNA isolated from lung andthyroids of 4 week old rats (Guazzi etal. 1990). In order

1094 D. Lazzaro and others

to determine if the expression of TTF-1 correlates withthe appearance of specific markers during thyroidorganogenesis, we carried out in situ hybridizationexperiments at several stages of rat development (days10.5 to 17p.c.) with cRNA probes derived from TTF-1,Tg and TPO. We also analyzed the appearance of thethyroid-stimulating hormone receptor (TSHR) mRNA.The cDNA for the rat TSHR has recently been isolated(Akamizu et al. 1990) and is expressed in differentiatedthyroid cells (FRTL-5) but not in several other tissuestested, thereby providing a third thyroid-specificmarker. It is not known if TTF-1 plays a role in thethyroid-specific expression of the TSHR gene.

We demonstrate that TTF-1 mRNA and protein aredetectable at the onset of thyroid organogenesis (day10.5 p.c.) whereas Tg, TPO and TSHR mRNAs appear5 days later. We also found TTF-1 transcripts andprotein in the lung rudiment and in discrete regions ofthe developing brain indicating that TTF-1 may play arole in the differentiation of more than one cell type.

Materials and methods

Northern blot analysisRNA preparation and northern blot analysis was carried outas described in Guazzi et al. 1990. The probe for the northernblot was the TTF-1 3', non-translated region cDNA probedescribed below and was labelled with 32P by random oligopriming.

Tissue preparationEmbryos and foetuses were obtained from natural matingsbetween Wistar rats. Midday of the day of vaginal plug wasconsidered day 0.5 p.c. Samples were staged according to theexternal criteria of Witshi (1962). The samples were fixedimmediately after dissection in 4% paraformaldehyde in l xPBS, pH7.0 for 12 h at 4°C. Subsequently samples werecryoprotected by immersion in 20% sucrose/lxPBS over-night at 4°C and embedded in tissue-teck OCT compound(Miles Scientific, Naperville, IL.). Sections of lOjan were cutusing a cryostat and collected on poly-L-lysine-coated slides,according to Toth et al. 1987.

Preparation of 3SS-labelled riboprobesSingle-stranded RNA probes were prepared and labelled with35S ([a-35S] UTP, >1000Cimmor1, Amersham), accordingto Toth et al. 1987 to a specific activity of S.SxlC^disintsmin~' ng~'. All probes were subcloned into Bluescript vector Results(Stratagene) and transcribed using T3 or T7 RNA polymerase(Stratagene). The TTF-1 antisense riboprobe was a 625 bpfragment derived from the 3' non-translated region of theTTF-1 cDNA (bp 1694-2319, Guazzi et al. 1990). Other TTF-1 probes described in Guazzi et al. 1990 (from the 5'-end of thecDNA and the homeobox) were used in in situ hybridizationexperiments. All the probes gave the same results and the 3'non-translated probe was selected for further use since it gavethe best signal-to-noise ratio. The Tg riboprobe consisted of afragment encoding sequence from the carboxy-terminus ofthe Tg gene (bp 2227-2916, in Di Lauro et al. 1985). The TPOriboprobe is a 500 bp insert from the 5'-coding region of therat TPO gene (bp 1-335 from the AUG plus 155 bp ofadjacent 5'-non-translated, H. F-L, M.P and R. D-L,unpublished). The TSHR riboprobe is an approximately

300 bp insert from the coding region of the rat TSHR cDNAclone (BspHI-EcoRV fragment) and contains a region withresidues unique to the TSHR which are not found in thestructurally related lutenizing hormone receptor (Akamizu etal. 1990). All probes were tested on northern blots before use.

In situ hybridizationIn situ hybridization was carried out as described in Toth et al.1987. After washing and RNAase treatment, the sectionswere dehydrated with graded ethanols containing 300 mMammonium acetate and processed for autoradiography usingKodak NTB2 emulsion (Eastmann Kodak, Rochester, NY)diluted 1:1 with water. Slides were developed after 2-4 weekswith D19 developer (Kodak), washed in distilled water andfixed in AL-4 fixer (Kodak). The slides were stained withtoluidine blue or eosin and coverslips were mounted in Eukitt(O. Kindler GmbH, Freibourg, Germany).

Sample preparation for immunohistochemistryEmbryos and foetuses were collected from the same litters asthose prepared for in situ hybridization and were processedidentically.

TTF-1 antibody preparationRabbits were immunized with a mixture of three peptides El,A2 and Fl, spanning amino acid residues 2-14, 92-104 and110-122 from the TTF-1 coding region, respectively (Guazziet al. 1990). The antibody was purified according to Harlowand Lane, 1988, and further purified by affinity chromatogra-phy on Affi-gel 10 (Bio Rad) coupled with the syntheticpeptides used for the immunization.

ImmunohistochemistryTissue sections were incubated for 20min in PBS/0.1 % triton(v/v) and washed in PBS for 5min, covered with 1.5% goatserum (Vector Lab. Inc., Burlingame, CA) in PBSfor 30min.The sections were then incubated with affinity-purified TTF-1antibody (diluted 1:5000 in PBS) supplemented with 1.5%goat serum for 30min, followed by a 30min incubation with a1:200 dilution of a biotinylated goat anti-rabbit IgG (VectorLab. Inc.). Endogenous peroxidases were inactivated bytreatment with 3 % hydrogen peroxide in water, sections werethen incubated for 30min with a biotin-avidin-peroxidasecomplex (Vector Lab. Inc.). Finally the sections were treatedfor 5min in 0.5mgmr' diaminobenzidine (Sigma), dissolvedin 10 mM TBS, pH7.6 and containing 0.01% hydrogenperoxide. All steps were separated by a wash in PBS/0.1%Triton at room temperature. Sections were dehydrated,cleared with xylol and mounted in Eukitt.

The pattern of expression of TTF-1 at late gestationTo determine the overall pattern of expression of TTF-1in the rat embryo, hybridizations were performed in situon serial sections of rat embryos from 13.5 to 14.5 dayspost coitum {p.c.) using 35S-labelled antisense cRNAprobes from the 3' non-translated region of the TTF-1cDNA (see materials and methods). This initial analysisrevealed the expression of TTF-1 in the developingthyroid and primitive bronchioli of the lung, asexpected from the previous northern blot analysis. Thisin situ hybridization also revealed the presence of TTF-1 transcripts in restricted areas of the brain: the

TTF-1 expression in rat thyroid and lung morphogenesis 1095

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Fig. 1. Expression of TTF-1 in the embryonic rat brain.Poly (A)+ RNAs were prepared from liver, lung, thyroidand day 14 p. c. embryonic heads and electrophoresed onformaldehyde agarose gels and transferred to nylonmembrane. Hybridization was carried out using the 3'-nontranslated region TTF-1 cDNA probe. The TTF-1transcript in thyroid, lung and day 14 p.c. embryonic heads(E14H) is indicated by an arrow (2.4kb). N, indicates thenormalization of the RNA on the filter after hybridizationto a glyceraldehyde-3-phosphate dehydrogenase DNAprobe.

diencephalon and neurohypophysis (see later). mRNAwas prepared from heads dissected from day 14.5 p.c.embryos to day 19 fetuses and analyzed on northernblots using TTF-1 cDNA probes. A transcript havingthe same size as the major TTF-1 transcript in thyroidmRNA is observed in all these embryonic head mRNAs(shown in Fig. 1 for day 14.5 p.c.) using three probesencompassing different regions of the TTF-1 cDNA(Fig. 1 and data not shown). We have also demon-strated that the major start of transcription is the samein all three adult tissues (R. Lonigro, M. De Felice andR. D. L., unpublished observations). Whether thepattern of TTF-1 mRNAs changes during developmentof the three positive tissues must await for a structuraldescription of the minor transcripts.

The distribution of TTF-1 transcripts during thyroiddevelopmentThe thyroid gland originates from the ventral wall of theprimitive pharynx (cranial gut), between day 10.5 to11.5 p.c. (Rugh, 1968; Hebel and Stromberg, 1986, seeFig. 7). The thyroid rudiment develops from a medianendodermal thickening in the floor of the primitivepharynx, forming a small button of cells just above thedilated aortic sac. This thickening, located beneath theposterior part of the tongue anlage, then migratescaudally as a downgrowth known as the thyroiddiverticulum (day 11.5 p.c). The diverticulum is

connected to the pharyngeal cavity by the thyroglossalduct.

Expression of TTF-1 before the appearance of thethyroid anlage (day 7.5 to day 9 p.c.) was undetectable(data not shown), indicating that the onset of TTF-1expression must occur between day 9 and day 10.5 p.c.Similar results have been obtained during mousedevelopment, where TTF-1 expression, measured byRNAase mapping, starts to be detectable around day9.5 p.c. ( M. P., D. L. and R. D. L., in preparation).Serial transversal sections of the cranial gut from days10.5 to 11.5 p . c , showed that, at the level of the thyroidanlage, TTF-1 expression is restricted to the thyroidrudiment (Fig. 2a). At day 11.5 p.c. the thyroiddiverticulum is shaped as a vesicle and TTF-1-specinclabelling seems to be distributed on the ventral edgeand not on the posterior remnant of the thyroglossalduct (Fig. 2b and see also Fig. 3a). At day 12.5 p.c. thethyroid has reached the arcus aortae and by day 13.5p.c, migration is completed. At this stage, the thyroidis still a solid complex of endodermal cells and the TTF-1 signal is homogeneously distributed on the immaturethyroid epithelium (not shown). As shown in Fig. 2c, atday 14.5 p.c, the parathyroids are joining the thyroidwhich at this stage is a lobular shaped structure andbegins to be broken up into a network of epithelialcords by invasion of the surrounding vascular mesen-chyme. TTF-1 transcripts are detected on the thyroidallobes which are located on both sides of the trachea andthere is clearly no signal on the parathyroids or on theepithelium of the upper respiratory airways (Fig. 2c,d).Between days 15 and 17p.c, a few primitive follicularstructures appear in the thyroid lobules between theepithelial cells. TTF-1 expression at day 17 p.c. isclearly restricted to the cords of epithelial cells whichare still arranged in an irregular interdigitating pattern(Fig. 2e).

Relationship between the expression of TTF-1 andthyroid-specific markersIn order to establish whether there is a correlationbetween TTF-1 expression and the onset of the thyroidfunctional markers, riboprobes were constructed fromthe coding regions of the Tg, TSHR and TPO cDNAs(see Materials and methods). Antisense cRNA probeswere transcribed in the presence of 35S-UTP andhybridized in situ to serial sections of embryos andfetuses from days 10.5 to 17 p.c. The results aresummarized in Fig. 3a—i. From day 10.5 to day 14.5p.c, TTF-1 expression is detectable in the developingthyroid (Fig. 3a,d,g), while hybridization with Tg orTSHR probes did not give any signal. As describedabove, from day 10.5 to 11.5 p.c. the thyroid rudimentbegins to migrate and appears as a homogeneous buttonof endodermal cells. At day 14.5 p.c., the first cords ofepithelial cells delineate and by day 15.5 p.c, Tg andTSHR transcripts are detectable in the epithelial cords(shown in Fig. 3e,h). Previous immunocytochemicalstudies in rat revealed immunoreactive Tg at day 15p.c.in the cytoplasm of immature thyroid epithelial cells(Kawaoi and Tsuneda, 1985). Two days later (day 17

1096 D. Lazzaro and others

THYROID

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Fig. 2. TTF-1 expression pattern during thyroiddevelopment. Sections through different axes of ratembryos at various stages of development hybridized witha TTF-1 cRNA probe, (a) Bright-field, transversal sectionthrough the pharyngeal cavity (Ph) of a day 10.5 p.c.embryo. Dark-field, TTF-1 transcripts are detected only inthe thyroid anlage (Th). (b) Transversal section throughthe pharyngeal cavity of a day 11.5 p.c. embryo. Dark-field, TTF-1 transcripts are restricted to the thyroiddiverticulum (Th). (c) Bright-field, transversal section of anembryo at day 14.5 p.c. Dark-field, TTF-1 hybridizationsignal is only on the thyroid lobes, and not detectable onthe parathyroid (Pt, arrow) fusing to the right thyroid lobeor on the larynx (L). (d) Bright-field, mid-sagittal sectionthrough the thyroid isthmus of an embryo day 15.5 p.c.Dark-field, TTF-1 transcripts are localized on the thyroidalconnecting isthmus, (e) Bright-field, frontal section of afoetus, day 17 p.c. Dark-field, silver grains are found in thecords of endodermal cells and the invading vascularmesenchyme is negative. L, larynx; Oe, oesophagus; Ph,pharyngeal cavity; Pt, parathyroid; Sc, spinal cord; Th,thyroid; Tr, trachea. Bars (a, b and e) 153/an; (c) 200/an;(d) 220/an.

p .c) , the first thyroid follicles begin to form and thyroidhormones (T3 and T4) are detectable in the lumen ofthese primitive follicles. At day 17, Tg and TSHRtranscripts are found in the cords of thyroid epithelialcells (Fig. 3f,i).

As this time course experiment demonstrates, thereis a lag of approximately 5 days between the appearanceof TTF-1 rnRNA and that of the thyroid-specificmarkers Tg and the TSHR. This lag also holds for theexpression of the TPO gene (unpublished data). Thelag in appearance of thyroid-specific marker RNAs isnot due to a slow accumulation of small amounts of theRNAs since we observe their abrupt appearance at day15 p.c.

The distribution of TTF-1 transcripts in the developinglungThe lower respiratory system is first observable as themedian laryngo-tracheal groove in the caudal end of theventral wall of the primitive pharynx (Rugh, 1968;Hebel and Stromberg, 1986, see Fig. 7). This grooveproduces a ridge on the external surface of the primitivepharynx. As this diverticulum grows, it becomesinvested with splanchnic mesenchyme and its distal endenlarges to form a globular lung bud at day 10.5 p.c.While the trachea is separating from the cranial gut, thebud rapidly divides into two branches that represent theanlage of the two main bronchi which elongate andgrow distally into the surrounding mesenchyme (day11.5 p.c) .

Transversal sections of an embryo at day 10.5 p.c. (asmentioned above TTF-1 expression is undetectablebefore this stage) showed that the TTF-1 expressionpattern is restricted to the ventrally migrating edge ofthe lung bud and no signal is detectable on the laryngo-tracheal groove (Fig. 4a, day 10.5p.c). One day later astrong signal is present in both branches of the primitivebronchi (day 11.5 p.c, Fig. 4b).

TTF-1 expression in rat thyroid and lung morphogenesis 1097

TTF-1 Tg TSKR

Th

Fig. 3. Appearance of TTF-1, Tg and TSHR mRNAs during thyroid development, (a) At day 11.5 p.c. a transversalsection through the pharynx (Ph) shows the thyroid diverticulum (Th, bright-field) and TTF-1 hybridization signal can beseen in the dark-field view to be confined to the thyroid diverticulum. In a higher magnification of the thyroid diverticulum(a') the silver grains are restricted to the thyroid diverticulum (Th) and TTF-1 transcripts are not seen in the trailingthyroglossal-duct (Td) which connects the diverticulum to the pharyngeal wall (Ph). No signal is apparent in adjacentsections for Tg or TSHR (d and g, respectively). At day 15.5 p.c. in a transversal section through the thyroid, the twolobes of the thyroid (Th) can be observed on either side of the trachea (Tr) in the bright-field. All three genes areexpressed at this stage, in adjacent sections, TTF-1, Tg and TSHR (b, e and h, respectively). At day 17 p.c, a frontalsection through the lobes of the thyroid demonstrates that the TTF-1, Tg and TSHR transcripts are localized in the cordsof epithelial cells in adjacent sections (c, f, i, respectively). L, larynx; Ph, pharynx; Oe, oesophagus; Td, thyroglossal-duct;Th, thyroid; Tr, trachea. Bright-field views: 11.5 dp.c., 15.5 dp.c, 17.0 dp.c. Bars 153 /mi.

Between days 13.5 and 15.5 p.c, the two mainbronchi continue elongating and branching dichoto-mously together with the surrounding splanchnicmesenchyme. Serial sagittal sections of embryos fromdays 13.5 to 15.5 p.c. (Fig. 4c,d) clearly show that TTF-1 is constantly expressed in the bronchial epitheliumduring all of its early stages of differentiation while thesurrounding splanchnic mesenchyme and the epi-

thelium of the upper respiratory airways do not containany detectable TTF-1 mRNA.

TTF-1 expression in the CNSSince TTF-1 transcripts were observed in the brain ofday 14.5 p.c. embryos, several embryos and fetusesfrom days 7.5 to 17 p.c. were sectioned along differentaxes and hybridized in situ with TTF-1 cRNA probes.

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LUNGFig. 4. TTF-1 expression pattern during lung development,(a) Bright-field, transversal section through the pharyngealcavity at day 10.5 p.c. Dark-field, TTF-1 signal is restrictedto the lung bud (Lb) and not in the connecting laryngo-tracheal groove (Lt). (b) Bright-field, transversal section atday 11.5 p.c., the lung bud is branching dichotomously,pre-forming the anlage of the two main bronchi (MB,arrowheads). Dark-field, TTF-1 signal is distributed overthe anlage. (c) Bright-field, a parasagittal section at day15.5 p.c. Dark-field, the signal is restricted to the growingand dividing bronchi (pulmonary primordium) and not inthe surrounding mesenchyme. (d) Bright-field, sagittalsection at day 16.5 p.c., the bronchial tree segmentation isshown at higher magnification. Dark-field, TTF-1transcripts are found in the bronchial cuboidal epithelium(B) and not in the neighboring mesenchymal cells (M).B, segmented bronchi; Lb, lung-bud; Lt, laryngo-trachealgroove; Lu, lung; M, surrounding mesenchyme; PV, pre-vertebral bodies; Sc, spinal-cord. Bars (a) 153 /*m; (b)200/an; (c) 220/an; (d) 60 /an.

Discrete areas of hybridization were detected, from day10.5 p.c., in the diencephalon and in the telencephalicfloor. In the developing diencephalon, TTF-1 tran-scripts are restricted to the hypothalamic area of thediencephalon and to the infundibulum at the earlieststages of their differentiation, day 10.5 p.c. (Fig. 5A,section a). The infundibulum will form the posteriorlobe of the pituitary, the neurohypophysis. The anteriorlobe (glandular part) originates from the Rathke'spouch, which migrates from the vault of the primitivemouth (Rugh, 1968). As is depicted in a transversalsection of an embryo at day 11.5 p.c. and in a sagittalsection of embryo at day 16.5 p.c. (Fig. 5A, sections b,c), TTF-1 transcripts are restricted to the developingneurohypophysis and no signal was ever detected in thepituitary glandular primordium (Rathke's pouch) in anyof the stages examined. In a sagittal section at day 14.5p.c. (Fig. 5B, section c), the overall pattern of TTF-1expression in the brain can be seen and it is apparentthat TTF-1 continues to be expressed in the hypothala-mic area of the diencephalon, in the infundibulum andin the recessus opticus. In the recessus opticus a strongsignal is detectable in the area corresponding to theoptic chiasma (Fig. 5B, section c). A frontal section ofan embryo at the same stage, (Fig. 5B, section b)clearly shows that the rostral boundary of TTF-1expression in the wall of the diencephalon is alwayscontained in the hypothalamic area spanning from thefloor of the third ventricle to the sulcus diencephalicusventralis, which represents the anatomical boundarybetween the hypothalamus and ventral thalamus.Although the TTF-1 transcripts are not always strictlycoincident with this boundary, they never cross theanatomical limit between these two structures. Thisboundary is already established by day 10.5 p.c. as canbe seen in Fig. 5B, section f. Also in this figure and oneday later (11.5 p.c, Fig. 5B, section g), TTF-1transcripts appear to be localized to the floor of thetelencephalic vesicles in an area that corresponds to thedeveloping striatum. At later stages (between days 14.5and 16.5 p.c), the development of the floor is

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Fig. 6. Distribution of TTF-1 protein. In the top of the figure, left corner, a diagram of a parasagittal section of a rat embryo atday 14.5 p.c. showing, boxed, the portion of the embryo displayed in a. (a) A parasagittal section of a 14 day p.c. embryo reactedwith the TTF-1 antibody. Only the lung (Lu), the thyroid (Th) and the diencephalon (Di) show clear positivity. Panels c and dshow higher magnification of the areas labeled i and ii, respectively, in panel a. (d) A transversal section of an embryo at day 10.5p.c, TTF-1 protein is localized in the thyroid anlage (Th) which is located in the anterior wall of the pharynx (Ph). (e) Thethyroid anlage has been counterstained with eosin and is shown at higher magnification. The immunostaining (now seen as darkbrown on a pale pink background) is clearly detected in the nuclei of the thyroid epithelial cells. In the adult rat thyroid (f),counterstained with toluidine, the peroxidase staining (which appears black with this stain) is restricted to the nuclei of theepithelial cells lining the colloid (C) containing follicles (F). The cells of the septa (S) are negative and appear light blue. In g, atransversal section through the lung bud at day 10.5 p.c. shows a specific nuclear staining in the migrating lung bud (Lb) while thelaringo-tracheal groove (Lt) is negative. At higher magnification (h), stained as in f, TTF-1 protein is clearly localized in theanterior edge of the lung bud (Lb) and not in the laryngo-tracheal groove (Lt). In i, a sagittal section of a fetus at day 17 p.c.shows the distribution of the protein in the cells of the cuboidal epithelia lining the bronchioli (B); in the same section, theepithelia of the main bronchi (MB) are negative. In 1, a frontal section (day 10.5 p.c.) through the diencephalic area of the brainwhich delimits the cavity of the third ventricle (III), shows the localization of TTF-1 protein to the hypothalamic region (Ht). Thearrows indicate the boundary of TTF-1 protein as was observed for the mRNA (see Fig. 5). In m, which is a higher magnificationof 1 and is counterstained as in e, the neuroblasts in the hypothalamic wall show an intense nuclear staining. In n, a sagittal sectionthrough the hypophysis of a new born rat, TTF-1 protein is restricted to the neurohypophysis (N). Ill, third ventricle; Ad,adenohypophysis; C, colloid; F, follicular lining epithelia; H, heart; Ht, hypothalamus; L, liver; Lb, lung bud; Lt, laringothrachealgroove; Lu, Lung; N, neurohypophysis; P, pars intermedia; Ph, pharynx; S, vascular mesenchyme of the septa; Te, Telencephalon;Th, thyroid. Bars (a,d,g) 150/an; (b,e,h,c,f,i) 60fan.

TTF-1 expression in rat thyroid and lung morphogenesis 1099

characterized by the appearance of the striated nuclei,lateral and median striatum (Fentress et al. 1981). Asshown in Fig. 5B, section d, in a frontal view at day 15.5p.c., the hybridization signal is restricted to the mediancorpus striatum, while the lateral striatum is clearlynegative. In the same section, it is also possible todetect another area of expression, which is in closeproximity to the median nucleus striatum and possiblycorresponds to the globus pallidum or paleostriatum, anucleus that originates from the median nucleusstriatum (Niimi et al. 1962).

Localization of TTF-1 proteinThe results of the in situ hybridization experiments arenot consistent with an exclusive role of TTF-1 in theactivation of thyroid-specific transcription, since TTF-1mRNA is detected in the thyroid five days beforetranscription of the target genes and, furthermore, it isdetected in tissues different from the thyroid. To assessbetter the significance of the in situ hybridizationresults, an affinity-purified polyclonal antibody wasused to detect the presence of the TTF-1 protein. Thespecificity of the antibody was demonstrated by theexclusive staining of thyroid, lung and diencephalon insections of whole, 14 day p.c., embryos (Fig. 6a, b, c).

To determine whether TTF-1 is detectable at theearliest stages in which we observed TTF-1 mRNA,serial transversal sections from embryos at day 10.5p.c.were analyzed. As expected, due to the absence ofTTF-1 mRNA, the antibody did not label any structureat earlier stages (data not shown). As shown in Fig. 6d,g, 1, TTF-1 is localized in the endodermal cells ofthyroid anlage, of the lung bud and in diencephalicneuroblasts. At higher magnification (Fig. 6e, h, m),the immunostaining is clearly localized to the nuclei. Atday 10.5 p.c, the neuroblasts are still arranged in asingle layer in the developing CNS called the ventricu-lar zone and in Fig. 61, m, a frontal section of thediencephalon shows that the immunolabelling is clearlydistributed in the nuclei of the neuroblasts belonging tothe hypothalamic area. At late gestational stages (day17.5 p.c), in the respiratory system, TTF-1 protein ispresent in the nuclei of epithelial cells of the bronchiolibut not in the trachea and in the main bronchi (Fig. 6i).In the thyroid, TTF-1 protein is localized in the nucleiof the thyroid follicular cells (Fig. 6f). No labelling wasever detected in the vascular mesenchyme of the septasurrounding the follicles (Fig. 6f). In brain at day 17.5p.c., TTF-1 was clearly detected in the neurohypohysis(Fig. 6n).

Discussion

TTF-1 was isolated as a molecule important for theexpression of genes expressed specifically in the thyroid(Civitareale etal. 1989; Guazzi et al. 1990). It contains anovel homeodomain, quite divergent from the class 1(Hox) homeodomains. The Hox genes show wide-spread expression during embryogenesis, being tran-scribed in distinct but overlapping domains along the

antero-posterior axis, and, on the whole, expressionpatterns are found to be region specific rather thantissue specific (e.g. see Kessel and Gruss, 1990; Gaunt etal. 1988). TTF-1, like some other genes that containhomeodomains divergent from the class I homeo-domain (e.g. Evx-1, Bastian and Gruss, 1990; Cdx-1,Duprey et al. 1988, GFHl/Pitl, Dolle" et al. 1990;Simmons et al. 1990), shows a more restricted ex-pression pattern during embryogenesis being expressedspecifically in the thyroid and lung and in restrictedregions in the brain.

TTF-1 expression and thyroid developmentSeveral lines of evidence (see introduction) indicatethat TTF-1 has an important role in the thyroid-specificexpression of both the Tg and TPO genes. Theexpression of TTF-1 in the thyroid anlage at day 10.5p.c. provides a marker for the early determination ofthyroid cells. In addition to its function as a transcrip-tional activator of Tg and TPO genes in differentiatedthyroid cell lines, TTF-1 may play an important role inthe commitment of thyroid cell precursors. The fivedays lag between the appearance of TTF-1 and theexpression of the Tg and TPO genes, which are knowntargets of TTF-1 action, suggests that the presence ofTTF-1 protein is not by itself sufficient for the initiationof transcription of these genes. It is possible that TTF-1is not active during this lag period but requires somesort of modification, which occurs at a time whenthyroid function is initiated. Another possibility is thatadditional factors are required to initiate the expressionof thyroid-specific genes and these appear at a laterstage than TTF-1 in thyroid development. A furtherpossibility is that Tg and TPO genes are not competentto be expressed until day 15 p.c. This last model couldalso explain the discrepancy between the data pre-sented in this paper and the observation that TTF-1 can,in transient co-transfection assays, activate transcrip-tion of both Tg and TPO promoters in non-thyroid cells(M. De Felice, H. Francis-Lang and R. D. L.,unpublished observations).

What could be the nature of the event(s) that, afterthe appearance of TTF-1, trigger the synthesis of Tgand TPO? Specific interactions between thyroid cellprecursors with the surrounding mesenchyme, whichhave been demonstrated to play an important role inthyroid morphogenesis (Hilfer and Stern, 1971), couldbe involved in the activation of thyroid-specific tran-scription. It has also been shown that the interactionbetween TSH and its receptor has a role in thedifferentiation of primary dog thyrocytes (Pohl et al.1990). It is relevant therefore that we find that themRNA for the TSHR appears together with Tg andTPO mRNAs, suggesting that they are subject to asimilar regulation. The notion that TSH is not involvedin the early stages of thryoid differentiation (day 10.5 to15.5 p.c.) is supported by the observation that foetalTSH-secreting cells appear in the anterior pituitarygland of the rat around day 16-17 p.c., (Watanabe andDaikoku, 1979; Begeot et al. 1981), two days after thetranscription of thyroid-specific genes and the appear-

1100 D. Lazzaro and others

So H'

O)

TTF-1 expression in rat thyroid and lung morphogenesis 1101

Fig. 5. TTF-1 expression pattern in the developing CNS(Panel A). Expression of TTE-1 in the developingneurohypophysis. (a) Bright-field represents a transversalsection through the diencephalon of an embryo day 10.5p.c. In the dark-field, the hybridization signal is detected inthe hypothalamic area (Ht, arrowheads) of thediencephalon (Di) and inthe developing infundibulum (In,arrowhead), (b) Bright-field, shows a transversal section(day 11.5 p.c.) through the diencephalon at the level of theoptic stalk (Os). Dark-field, TTF-1 expression is restrictedto the hypothalamus (Ht) and the infundibulum (In). TheRathke's pouch (R) and optic stalk are negative,(c) Bright-field shows the developing neurohypophysis (N,arrowheads) in a sagittal section of a fetus day 16.5 p.c. Inthe dark-field silver grains are restricted to the parsnervosa or neurohypohysis (arrows) and no signal isdetected on the adjacent Rathke's pouch (R, arrowheads,bright-field). Di, diencephalon; Ht, hypothalamus; In,infundibulum; N, neurohypohysis; Os, optic stalk; Ov,optic vesicle; R, Rathke's pouch. Bars (a) 153/an; (b)200/an; (c) 220/an. (Panel B) TTF-1 expression in thediencephalon and the telencephalic floor, (a) A diagram ofa parasagittal section of an embryo at day 14.5 p.c. Twoplanes of section are indicated, (b) a frontal sectionthrough the diencephalon and (d) a frontal section throughthe floor of the telencephalon and the diencephalon, c isthe corresponding dark-field view of the parasagittalsection, and TTF-1 transcripts are restricted to the lungbronchioli (Lu), the diencephalon (Di), optic chiasma (Oc)and the median corpus striatum (Cs). (b) (plane of sectionindicated in a), the bright-field shows the walls of thediencephalon at higher magnification, indicating thecompartmentalization (dotted lines) into four zones: Ht,hypothalamus; Tv, ventral thalamus; Td, dorsal thalamus,and Et, epithalamus, surrounding the third ventricle (m).In the dark-field view, grains are detected in thehypothalamus; the anterior border of expression is in close

proximity to the subthalamic groove (Sg), which representsthe anatomical border between the hypothalamus and theventral thalamus. In d, a frontal section (represented by anarrowhead in a) of an embryo at day 16.5 p.c, thehypothalamic (Ht)-thalamic (T) boundary is indicated bywhite lines and positive signal is detectable in the floor ofthe telencephalon in an area corresponding to the mediancorpus striatum (Csm). At this stage, close to the Csm,another positive nucleus is observable which corresponds tothe paleostriatum (Ps, arrow), a special nucleus ofdiencephalic origin which develops from the median corpusstriatum. The lateral corpus striatum (Csl) is observablelateral to the Csm and no grains were ever detected in thisstructure, e is a diagram showing a parasagittal section ofan embryo at day 11.5 p.c., arrowheads indicate thedifferent planes of section, f represents a transversalsection through the floor of the telencephalon and thehypothalamus at the level of the optic stalk and grepresents a frontal section through approximately thesame area, (f) Dark-field, the distribution of TTF-1transcripts is clearly restricted to the floor of thetelencephalic vesicles which at this stage constitutes thecorpus striatum (Cs, arrowheads) and in the hypothalamicarea (Ht) of the diencephalon. (g) Bright-field (sectiondescribed in e) shows the telencephalic vesicles (Te), thecorpus striatum (Cs) and the diencephalon. In the dark-field view, TTF-1 transcripts are restricted to the corpusstriatum and the hypothalamus (Ht). Arrowheads indicatethat the thalamus (T) is clearly negative. HI, thirdventricle; Cs, corpus striatum; CsL, lateral corpus striatum;CsM, median corpus striatum; Di, diencephalon; Et,epithalamus; Ht, hypothalamus; Lu, lung; My,myelencephalon; Oc, optic chiasma; Or, optic recess; Ps,paleostriatum; Sg, sulcus diencephalicus ventralis; T,thalamus; Td, dorsal thalamus; Te, telencephalic vesicles;Tv, ventral thalamus. Bars (b) 200/an; (g) 153/an.

ance of Tg protein (our work and Kawaoi and Tsuneda,1985).

The simultaneous appearance of TTF-1 mRNA andprotein contrasts with the situation of GHFl/Pitl, atranscription factor involved in the regulation of growthhormone (GH) gene expression in the developingpituitary of the mouse. In this system, althoughGHFl/Pitl transcripts appear between days 12.5 and13.5 p.c. in the developing pituitary, GHFl/Pitl is post-transcriptionally regulated and no protein is detectableuntil day 15.5 p.c, exactly with the appearance of theGH target gene transcripts (Doll£ et al. 1990).However, in the developing rat pituitary, there is noevidence for the same translational regulation and agood temporal correlation is observed between theappearance of GHFl/Pitl and GH mRNA (Simmons etal. 1990).

TTF-1 expression in the lungLung and thyroid develop both from endodermalthickenings along the anterior wall of the pharynx andTTF-1 transcripts are detected at the beginning of theirmigration towards the surrounding splanchnic mesen-chyme. The hybridization signal is restricted to the

anterior migrating edge of the two diverticula and boththe connecting stalks (thyroideal-groove and laryngo-tracheal groove) are negative (see Fig. 7). The detec-tion of TTF-1 transcripts in restricted areas of thethyroid diverticulum and of the lung bud (i.e. in theanterior migrating edge) suggests that during the earlymorphogenesis of these two organs there are subsets ofcells that are already committed. This hypothesis issupported by the finding that, in adult rabbit lung thetrachea and bronchi and the bronchioli contain twodifferent stem cell populations which are responsiblefor the renewal of the lining epithelia (Nettesheim et al.1990). It is also of interest that two types of lungmesenchyme have been described: bronchial, whichpromotes formation of bronchial buds from trachealepithelium, and tracheal, which inhibits bronchialformation (Wessels, 1970; Alescio and Cassini, 1962).The expression of TTF-1 in the bronchiolar epitheliumand its absence in the upper respiratory airwayscorrelates with the presence of these two differentmesenchymes and could be the result of specificinteractions of endodermal cells with regionally special-ized mesoderm (see for example, Taderera, 1967;Spooner and Wessels, 1970).

1102 D. Lazzaro and others

TTF 1 Expression

Fig. 7. TTF-1 transcripts are asymmetrically distributed inthe thyroid diverticulum and lung bud. A schematicdiagram of the cranial gut (primitive pharynx, Ph)depicting the onset of migration of the thyroid diverticulum(Th) and the lung bud (Lb) from the anterior wall of thepharynx. The thyroid and lung develop in close proximityas two endodermal thickenings along the same median axisin the anterior wall of the pharynx, projecting into thesurrounding splanchnic mesenchyme. Note that the TTF-1transcripts (represented by shading) are restricted to theanterior migrating edge of the thyroid diverticulum and ofthe lung bud while the thyroglossal-duct (td) and thelaryngo-tracheal groove (It) are negative.

TTF-1 expression in the brain is restricted to structuresof diencephalic originThe expression ofTTF-1 in the brain is specific for somediencephalic (e.g. hypothalamus, neurohypophysis,optic chiasma) or telencephalic structures of diencepha-lic origin (e.g. median corpus striatum, globus palli-dus). There are not many examples of homeobox-containing genes that are expressed in such restrictedareas of the CNS; En-2 and Hox-2.9 are expressed inthe midbrain-hindbrain junction and in rhombomere 4,respectively (Davis and Joyner, 1988; Murphy et al.1989; Wilkinson et al. 1989, for review see Lobe andGruss, 1989).

An important aspect of the domains of TTF-1expression in the brain is the correlation of someexpression boundaries with precise anatomical ordevelopmental landmarks. Although the segmentalorganization of the forebrain is unknown, in some casesmolecular markers have been identified that may

correspond to forebrain segmental territories (e.g. seePuelles et al. 1987; Mori et al. 1987; Layer et al. 1990).The hypothesis that the TTF-1 expression boundariesmay correspond to segmental patterns in the forebrainis supported by the observation that genes related toTTF-1 are expressed in overlapping domains along therostro-caudal axis of the developing forebrain (Price etal. 1991 and unpublished data).

Conclusion

The experiments presented in this paper suggest thatTTF-1, isolated and defined as a maintenance factor forthyroid differentiation, may have a role in thecommitment of the precursors for thyroid follicularcells, bronchiolar epithelium and diencephalic neuronalsub-populations. Furthermore, these experiments indi-cate that other factor(s), in addition to TTF-1, may berequired for in vivo activation of the thyroglobulinpromoter. In this context, it is interesting that themRNA encoding Pax-8, a putative transcription factor,has been detected in the developing mouse thyroid, inaddition to kidney and, transiently, in the CNS(Plachov etal. 1990). Furthermore, a specific set of Hoxgenes has been reported to be expressed in mousethyroid at day 12.5 p.c. (Gaunt et al. 1988), but it is notclear whether their expression is restricted to endoder-mal cells. We are currently testing whether any of thesefactors may cooperate with TTF-1 in the activation ofthyroid-specific transcription.

We are very grateful to Drs, D. Duboule, 1. Mattaj, P.Doll6, J. Zakany and H. Francis-Lang for critical reading andhelpful discussion of the manuscript and Dr T. Graf, forcomments. We thank Dr L. Kohn for giving us the TSHRprobe.

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{Accepted 20 August 1991)