The thyroglobulin gene evolutionary and regulatory issues

cific enhancer of the human glycoprotein

hormone a-subunit gene: dependence on cyclic AMP-inducible elements. Mol Cell Biol Deutsch ner

1987; 7:3994. P, Hoeffler J: Structural

J, Jameson

J, Lin J, Habe-

determinants

scriptional activation DNA elements. J 263:18,466.

for

tran-

by CAMP-responsive Biol Chem 1988;

Jameson J, Jaffe R, Deutsch P, Albanese C, Habener J: The gonadotropin a-gene contains multiple protein that interact to modulate responsive transcription. 1988; 26319879.

binding domains basal and CAMPJ Biol Chem

Jantzen H. Strahle U. Gloss B. et al.: Coonerativity of glucocorticoid response elements located far upstream of the tyrosine amino. transferase gene. Cell 1987; 49:29. Maniatis T, Goodbourn S, Fischer J: Regulation of inducible and tissue-specific gene expression. Science 1987; 236: 1237. Quinn P, Wong T, Magnuson M, Shabb J, Granner D: Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol 1988; 8:3467.

The Thyroglobulin

Roesler W, Vandenbark G, Hanson R: Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 1988; 263:9063. Roesler W, Vandenbark G, Hanson R: Identification of multiple protein binding domains in the promoter-regulatory region of the phosphenolpyruvate carboxykinase (GTP) gene.

J Biol Chem

1989; 264:9657.

Schule R, Muller M, Otsuka-Murakami H, Renkawitz R: Cooperativity of the glucocorticoid receptor and the CACCC-box binding factor. Nature 1988; 332:87. Short J, Wynshaw-Boris A, Short H, Hanson R: Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoterregulatory region. J Biol Chem 1986; 261:9721. Strahle U, Schmid W, Schutz G: Synergistic action of the glucocorticoid receptor with transcription factors. EMBO J 1988; 713389. Tsai S, Tsai M, O’Malley B: Cooperative binding of steroid hormone receptors contribute to transcriptional synergism to target enhancer elements. Ceil 1989: 57:443. TEM

Gene

Evolutionary and Regulatory Issues Daniel Christophe

and Gilbert

Vassart

The coding information for thyroglobulin synthesis is contained in a large transcription unit, which is made from the juxtaposition of short repetitive gene modules and of a copy of an ancient acetylcholinesterase homologue. Minor alternatively spliced transcripts with conservation of the reading frame seem to be common. Whether they have a role or represent noise in the splicing phenomena is unknown. Transcription of the thyroglobulin gene is controlled by CAMP through a pathway involving sequence motifs and trans-acting factors that differ from those identified so far in other systems.

Thyroglobulin is a high molecular weight dimeric glycoprotein (2 X 330 kDa) that constitutes the precursor for thyroid hormone synthesis (Van HerlC Daniel Christophe is at the IRIBHN, Fact&C de MCdecine, UniversitC Libre de Bruxelles; Gilbert Vassart is at the IRIBHN and the Service de Genetique Medicale, HBpitaI Erasme, Campus Erasme, Bat. C, 808 route de Lennik, 1070 Bruxelles, Belguim.

TEM SeptemberlOctober

et al. 1979). Thyroglobulin production is restricted to the follicular cells of the thyroid gland and is controlled by thyrotropin (TSH). Analysis of the primary structure of thyroglobulin protomer (2748 amino acids) reveals a complex structure made up of four distinct regions (Mercken et al. 1985; Malthiery and Lissitzky 1987) (Figure 1). Domain A extends over 1200

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residues

from

(about one-third

the

amino

terminus

of the peptide) and con-

tains 10 repeats (type 1) having a consensus sequence dividual

-60

repeat

amino acids long. Inmembers,

however,

display variable spacing between some blocks of conserved residues at precise positions, the amount of inserted material varying from a few to up to 250 residues. Domain B (50 residues) is composed of three repeats of a shorter motif (type 2), and domain C (520 residues) consists of the juxtaposition of five copies of two related motifs (types 3a and 3b). The landmarks of these three types of motifs are highly conserved cysteine residues. The D domain, which contains the 600 carboxy-terminal residues, shows no internal homology, but displays a significant sequence similarity (28%) with the whole sequence of acetylcholinesterase from Torpedo califomica (Schumacher et al. 1986). The so-called”acceptor” tyrosine residues involved in the coupling reaction leading to hormone formation have been localized close to both extremities of the peptidic chain (Figure 1). The thyroglobulin gene has been mapped to chromosome 8q24, distal to the c-myc locus, in humans (Baas et al. 1985). This synteny between c-myc and thyroglobulin is conserved in rat and mouse, on chromosomes 7 and 15, respectively (Brocas et al. 1985). The use of thyroglobulin probes allows the detection of several restriction fragment length polymorphisms, making the thyroglobulin locus a useful marker for this region of the human chromosome 8 (Baas et al. 1985; Simon et al. 1987), and providing the tools to study cosegregation of hereditary ulin alleles.

??

Structure

goiter with thyroglob-

of the Thyroglobulin

Gene

So far, data are available on the structure of human (Baas et al. 1986), bovine (de Martynoff et al. 1987), and rat (Musti et al. 1986) thyroglobulin genes. All studies point to a common picture of the gene in the different species. The thyroglobulin gene appears to be more than 250 kb long, the 8450-base messenger sequence being distributed among 42 exons. All exons, except exons 9 and 10 (- 1100 and 600 bp, respectively) have a size of 100-200 bp. The proportion of intronic material clearly distinguishes two domains in the gene itself. The 5’

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351

lwww

I

type 1 homology

I

w

type 2 homology

KXq

type 38 homology

m

type 3b homology

m

AChE-like domain

1

hormonogenic site

-

1

1

0

P

Figure 1. Subdomain of individual repeated

to

100 aa

3

CWCVD--G-E--GTR n

to 4 aa

4

to 124 aa

organization of thyroglobulin protomer. motifs, of the acetylcholinesterase(AChE)-like

The approximate domain,

6

portion (30 kb, about one-third of the coding information) has an average intron-exon ratio of 10, whereas this ratio rises to nearly 50 for the remaining part of the gene. Accordingly, a few extremely large introns (of a size up to 60 kb) have been identified in the latter region of the gene.

Evolutionary Thyroglobulin

Aspects of Gene Organization

Intron-exon junctions were precisely localized in the 5’ portion of the gene (Parma et al. 1987). The part of the gene corresponding to the A domain of the thyroglobulin protomer (the first aminoterminal third of the peptide) contains 16 exons. When related to the repetitive organization of this region of the protein (10 type 1 repeats), intron positions fall either between the repeat motifs or within the repeats, at positions where they may contain inserted material. Intron 2 constitutes an exception to this rule, as it interrupts an invariant block of sequence of the repeats, but falls at a position on the boundary of an internal homology in the repeat itself. This may reflect an initial duplication event at the

352

to 47 aa

positions and of the

hormonogenic tyrosines are indicated. The position of introns (awows) are indicated with their number on a blowup of a consensus sequence of type I repeat (one-letter acid code). Triangles indicate the position and the size range of the inserts disrupting ual type 1 repeats.

??

C

n

n

n aa 6

PVQC

15.9.1610 3.13. 4. 7.

6 11

l”*”

1’

G-F

Y-PQC

CE

n 2 to 240

1’”

11

together aminoindivid-

origin of a primordial thyroglobulin gene type 1 motif. Whereas the introns that fall at the borders of the repeats are obvious traces of the serial duplication events responsible for the evolution of this region of the gene, the significance of the introns found at a few defined positions inside some repeats is less evident. Based on the observations that (a) the positions always coincide with places where the repeats contain a variable amount of inserted material; (b) the DNA sequences coding for inserted residues have a lower GC content; and (c) some sequence similarity can be found between sequences that are inserted at homologous positions in different repeats, we have proposed the hypothesis that these introns reflect a highly complex structure of the original repeat unit. The latter would have been composed of several exonic portions, most of the introns having been lost progressively during evolution, but some of them becoming partially”exonized” by sliding of intronic material into the coding sequence repeat. Interestingly, both a dispensable short exon of the gene for the murine Ia-associated invariant chain (Koch et al. 1987) and a segment of a

major

gastrointestinal

tumor-associ-

ated antigen (GA733; Linnenbach et al. 1989) encode a protein domain presenting a significant sequence similarity with the thyroglobulin type 1 repeat, which suggests that the same basic coding unit has been used many times in gene evolution. In the 3’ part of the gene, on the other hand, most of the few intron-exon junctions that were determined perfectly match those found in the acetylcholinesterase gene (Chatonnet and Lockridge 1989), which indicates that present-day thyroglobulin results from the fusion of a shorter ancient thyroglobulin gene with an ancestor of the acetylcholinesterase gene.

??

Alternative Splicing of the Thyroglobulin Primary Transcript

The intricate structure of the thyroglobulin gene is provocative with respect to the processing of the primary transcript. Not only the presence of some unusually large introns, but also the recent (in terms of evolution) remodeling of intron-exon boundaries may be sources of aberrations in the maturation process. Several observations, indeed, point to the existence of alternative splicing leading to minor thyroglobulin molecular species, whose function, if any, is not clear. The most remarkable case is found in the hereditary goiter of Afrikander cattle, where a mutation, creating a stop

0 1990, ElsevierScience PublishingCo.,Inc. 1043.2760/90/$2.00

TEM SeptemherlOctober

codon in exon 9, is partially missplicing

tion of the defective transcripts Clearly,

silenced

by

of the message and eliminaexon in some of the

(Ricketts

alternative

et splicing

al.

1987).

of exon 9

exists in normal thyroid also, leading to a minor, but detectable, shorter mRNA species. Other alternatively spliced mature mRNA molecules have been identified both in beef (Mercken et al. 1989) and in humans (Malthihy et al. 1989). For example, minor transcripts were constantly observed in beef thyroglobulin complementary DNA preparations. They were shown to correspond to the deletion of a block of two exons in the 3’ part of the primary transcript. In this case, the two exons that are “skipped over” are separated by an intron of moderate size, while both introns on the borders are very large (20-60 kb). These characteristics make the thyroglobulin gene an interesting model to investigate the limits in precision of the splicing machinery and the fate or possible role, of misspliced transcripts. In terms of evolution, some noise in the

Thyroglobulin be controlled, of transcription

production

appears

to

at least in part, at the level (Van Heuverswyn

et al.

controlled

tein synthesis

excess circu-

lating TSH does not increase transcription rate of the gene over normal values, transcription is dramatically reduced if TSH is withdrawn. Injected TSH is subsequently able to restore transcriptional activity rapidly (in -1 h). The situation seems somehow different in cultured thyroid cells, as both in the differentiated cell line FRTL-5 (Lee et al. 1989) and in primary cultures of thyrocytes (Gerard et al. 1989) TSH action on thyroglobulin gene expression is much slower (several hours). In these systems, it has also been shown that the TSH effect requires ongoing protein synthesis, as it can be blocked by addition of the protein synthesis inhibitor cycloheximide. An obvious difference between in vivo and in vitro situations is

roglobulin mRNA has the potential to encode a 243 amino-acid polypeptide containing the major hormonogenic domain of the molecule. Future work will tell us the functional significance of this transcript and whether a similar situation exists in humans.

??

Control of Thyroglobulin Gene Expression: The Role of CAMP

The expression of the differentiated functions that participate in hormone production in the thyroid cell are positively controlled by TSH, whose intracellular effects are mediated by the CAMP second messenger pathway (Dumont et al. 1989). Indeed, most of the TSH effects on these cells can be mimicked in vitro by CAMP agonists (forskolin, cholera toxin).

TEM SeptemberlOctober

the existence

in the former

of a highly

ordered follicular structure, which involves orderly contacts between individual ceils. Whether the differences in cell-cell contacts (which have been shown to be of some importance regarding the expression of differentiated functions in related systems; Clayton et al. 1985; Bosco et al. 1989) are responsible for the observed discrepancy remains to be elucidated. Although CAMP alone can elicit transcription of the thyroglobulin gene in cultured thyrocytes, insulin also appears to have an independent effect on the transcription rate of the gene (Santisteban et al. 1986; Gerard et al. 1989). The genes whose transcription is controlled by CAMP can be subdivided into two classes according to the kinetics of transcriptional induction (Roesler et al. 1988). Genes that are controlled in a rapid fashion (usually within minutes) harbor a “classic” CAMP responsive element (CRE) in their promoter region

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phosphoenol pyrgenes). On the

other hand, a few genes are known to be

have shown that whereas

thal otherwise. Very recently a new splice variant of thyroglobulin mRNA has been reported in FRTL-5 cells and in rat thyroid fol-

no relation to any known thyroglobulin sequence. This alternatively spliced thy-

carboxykinase

1984 and 1985). In vivo studies in the rat

splicing phenomena would allow the “testing” of mutations that would be le-

lowing stimulation by TSH (Graves and Davies 1990). It consists of a 0.9-kb mRNA containing the first five exons of the gene spliced to a 3’ sequence with

(e.g., somatostatin, uvate

at a slower rate (active is also required

pro-

for most

of them): genes for lactate dehydrogenase, p-subunit of chorionic gonadotropin, adrenal steroid hydroxylases (Waterman et al. 1986), adipocyte aP2 (Cook et al. 1988), fibronectin (in HT-1080 cells; Dean et al. 1989), and thyroglobulin. They generally do not contain a canonical CRE in their 5’-flanking regions [lactate dehydrogenase, fibronectin, and steroid ilp-hydroxylase (Mouw et al. 1989) genes are exceptions].

??

Study of Thyroglobulin Gene Promoter Sequences: Toward a Consensus Sequence for Some Genes Slowly Responding to CAMP?

The DNA sequences of 5’-flanking regions of thyroglobulin genes have been determined in three species: human (Christophe et al. 1985), beef (de Martynoff et al. 1987), and rat (Musti et al. 1987). When aligned, they point to two conserved features (Figure 2): a highly conserved (73%) region extending from about - 80 to - 140 bp (relative to transcription start), and the presence of a block of repetitive DNA not far upstream (a long homopurine-homopyrimidine stretch in both human and rat, a shorter homopurine-homopyrimidine region and a member of the bovine monomer family of repeats in the beef). None of the three sequences exhibits a CRE consensus motif. Transient expression experiments, involving either the rat promoter in FRTL5 cells (Musti et al. 1987; Lee et al. 1989) or the bovine promoter in dog (Christophe et al. 1989) thyrocytes in primary culture, indicate that only -200 bp of proximal 5’-flanking DNA are required to confer thyroid-specific expression and proper transcriptional control by TSH and CAMP. Replacing the bovine -801 - 140 box by its human counterpart does not significantly alter the control by CAMP in the transient assay (our unpublished results). Comparison of the thyroglobulin gene’s proximal promoter sequences with those of the other genes that respond slowly to CAMP reveals a striking similarity between a portion of the - 80/ - 140 conserved region and both the FSE 2 element found in adipocytes genes

Co., Inc. 1043.2760/90/$2.00

353

aP2 and glycerol-3-phosphate

dehydro-

genase (Cook et al. 1988), and a segment of the fibronectin promoter (Dean et al. 1989) (Figure 2). A similar resemblance also exists with a segment of one of the

a -142

Human -512

Identification Trans-Acting

of Thyroid-Specific Factors

I +i -304

73 % -136 I -77

Ir+ x +i

BMF -556

-519

-409

-221

73 % -130:-70

Rat

Ir'

Pu/Py -410

??

xl+

Pu/Py

6-phosphofructo-2-kinaselfructose-2,6bisphosphatase gene promoters (Figure 2), although it is not known at present if transcription initiation from this promoter is controlled by CAMP (Darville et al. 1989). Whether these sequence similarities reflect the existence of a common mechanism of transcriptional control, involving identical, or, more likely, closely related, trans-acting factors, remains to be investigated.

-El

I: +1

-295

b

Studies in cultured thyroid cells have shown that CAMP elicits hypersensitivity to deoxyribonuclease I digestion in the chromatin of the proximal 5’-flank-

FS;a;2e;i;;;t

ing sequences of the thyroglobulin gene (Hansen et al. 1988). Nuclear factors recognizing this part of the bovine or rat

fibronectin

-122

-106

T G A C T C A G A G G A A A A C A

;~WJJyp,y/1;$T G A C f A G C A G A G A A A A C A TGACCGCAAAGGAAACC -249

-265

thyroglobulin promoter have been identified and are currently being characterized. A nuclear thyroid

protein

tissue

identified

and primary

in beef

cultured

dog

-801 - 140 conserved region (- 107 to - 126) in the bovine promoter (Hansen et al. 1989). Introduction of point mutations in, or deletion of, the - 107/- 126 region

thyrocytes

binds

to a part

of the

of the bovine promoter drastically reduces, or abolishes, the transcriptional induction by CAMP in the transient transfection assay (Donda et al. in preparation). Two thyroid-specific nuclear factors have been found in FRTL-5 cells (Civitareale et al. 1989). Thyroid transcription factor I binds to a sequence motif that is repeated three times in the rat promoter. One of these repeats is contained in the - 80/- 140 conserved region shared by the other promoters, whereas the two additional copies are specific for the rat promoter. Replacing either of the two rat-specific repeats with unrelated sequences results in a dramatic decrease of promoter activity in transfected FRTL-5 cells. Unexpectedly, this is not observed when the motif contained in the - 80/- 140 conserved region is mutated. Thyroid transcription factor II recognizes a portion of the -go/140 conserved region in the rat sequence, at the exact position where

354

PFK-2/FBPase-2

T G G A C T G A A G A G A A A A -90

-75

Figure 2. (a) Schematic representation of thyroglobulin gene 5’-flanking sequences organization in three species. PuiPy, homopurine-homopyrimidine segment; and BMF, bovine monomer family repeat unit. The empty box represents the conserved region. (b) Sequence similarities between part of the thyroglobulin promoter conserved region (represented as the consensus sequence found between - 90/- 95 to - 107/ - 112 in the three species), the FSE 2 element from adipocyte gene aP2, the fibronectin promoter, and one of the 6-phosphofructo-2-kinasei fructose-2,6-bisphosphatase (PFK-ZiFBPase-2) gene promoters.

similarities are found with the adipocytes genes, fibronectin, and 6-phosphofructo - 2 - kinase I fructose - 2,6 - bis phosphatase promoters. No data on the role of this interaction with regard to the control of transcription by CAMP are currently

??

available.

Prospects

The main current research aims at the understanding of the mechanism by which CAMP acts on the transcription of the thyroglobulin gene. The identification of the precise DNA sequences and trans-acting factors involved in this regulation could shed light on the way some other genes responding slowly to CAMP are transcriptionally controlled.

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The situation seems, however, still obscure at the present time, since no common picture emerges from the data collected in the two systems studied so far. Whereas studies involving the bovine promoter in primary cultured thyrocytes point to a dominant role of the - 801- 140 conserved region, the data obtained by transfecting the rat promoter into FRTL-5 cells do not support the same conclusion. Thyroid transcription factors I and II from FRTL-5 cells do not bind to the rat counterpart of the bovine sequence recognized by the nuclear factor identified in vivo and in primary cultures. Functional data also indicate that both the basal activity and the transcriptional response to CAMP of the rat promoter are determined pri-

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TEM September/October

marily

by sequences

that lie outside

the evolutionarily conserved promoter. Does this reflect

of

part of the the differ-

ences between the experimental

D.C. is a Research

Associate

at the Na-

tional

Fund

Scientific

Research

(FNRS,

Belgium).

for

its 5’-flanking

systems

tempting to speculate that these differences reflect the response to CAMP of thyrocytes in three different stages of differentiation. In vivo, or in tissue slices, transcription of the gene (as well as translation of the messenger RNA; Davies et al. 1978) could be modulated rapidly, by activation (possibly through

References Baas F, Bikker

H. Geurts van Kessel, A et al.: The human thyroglobulin gene: a polymorphic marker localized distal to c-MZ~C on chromosome 8 band q24. Hum Genet 1985; 69:138.

Baas F, van Ommen G-JB, Bikker A, Arnberg AC, de Vijlder JJM: The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb. Nucleic Acids Res 1986; 14:5171. Bosco D. Orci L, Meda P: Homologous but not heterologous contact increases the insulin secretion of individual pancreatic B-cells. Exp Cell Res 1989; 184:72. Brocas H, Szpirer J, Lebo RV, et al.: The thyroglobulin gene resides on chromosome 8 in man and on chromosome 7 in the rat. Cytogenet Cell Genet 198.5; 39: 150.

phosphorylation) of preexisting factors, whereas some redifferentiation of the cell would be required for thyrocytes in

Chatonnet A, Lockridge 0: Comparison of butyrylcholinesterase and acetylcholinesterase. Biochem J 1989; 260:625.

primary culture and, even more so, for the FRTL-5 cell line.

Christophe D, Cabrer B, Bacolla A, Targovnik H, Pohl V, Vassart G: An unusually long polv(purine)-poly(pyrimidine) sequence is located upstream from the human thyroglobulin gene. Nucleic Acids Res 1985; 13:5127.

Recently,

mice harboring

thyroglobu-

lin-chloramphenicol-acetyltransferase transgenes have been obtained (Ledent et al. in preparation). As expected, expression of the reporter gene was observed in the thyroid only. Apart from their importance in identifying the sequences involved in the tissue specificity of thyroglobulin gene expression, these experiments raise the interesting possibility of using the thyroglobulin promoter to specifically target expression of any given gene to the thyroid in a living animal.

??

Acknowledgments

The continuous interest and support of Dr. J.E. Dumont are deeply acknowledged. The framework of this review article makes constant use of the work of our past or present colleagues A. Bacolla, H. Brocas, B. Cabrer, C. Christophe-Hobertus, G. de Martynoff, A. Donda, C. Gerard, C. Hansen, F. Javaux, G. Juvenal, C. Ledent, A. Leriche, L.Mercken, J. Parma, V. Pohl, H. Targovnik, and B. Van Heuverswyn. Studies conducted in the authors’ laboratory were supported by grants from the Belgian Minis&e de la Politique Scientifique (Sciences de 2u Vie), FRSM, FNRS, NIH, the Solvay Company, and ARBD (asbl).

TEM SeptemberlOctobrr

region.

L, van Omorganizagene and of

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164:591.

used (a differentiated but immortal cellline, FRTL-5, vs. thyrocytes in primary culture), the existence of species-specific mechanisms of transcription control, or merely our still too limited understanding of the phenomenon? Another important point to be clarified concerns the dramatic difference observed in the kinetics of transcriptional induction of the gene in vitro (-10 h) as compared to in vivo or in tissue slices (1 h). It is

de Martynoff G, Pohl V, Mercken men G-JB, Vassart G: Structural tion of the bovine thyroglobulin

Christophe D, Gerard C, Juvenal Identification of a cAMPresponsive in thyroglobulin gene promoter. Endocrinol 1989; 64:s.

G, et al.: region Mol Cell

Civitareale D, Lonigro R, Sinclair AJ, Di Lauro R: A thyroid-specific nuclear protein essential for tissue-specitic expression of the thyroglobulin promoter. EMBO J 1989; 8:2537. Clayton DF, Harrelson AL, Darnell JE: Dependence of liver-specific transcription on tissue organization. Mol Cell Biol 1985; 5:2623. Cook JS, Lucas JJ, Sibley E, et al.: Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and CAMP. Proc Nat1 Acad Sci USA 1988; 85:2949. Darville MI, Crepin KM, Hue L, Rousseau GG: 5’-Flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinaseifructose-2,6-bisphosphatase. Proc Natl Acad Sci USA 1989; 86:6543. Davies E, Dumont JE, Vassart G: Thyrotropin-stimulated recruitment of free monoribosomes on to membrane-bound thyroglobulin-synthesizing polyribosomes. Biochem J 1978; 172:227. Dean DC, Blakeley P, Hennighausen inducibility and of the fibronectin 1989; 9: 1498.

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Dumont JE, Vassart G, Refetoff S: Thyroid disorders. In Striver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease, 6th ed, ~012. New York, McGraw-Hill, 1989, p 1843. Gerard CM, Lefort A, Christophe D, et al.: Control of thyroperoxidase and thyroglobulin transcription by CAMP: evidence for distinct regulatory mechanisms. Mel Endocrinol 1989; 3:2110. Graves PN, Davies TF: A second thyroglobulin mRNA species (rTg-2) in rat thyrocytes. Mol Endocrinol 1990 (in press). Hansen C, Gerard C, Vassart G, Stordeur P, Christophe D: Thyroid-specific and CAMPdependent hypersensitive regions in thyroglobulin gene chromatin. Eur J Biochem 1988; 178:387. Hansen C, Javaux F, Juvenal G, Vassart G, Christophe D: CAMP-dependent binding of a trans-acting factor to the thyroglobulin promoter. Biochem Biophys Res Commun 1989; 160:722. Koch N, Lauer W, Habicht J, Dobberstein B: Primary structure of the gene for the murine la antigen-associated invariant chains (Ii): an alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin. EMBO J 1987; 6:1677. Lee N-T, Nayfeh SN, Chae nuclear protein factors mone-responsive region of thyroglobulin gene by thyroid FRTL-5 cells. J 264~7523.

C-B: Induction of specific for horduring activation thyrotropin in rat Biol Chem 1989;

Linnenbach AJ, Wojcierowski J, Wu S, et al.: Sequence investigation of the major gastrointestinal tumor-associated antigen gene family, GA733. Proc Nat1 Acad Sci USA 1989; 86:27. Malthiery Y, Lissitzky S: Primary structure of human thyroglobulin deduced from the sequence of its 8448-base complementary DNA. Eur J Biochem 1987; 165:491. Malthiery Y, Marriq C, Berg&Lefranc J-L, et al.: Thyroglobulin structure and function: recent advances. Biochimie 1989; 71:195. Mercken L, Simons M-J, Swillens S, Massaer M, Vassart G: Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431-base complementary DNA. Nature 1985; 316:647. Mercken L, Simons M-J, Brocas H, Vassart G: Alternative splicing may be responsible for heterogeneity of thyroglobulin structure. Biochimie 1989; 71:223. Mouw AR, Rice DA, Meade JC, et al.: Structural and functional analysis of the promoter region of the gene encoding mouse steroid 1 lp-hydroxylase. J Biol Chem 1989; 264:1305.

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specific factor recognizes the rat thyroglobulin promoter. Nucleic Acids Res 1987; 15:8149.

Parma J, Christophe D, Pohl V, Vassart G: Structural organization of the 5’ region of the thyroglobulin gene: evidence for intron loss and “exonization” during evolution. J Mol Biol 1987; 196:769. Ricketts

MH, Simons

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L, Dong Q, Vassart G: A nonsense mutation causes hereditary goiter in the Afrikander cattle and unmasks alternative splicing of thyroglobulin transcripts. Proc Nat1 Acad Sci USA 1987; 84:3181.

Roesler WJ, Vandenbark GR, Cyclic AMP and the induction gene transcription. J Biol 263:9063.

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Van Heuverswyn B, Leriche A, Van Sande J, Dumont JE, Vassart G: Transcriptional control of thyroglobulin gene expression by cyclic AMP. FEBS Lett 1985; 188:192. Waterman MR, Mason JI, Zuber MX, John ME, Rodgers RJ, Simpson ER: Control of gene expression of adrenal steroid hydroxylases and related enzymes. Endocr Res TEM 1986; 12:393.

systems can be found in other recent re-

main emphasis of this article will be on the human fetus and neonate, but appropriate corollaries in other species are also presented.

ment independently, and become fully interactive as the last link of their maturation. Although we know a great deal about the development of morphology and endogenous hormone levels in fetal endocrine tissues, maturation of the functional interactions has remained a secret of the well-sheltered intrauterine environment. This minireview deals with some of the recent findings and persisting controversies of the endocrine physiology of the fetal hypothalamic-pituitary-gonadal axis. More comprehensive the presentations on ontogeny of the reproductive endocrine

0 1990, Elsevier

Nucleic

views (Mulchaney et al. 1987; George and Wilson 1988; Reyes et al. 1989). The

The different compartments of the fetal hypothalamic-pituitarygonadal axis, the hypothalamus, anterior pituitary, and gonads, probably start their embryonic development independently, and become fully interactive as the last link of their maturation. The developing hypothalamic-pituitary-gonadal axis offers a good model fbr studies on the mechanisms of regulation of fetal hormonal systems. It is evident that fetal hormonal functions are not the same as those of the adult on a smaller scale, but that there are fundamental differences between the fetus and adult in basic features of the mechanisms of reproductive hormone action.

356

probe.

Van Heuverswyn B, Streydio C, Brocas H, Refetoff S, Dumont J, Vassart G: Thyrotropin controls transcription of the thyroglobulin gene. Proc Nat1 Acad Sci USA 1984; 81:5941.

Simon P, Brocas H, Rodesch C, Vassart G: RFLP detected at the 8q24 locus by a thyro-

and Dwight W. Warren

Ilpo T. Huhtaniemi is at the Department of Physiology, University of Turku, 20520 Turku, Finland: Dwight W. Warren is at the Department of Physiology and Biophysics, University of Southern California, Los Angeles, CA 90033, USA.

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Schumacher M, Camp S, Maulet Y, et al.: Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence. Nature 1986; 319:407.

Current Advances and Controversies

The complex network of regulatory interactions within the hypothalamic-pituitary-gonadal axis is attained gradually during developmental maturation from fetus to adult. The different compartments of this system, the hypothalamus, anterior pituitary, and gonads, probably start their embryonic develop-

1987;

Santisteban P, Kohn LD, Di Lauro R: Thyroglobulin mRNA is regulated by insulin and IGF-I as well as by thyrotropin in the FRTL5 cell line. In Puett D, Ahmad F, Black S, et al., eds. Advances in Gene Technology: Molecular Biology of the Endocrine System (ICSU Short Reports, vol 4). Cambridge, Cambridge University Press, 1986, p 322.

Ontogeny of Pituitary-Gonadal Interactions Ilpo T. Huhtaniemi

globulin

Hanson RW: of eukaryotic Chem 1988;

Co., Inc.

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Ontogeny of the Hypothalamic Control of Gonadotropin Secretion

The development of the anterior pituitary in the human [for a review, see Mulchaney et al. (1987)] starts between 4 and 5 weeks of fetal life, and the floor of the sella turcica is discernible by week 7. The median eminence can be distinguished by week 9, and the hypothalamohypophyseal vascular system is functional by week 12 of gestation. However, hypothalamohypophysial connections by local diffusion may be possible even earlier. The origin of the neurons secreting gonadotropin-releasing hormone, GnRH, has remained an enigma for a long time. The main sites of these neurons in the adult brain are in the septal-preoptic nuclei and the hypothalamus. GnRH-expressing neurons are also present in the nervus terminalis, a cranial nerve that is a part of the accessory olfactory system and projects directly from the nose to the septal-preoptic nuclei in the brain, Schwanzel-Fukuda and Pfaff (1989) recently showed that in fetal mice the GnRH neurons originate in the medial olfactory placode and enter the forebrain with the nervus terminalis. These

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