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Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
A FAMILY OF POU-DOMAIN AND PIT-1 TISSUE-SPECIFIC TRANSCRIPTION FACTORS IN PITUITARY AND NEUROENDOCRINE DEVELOPMENT Holly A. Ingraham, Vivian R. Albert, Ruoping Chen, E. Bryan Crenshaw III, Harry P. Elsholtz, Xi He, Michael S. Kapiloff, Harry]. Mangaiam, Larry W. Swanson, Maurice N. Treacy and Michael G. Rosenfeld Howard Hughes Medical Institute and Eukaryotic Regulatory Biology Program, School of Medicine, University of California, San Diego, La Jolla, California 92093
KEY WORDS:
development, somatotroph, lactotroph, gene expression, DNA-binding proteins
INTRODUCTION The sequential activation of a hierarchy of regulatory genes is a prominent mechanism for dictating the precise temporal and spatial patterns of develop ment (23, 50) and the specific patterns of gene expression that will ultimately dictate organ identity. Considerable evidence has supported the existence of tissue-specific factors critical for the transcriptional activation of the genes that define cellular phenotype in mammals (e.g. 58, 55, 47, 6, 27, 28, 15,14, 46, 53). We have utilized the development of the anterior pituitary gland as an excellent model system in which to study cell-specific gene activation. Pitu itary development results in the temporally precise appearance of five distinct cell types derived from a common lineage that are distinguished on the basis of the trophic hormone elaborated. Somatotrophs and lactotrophs are the last 773
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Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
774
INGRAHAM ET AL
two phenotypically distinct cell types to appear during development, which produce the recently diverged hormones, growth hormone and prolactin, respectively (10,59,13,32). Transient co-expression of prolactin and growth hormone occurs in a subset of somatotrophs during pituitary development prior to the appearance of lactotrophs (l0, 59, 32), which suggests a common lineage for somatotrophs and lactotrophs; a variable number of such cells (somatomammotrophs) persist in the developed gland. Understanding the molecular basis of the binary decisions that ultimately lead to generation of distinct cell types is critical to understanding pituitary development. Cell-specific expression of the rat prolactin gene is dictated by two separate regions, a distal enhancer ( - 1831 to - 1530) (46) and a proximal region (-422 to +33) (see Figure 1) (47, 26, 7, 41, 46). Transfectional analyses of various pituitary cell lines in our laboratory have indicated that the distal enhancer is predominately responsible for the high levels of cell-specific expression, which suggests that both the distal and promoter regions are critical (46, 47), while others have suggested that cell-specific expression in cell culture reflects the actions of only the promoter proximal region (26, 4 1) . Based on studies of DNA-mediated gene transfer into pituitary cells (GC or GH3 cells), mutagenesis revealed that both the distal and proximal regulatory regions contained multiple related sequences that appeared to bind tissue specific, nuclear protein(s) and exhibit synergistic interactions (46).
Determinants of Development Patterns of Prolactin and Growth Hormone Gene Expression Because of the potential action of two discrete genomic regions in vitro, a critical issue was the basis for determination of physiologic patterning expres sion during normal development. This question has been tested in physiologic development by the introduction of prolactin fusion genes into fertilized
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POU-DOMAIN DEVELOPMENTAL REGULATION
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mouse oocytes and analysis of the resultant transgenic animals. Fusion genes
containing three kb of 5' flanking region or the proximal region (-422 to +33 bp) alone directed strict cell-type specific expression (16). Prolactin proximal elements alone conferred low levels of reporter gene expression in a small percentage of the transgenic pedigrees that were established. Inclusion of both the distal enhancer and the proximal promoter region dramatically
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increased the expression of the transgene. The distal enhancer was also
capable of directing tissue-specific expression when fused to the herpes virus thymidine kinase (tk) promoter, but the reporter gene expression in the pituitary was low, and thyrotroph as well as lactotroph expression was observed (16). The distal enhancer, therefore, specifically directed expression to the correct tissue, but it apparently required specific flanking sequences to r estrict its expression to the correct cell type within the pituitary of transgenic
mice. These results indicated that while the distal and proximal regions were each capable of directing tissue-specific expression, they acted synergistically to generate high penetrance and high expression levels of rat prolactin fusion genes in transgenic mice (16) (Figure 2). Transgenes containing prolactin promoter constructs showed strict tissue specificity of expression with no
detectable expression « 3 orders of magnitude) outside of the pituitary gland. Mutation of even single cis-active elements of the distal enhancer reduced prolactin gene expression by 80-90% (46). Expre ssi on of growth hormone in somatotrophs of transgenic mice (4, 40) was specified by as little as 180 bp of rat growth hormone 5' flanking genomic information (40). This -
region contained two cis-active elements required for cell-specific expression
in vitro (47,61,63,46), The possibility that a single or twp related cell specific positive transcription factors could bind to sites in both the rat
Synergism
DISTAL ENHANCER Activating
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the prolactin gene based on transgenic animal analyses. Prolactin distal enhancer and proximal region are synergistic, and additional sequences appear to restrict expression out of thyrotrophs.
776
INGRAHAM ET AL
prolactin and growth hormone genes was suggested by competition analyses of DNase 1 footprints and in vitro transcription (46). In examining the role of the distal and proximal regions in prolactin expression, it was established that the context of the distal enhancer was important for the strict cell-type specific expression of the prolactin gene (16).
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Pit-i, a Pituitary Specific Transcription Factor
To characterize factors responsible for prolactin and growth hormone gene activation, purification employing conventional separation techniques com bined with DNA affinity chromatography was performed using nuclear ex tracts from a rat pituitary cell line (GC). A highly purified (> 4000-fold) preparation of biologically active protein (Pit-I) (33,000,31,000 Mr protein doublet) that activated both prolactin and growth hormone fusion genes (35, 42) was obtained. The ability of the purified Pit-l to bind to cell-specific cis-active elements of the prolactin and growth hormone genes permitted successful utilization of this property for screening of rat pituitary and GC cell eDNA expression libraries (35). Pit- l had similar properties to proteins isolated in other laboratories, referred to as PUF-l or GHF- l (7, 6). The coding sequence for Pit-l (Figure 3) predicted an 873 nucleotide open reading frame corresponding to an encoded protein of 291 amino acids and a
A
B
c
o
Figure 3 Expression of Pit-l transcripts in the pituitary gland. In situ hybridization histochemis try reveals selective expression in the anterior pituitary (panel B), but not in intennediate (J) or posterior (P) lobes. Pit-l is expressed in somatotrophs and lactotrophs, colocalizing with growt h honnone (panel C) and prolactin (panel D).
777
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
POU-DOMAIN DEVELOPMENTAL REGULATION
predicted Mr=32,900 (35). Pit-l mRNA is expressed only in the anterior pituitary gland and is present in lactotrophs and somatotrophs (35, 42) (Figure 3). Cotransfectional analyses using Pit-I transcription units in HeLa cells resulted in expression of both prolactin and growth hormone fusion genes (Figure 4). A critical issue was whether, at physiologic levels of expression, Pit-l was capable of activating prolactin or growth hormone fusion gene expression in heterologous cells; in permanent HeLa cell transfectants ex pressing levels of Pit-l at tenfold less than those present in pituitary cells, preferential transcriptional activation of the prolactin gene promoter was observed (42). In contrast to reported results with a fusion GHF (Pit-I) protein (6) or in a partially purified protein preparation (9), Pit-l expressed in bacteria effectively bound to and activated in vitro transcription of both the prolactin and growth hormone promoters (42). Unexpectedly, Pit-l shared significant homology with a 60 amino acid region, referred to as the homeodomain, initially described in three gene products regulating early development in Drosophila antennapedia, VI trabithorax, and lushi tarazu (51, 43) and present in over 20 related gene
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235-1 putative lactotroph cell line show similar results.
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
778
INGRAHAM ET AL
products that dictate position-specific determination in Drosophila (1, 23, 51) (Figure 4). Multigene families containing highly conserved homeobox do mains appear to direct early developmental events and have been described in C. elegans, amphibians, mice, and humans (8, 39, 12, 1). Their shared homology with the yeast al and a2 regulatory gene products, known to individually and in combination with other transcription factors regulate gene expression that determines yeast cell mating type (52; see review 45), sug gested similar functional roles for the homeotic genes. It was observed that Pit-l directly activated prolactin and growth hormone fusion gene transcrip tion as a consequence of binding to cis-active elements, and in heterologous cells in mammals formally linked homeodomain-containing proteins to tissue specific transcriptional activation; subsequently, data confirming a transcrip tional role for Drosophila homeodomain proteins have been reported (e.g. 17, 36, 29). Pit-1 exhibited an identity of 7 of the 9 invariant and 15 of the 20 most highly conserved amino acids present in the Drosophila homeodomains (see Figure 5). The sequence variations modify only several of the conserved amino acids predicted to be on one side of the putative a helical regions (44, 4 9) . An apparently critical divergence is observed in the putative recognition region where cysteine, glutamine, and glutamic acid are uniquely present in residues 50, 54, 56. Recently a number of genes with highly diverged homeobox domains, and often exhibiting tissue-specific and .cell-specific patterns of expression, have been identified (e.g. 60, 2, 5); however, the WFC motif is unique to the POU-domain gene family. A lymphoid B cell-specific factor that activates the transcription of im munoglobulin genes (Oct-2) as a consequence of binding to an octamer recognition element and a more generally distributed octamer binding protein (Oct-I) have recently been sequenced based on cDNA analysis (11, 56). A comparison of Pit-I, Oct-2, and Oct-I with a C. elegans regulatory gene
.
Pit-J Antp Ftz Prd En Ubx Cut Hox 3.1 Mat a1
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Figure 5 Pit-I: Identification of a 60 amino acid region similar to homeodomains of several Drosophila regulatory proteins.
POU-DOMAIN DEVELOPMENTAL REGULATION
779
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product, unc-86 (21) genetically defined as a detenninant of cell-specific development fate, revealed that the four proteins share an extended region of homology (see Figure 6a). Amino-tenninal to the 60 amino acid divergent homeodomain, referred to as the POU-homeodomain, is a 76-78 amino acid region that is unique to these four proteins, referred to as the POU-specific domain (31). These two regions, together with a nonconserved region be tween them, constitute the POU-domain (Figure 6); the evolutionary con servation suggests critical functions are subserved by each region.
POU-Domain Protein Recognition Sites The Pit-l DNA recognition element is an A,T-rich sequence (T/A T/A T/A ATANCAT); a core sequence differing by only a single nucleotide from the TRANS-ACTING FACTORS CONTAINING THE PQU-DOMAIN Pit-, Oct-2
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Jm-l T.r;·l,
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Identification of four new mammalian POU-domain regulatory genes expressed in the
neuroendocrine system. The POD-specific and POD homeodomains encoded by four transcripts
expressed in brain and endocrine tissues (Bm-I, Brn-2, Tst-I , and Brn-3). Tst-l is expressed in
both brain and testes. Bro-I is expressed in both the central nervous system and medulla of kidney. The eight POD-domain proteins appears to fall into four classes (I-IV).
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
780
INGRAHAM ET AL
octamer sequence (ATTTGCAT) that binds different factors in lymphoid and non-lymphoid cells. This binding site is sufficient to impart lymphoid-specific promoter activity (58, 62, 53). While Pit-l can bind at tenfold lower affinity to the octamer sequence, Pit-l fails to activate transcription units containing the octamer, rather than Pit-l elements. The binding sites for Drosophila homeodomain gene products are AIT-rich (33), and the consensus yeast MATa2 product binding site (37) is remarkably similar to the Pit-l recognition element. Therefore, it is possible that the members of the homeodomain and POU-domain families responsible for developmental activation of gene transcription bind to highly related A/T-rich elements. This would be analo gous to the steroid hormone T3 receptor gene family that exhibits remarkable similarity of their binding sites (reviewed in 3; 20, 24, 25). The presence of multiple required elements, many of which bind Pit-l in the prolactin gene, appears to be an important aspect of its developmental activation because a single element is insufficient to produce marked in creases in gene expression. Therefore, combinatorial effects of multiple Pit-l elements and sites for other transcription factors must constitute the code responsible for the temporal and quantitative patterns of prolactin gene ex pression. In lactotrophs, additional factors may also bind to Pit-l sites and functionally effect prolactin gene transcription.
Ontogeny of Pit-l Expression in Somatotrophs, Lactotrophs, and Thyrotrophs Because the distal enhancer contains Pit-l binding sites, at least two of which are necessary for expression in prolactin-producing cells (46), possibly Pit-l was responsible for the activation of the distal enhancer-containing fusion genes in thyrotrophs (16). Hybridization histochemistry of sections im munostained for the TSH peptide revealed that Pit-l mRNA colocalized with TSH immunostaining in some thyrotrophs (Figure 7). The ontogeny of Pit-l expression correlates with the appearance of pituitary cell types during de velopment. Expression of Pit-l appears to be restricted to the developing anterior pituitary on embryonic day (16) (e16) with hybridization for Pit- l more difficult to detect in el 5 embryos. Pit-l gene expression is, therefore, found early in the ontogeny of the pituitary prior to the reported appearance of TSH, growth hormone, and prolactin. Many reports suggest that prolactin is expressed postnatally many days after the initial expression of Pit-1, but reports vary widely in the initial appearance of prolactin from e21 to just after birth in the rat (59, 10) and 8 days postpartum in the mouse (54), This apparent discrepancy between the appearance of Pit-l and the initial expres sion of prolactin might indicate that low levels of prolactin expression could be activated by Pit-l very early in development, but that other factors either cooperate with or replace Pit-l to generate detectable levels of expression at
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
POU-DOMAIN DEVELOPMENTAL REGULATION
781
RL
TSH Figure 7
Expression of Pit-l in lactotrophs
and thyrotrophs based on colocalization of Pit-I, by
hybridization histochemistry with immunostaining for PRL and TSH.
later times coincident with birth of the lactotroph. Consistent with this explanation, we found the expression of prolactin transgenes on mouse day e16 (rat day el7) (16). These analyses indicated that prolactin gene expression was activated at low levels in the rodent fetus, coordinate with or closely following the expression of Pit-l during ontogeny. The progressive increase in expression of two orders of magnitude, however, suggests that additional factors must be involved in establishing the patterns of cell-specific restriction and physiologic levels of gene expression. Because Pit-l at levels tenfold lower than those in pituitary cells
(42) is
capable of activating the expression of both the growth hormone and prolactin fusion genes and because the Pit-l gene is expressed in somatotrophs, Iacto trophs
(35, 6), and thyrotrophs (16), there are likely to be restrictive mech
anisms that prevent the expression of growth hormone and prolactin genes in
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782
INGRAHAM ET AL
lactotrophs and thyrotrophs, respectively. Sequences flanking the distal en hancer are likely to be required for restricting prolactin gene expression from thyrotrophs. Restrictive mechanisms could represent either negative regula tion or the lack of complementing factors that interact with or displace Pit-l to give physiologic levels of prolactin expression. On this basis, it is expected that there are additional factors that bind to the prolactin and growth hormone 5' flanking regions and play pivotal roles in regulating the physiologic levels of prolactin and growth hormone gene expression. The role of Pit-l during the organogenesis of the pituitary may be to activate initial transcription of specific genes in a pituitary-specific manner, while additional activation and restrictive mechanisms are responsible for delineating specific cell type within the pituitary gland. These additional mechanisms that regulate the differentia tion into lactotrophs and somatotrophs should provide an important paradigm for differentiation during mammalian development.
A Large Family of POU-Domain Regulatory Genes in Mammalian Brain Development Brain development involves an intricate program of gene expression that leads to the establishment of many neuronal phenotypes and a precise complex pattern of connections between them. The nuclear events underlying neuronal development and the accompanying diverse patterns of specific gene expres sion remain largely unknown. It is likely, however, that the precise temporal and spatial patterns characteristic of mammalian brain development reflect sequential activation of a complex network of regulatory factors similar to those presumed to account for establishing structural patterns in Drosophila (51,23). The specification of a variety of neuronal phenotypes derived from an apparently homogeneous population of neuroepithelial cells are pre requisites for brain development. The possibility that novel POD-domain proteins were expressed during establishment of the nervous system with distinct spatial and temporal pat terns, potentially exerting some roles in specifying neuronal phenotypes, was investigated using a strategy based on the structure of the initial four members of the POU-domain gene family. Degenerate oligonucleotides based on two highly conserved amino sequences in the A region of the POU-specific domain and in the C-terminal portion of the POU homeodomain were used as primers for the polymerase chain reaction employing DNA complementary to brain and testes mRNAs as templates. Four new members of the POU-domain gene family were identified; three from brain cDNA (Brn-l, Brn-2, and Bm-3) and one from rat testes cDNA (Tst-l), although this gene is pre dominantly expressed in the brain. Predicted amino acid residues for each new member are shown in Figure 6b. Structural comparisons of the four new POU-domain proteins revealed that
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POU-DOMAIN DEVELOPMENTAL REGULATION
783
they are all highly related to Pit-I, Oct-I, Oct-2, and unc-86. Recently another POD-protein has also been identified in C. elegans (ceh-6) (6a). These nine proteins constitute a distinct POD-domain protein family. Three of the new proteins (Brn- l , Brn-2, and Tst- l ) are highly homologous, with> 94% of the amino acid residues being identical among them throughout the entire POD-domain. Even the variable region between the POD-specific domain and the POD-homeodomain is well conserved among these proteins. Brn-3 is highly related to unc-86, including three characteristic additional amino acid residues in the region between the A and B portions of the POD-specific domain. Pit-l is distinct from the other eight proteins in the POD-family domain at several amino acid positions that otherwise would be totally conserved, and it is thus far the most divergent member of the family. The known POD-domain proteins appear to segregate into four classes arbi trarily referred to as POD-I (Pit-I), POD-2 (Oct-I, Oct-2), POD-3 (Brn-l, Brn-2, Tst-I), and POU-4 (Brn-3, unc-86). The N' terminal basic part of the POD-homeodomain is identical for the first 11 or 12 amino acids, and 15-18 amino acids in the C-terminal WFC region (see Figure 5) are particularly well-conserved between all members of each class. A consensus sequence for the POD-domain emphasizes the degree of primary amino acid sequence conservation among all nine family members. It is predicted that the POD III gene family will contain the most members. Each gene is expressed in at least one tissue other than brain and generates transcripts of different size. Thus Bm-l and Bm-2 exhibit virtually identical patterns of expression in the central nervous system, but Brn-l is clearly expressed in the medullary zone of the kidney, while Brn-2 is not (30). Hybridization for Brn-l and Brn-2 is found in at least some classes of neurons at all levels of the neuraxis. Almost all regions of the cerebral (layers II-V) and cerebellar (Purkinje cells) cortices are labeled, as are the basal forebrain cholinergic system, ventral midbrain dopamine system, paraventricular and supraoptic neuroendocrine system, somatic motoneurons in the cranial nerve nuclei and ventral hom, and tectum (Figure 8). In contrast, the Brn-3 gene transcript exhibits a considerably more restricted pattem with relatively dense hybridization limited to the habenula, posterior hypothalamic area, infer ior olive, inferior colliculus, and nucleus ambiguus. Brn-3 transcripts are uniquely present in sensory ganglion cells (in the trigeminal and dorsal root ganglia), which are derived from the neural crest. The Tst-I gene, while expressed in testes, is particularly well-expressed in brain and displays a characteristic pattern of expression in the cerebral (layers V-VI) and cere bellar (granule cells) cortices, as well as in the striatum. medial habenula, su perior colliculus and parabigeminal nucleus, and dorsal motor nucleus of the vagus nerve. Thus each POD-domain gene exhibits a unique, restricted pattern of expression, though not obviously correlated with known de-
INGRAHAM ET AL
784
Table 1 Distribution of POU-domain mANA expression in the adult rat neNOUS system determined by in situ hybridization
SENSORY GANGLIA
Pit·,
Brn-'
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•
•
•
+ +
CORTEX
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+
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+
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+
+ +
+ ++
+ +
... +
presubiculum. layer 2 CA1/Subiculum dentate gyrus subependymal zone AMYGDAlA
SEPTUM
BASAL GANGLIA striatum substantia innominata
s. nigraJyentral tegmental a.
THALAMUS medial habenula lateral habenula parafaseicular n. reticular n. zona incerta
HYPOTHALAMUS suprachiasmatiC n.
supraoptic/parallenltieular n. medial preoptic n.
tuberomammiUa'Y n. lateral mammillary n. medial rnammill9.ry n.
posterior hypothalamic a.
B�NSTEM-SENSORY
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area postrema
B�NSTEM - MOTOR motor
n. trigeminal
facial n.
hypoglossal n. n. ambiguuA
+
+
n. incertus
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+ ... +
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... ++
...
+
+
•
periaqueductal gray
interpeduncular n. CEREBELLUM & RELATED
granule redo.
inferior olive
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+ +++
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++
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+ ++
.+
+
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+ ...++
+
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Oct·,
•
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•
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islands of Galleja
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
Brn·3
++1-+
+++
...... +
r te i m ode a ; + ++: h gh:
Distributors of POU-domain mRNA expression in the adult rat nervous system
detennined by
in situ hybridization. The hypothalmic pattern of expression is noted.
POU-DOMAIN DEVELOPMENTAL REGULATION
785
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
velopmental, functional, or neurotransmitter-related systems in the nervous system. All four new POU-domain proteins are widely expressed in all levels of the neural tube during at least some part of this period. The anatomical restriction for each gene product in the developing neural tube is distinct however, and reflects the adult loci of expression. Expression is observed in the ventricular (proliferative) zone of the neuroepithelium, the mantle layer, the early cortical plate (Bm-l, Bm-2), and external granular layer of the cerebellum (Tst-l) (Figure 9). These data suggest that the genes are expressed
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Further
Quick links to online content Annu. Rev. Physiol. 1990. 52:773-91 Copyright © 1990 by Annual Reviews Inc. All rights reserved
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
A FAMILY OF POU-DOMAIN AND PIT-1 TISSUE-SPECIFIC TRANSCRIPTION FACTORS IN PITUITARY AND NEUROENDOCRINE DEVELOPMENT Holly A. Ingraham, Vivian R. Albert, Ruoping Chen, E. Bryan Crenshaw III, Harry P. Elsholtz, Xi He, Michael S. Kapiloff, Harry]. Mangaiam, Larry W. Swanson, Maurice N. Treacy and Michael G. Rosenfeld Howard Hughes Medical Institute and Eukaryotic Regulatory Biology Program, School of Medicine, University of California, San Diego, La Jolla, California 92093
KEY WORDS:
development, somatotroph, lactotroph, gene expression, DNA-binding proteins
INTRODUCTION The sequential activation of a hierarchy of regulatory genes is a prominent mechanism for dictating the precise temporal and spatial patterns of develop ment (23, 50) and the specific patterns of gene expression that will ultimately dictate organ identity. Considerable evidence has supported the existence of tissue-specific factors critical for the transcriptional activation of the genes that define cellular phenotype in mammals (e.g. 58, 55, 47, 6, 27, 28, 15,14, 46, 53). We have utilized the development of the anterior pituitary gland as an excellent model system in which to study cell-specific gene activation. Pitu itary development results in the temporally precise appearance of five distinct cell types derived from a common lineage that are distinguished on the basis of the trophic hormone elaborated. Somatotrophs and lactotrophs are the last 773
0066-4278/90/0315-0773$02.00
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
774
INGRAHAM ET AL
two phenotypically distinct cell types to appear during development, which produce the recently diverged hormones, growth hormone and prolactin, respectively (10,59,13,32). Transient co-expression of prolactin and growth hormone occurs in a subset of somatotrophs during pituitary development prior to the appearance of lactotrophs (l0, 59, 32), which suggests a common lineage for somatotrophs and lactotrophs; a variable number of such cells (somatomammotrophs) persist in the developed gland. Understanding the molecular basis of the binary decisions that ultimately lead to generation of distinct cell types is critical to understanding pituitary development. Cell-specific expression of the rat prolactin gene is dictated by two separate regions, a distal enhancer ( - 1831 to - 1530) (46) and a proximal region (-422 to +33) (see Figure 1) (47, 26, 7, 41, 46). Transfectional analyses of various pituitary cell lines in our laboratory have indicated that the distal enhancer is predominately responsible for the high levels of cell-specific expression, which suggests that both the distal and promoter regions are critical (46, 47), while others have suggested that cell-specific expression in cell culture reflects the actions of only the promoter proximal region (26, 4 1) . Based on studies of DNA-mediated gene transfer into pituitary cells (GC or GH3 cells), mutagenesis revealed that both the distal and proximal regulatory regions contained multiple related sequences that appeared to bind tissue specific, nuclear protein(s) and exhibit synergistic interactions (46).
Determinants of Development Patterns of Prolactin and Growth Hormone Gene Expression Because of the potential action of two discrete genomic regions in vitro, a critical issue was the basis for determination of physiologic patterning expres sion during normal development. This question has been tested in physiologic development by the introduction of prolactin fusion genes into fertilized
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POU-DOMAIN DEVELOPMENTAL REGULATION
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mouse oocytes and analysis of the resultant transgenic animals. Fusion genes
containing three kb of 5' flanking region or the proximal region (-422 to +33 bp) alone directed strict cell-type specific expression (16). Prolactin proximal elements alone conferred low levels of reporter gene expression in a small percentage of the transgenic pedigrees that were established. Inclusion of both the distal enhancer and the proximal promoter region dramatically
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
increased the expression of the transgene. The distal enhancer was also
capable of directing tissue-specific expression when fused to the herpes virus thymidine kinase (tk) promoter, but the reporter gene expression in the pituitary was low, and thyrotroph as well as lactotroph expression was observed (16). The distal enhancer, therefore, specifically directed expression to the correct tissue, but it apparently required specific flanking sequences to r estrict its expression to the correct cell type within the pituitary of transgenic
mice. These results indicated that while the distal and proximal regions were each capable of directing tissue-specific expression, they acted synergistically to generate high penetrance and high expression levels of rat prolactin fusion genes in transgenic mice (16) (Figure 2). Transgenes containing prolactin promoter constructs showed strict tissue specificity of expression with no
detectable expression « 3 orders of magnitude) outside of the pituitary gland. Mutation of even single cis-active elements of the distal enhancer reduced prolactin gene expression by 80-90% (46). Expre ssi on of growth hormone in somatotrophs of transgenic mice (4, 40) was specified by as little as 180 bp of rat growth hormone 5' flanking genomic information (40). This -
region contained two cis-active elements required for cell-specific expression
in vitro (47,61,63,46), The possibility that a single or twp related cell specific positive transcription factors could bind to sites in both the rat
Synergism
DISTAL ENHANCER Activating
Regions
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the prolactin gene based on transgenic animal analyses. Prolactin distal enhancer and proximal region are synergistic, and additional sequences appear to restrict expression out of thyrotrophs.
776
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prolactin and growth hormone genes was suggested by competition analyses of DNase 1 footprints and in vitro transcription (46). In examining the role of the distal and proximal regions in prolactin expression, it was established that the context of the distal enhancer was important for the strict cell-type specific expression of the prolactin gene (16).
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Pit-i, a Pituitary Specific Transcription Factor
To characterize factors responsible for prolactin and growth hormone gene activation, purification employing conventional separation techniques com bined with DNA affinity chromatography was performed using nuclear ex tracts from a rat pituitary cell line (GC). A highly purified (> 4000-fold) preparation of biologically active protein (Pit-I) (33,000,31,000 Mr protein doublet) that activated both prolactin and growth hormone fusion genes (35, 42) was obtained. The ability of the purified Pit-l to bind to cell-specific cis-active elements of the prolactin and growth hormone genes permitted successful utilization of this property for screening of rat pituitary and GC cell eDNA expression libraries (35). Pit- l had similar properties to proteins isolated in other laboratories, referred to as PUF-l or GHF- l (7, 6). The coding sequence for Pit-l (Figure 3) predicted an 873 nucleotide open reading frame corresponding to an encoded protein of 291 amino acids and a
A
B
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Figure 3 Expression of Pit-l transcripts in the pituitary gland. In situ hybridization histochemis try reveals selective expression in the anterior pituitary (panel B), but not in intennediate (J) or posterior (P) lobes. Pit-l is expressed in somatotrophs and lactotrophs, colocalizing with growt h honnone (panel C) and prolactin (panel D).
777
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
POU-DOMAIN DEVELOPMENTAL REGULATION
predicted Mr=32,900 (35). Pit-l mRNA is expressed only in the anterior pituitary gland and is present in lactotrophs and somatotrophs (35, 42) (Figure 3). Cotransfectional analyses using Pit-I transcription units in HeLa cells resulted in expression of both prolactin and growth hormone fusion genes (Figure 4). A critical issue was whether, at physiologic levels of expression, Pit-l was capable of activating prolactin or growth hormone fusion gene expression in heterologous cells; in permanent HeLa cell transfectants ex pressing levels of Pit-l at tenfold less than those present in pituitary cells, preferential transcriptional activation of the prolactin gene promoter was observed (42). In contrast to reported results with a fusion GHF (Pit-I) protein (6) or in a partially purified protein preparation (9), Pit-l expressed in bacteria effectively bound to and activated in vitro transcription of both the prolactin and growth hormone promoters (42). Unexpectedly, Pit-l shared significant homology with a 60 amino acid region, referred to as the homeodomain, initially described in three gene products regulating early development in Drosophila antennapedia, VI trabithorax, and lushi tarazu (51, 43) and present in over 20 related gene
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Effects of Pit-l on prolactin and growth hormone fusion genes in HeLa cells and the
235-1 putative lactotroph cell line show similar results.
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
778
INGRAHAM ET AL
products that dictate position-specific determination in Drosophila (1, 23, 51) (Figure 4). Multigene families containing highly conserved homeobox do mains appear to direct early developmental events and have been described in C. elegans, amphibians, mice, and humans (8, 39, 12, 1). Their shared homology with the yeast al and a2 regulatory gene products, known to individually and in combination with other transcription factors regulate gene expression that determines yeast cell mating type (52; see review 45), sug gested similar functional roles for the homeotic genes. It was observed that Pit-l directly activated prolactin and growth hormone fusion gene transcrip tion as a consequence of binding to cis-active elements, and in heterologous cells in mammals formally linked homeodomain-containing proteins to tissue specific transcriptional activation; subsequently, data confirming a transcrip tional role for Drosophila homeodomain proteins have been reported (e.g. 17, 36, 29). Pit-1 exhibited an identity of 7 of the 9 invariant and 15 of the 20 most highly conserved amino acids present in the Drosophila homeodomains (see Figure 5). The sequence variations modify only several of the conserved amino acids predicted to be on one side of the putative a helical regions (44, 4 9) . An apparently critical divergence is observed in the putative recognition region where cysteine, glutamine, and glutamic acid are uniquely present in residues 50, 54, 56. Recently a number of genes with highly diverged homeobox domains, and often exhibiting tissue-specific and .cell-specific patterns of expression, have been identified (e.g. 60, 2, 5); however, the WFC motif is unique to the POU-domain gene family. A lymphoid B cell-specific factor that activates the transcription of im munoglobulin genes (Oct-2) as a consequence of binding to an octamer recognition element and a more generally distributed octamer binding protein (Oct-I) have recently been sequenced based on cDNA analysis (11, 56). A comparison of Pit-I, Oct-2, and Oct-I with a C. elegans regulatory gene
.
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Figure 5 Pit-I: Identification of a 60 amino acid region similar to homeodomains of several Drosophila regulatory proteins.
POU-DOMAIN DEVELOPMENTAL REGULATION
779
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
product, unc-86 (21) genetically defined as a detenninant of cell-specific development fate, revealed that the four proteins share an extended region of homology (see Figure 6a). Amino-tenninal to the 60 amino acid divergent homeodomain, referred to as the POU-homeodomain, is a 76-78 amino acid region that is unique to these four proteins, referred to as the POU-specific domain (31). These two regions, together with a nonconserved region be tween them, constitute the POU-domain (Figure 6); the evolutionary con servation suggests critical functions are subserved by each region.
POU-Domain Protein Recognition Sites The Pit-l DNA recognition element is an A,T-rich sequence (T/A T/A T/A ATANCAT); a core sequence differing by only a single nucleotide from the TRANS-ACTING FACTORS CONTAINING THE PQU-DOMAIN Pit-, Oct-2
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Identification of four new mammalian POU-domain regulatory genes expressed in the
neuroendocrine system. The POD-specific and POD homeodomains encoded by four transcripts
expressed in brain and endocrine tissues (Bm-I, Brn-2, Tst-I , and Brn-3). Tst-l is expressed in
both brain and testes. Bro-I is expressed in both the central nervous system and medulla of kidney. The eight POD-domain proteins appears to fall into four classes (I-IV).
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
780
INGRAHAM ET AL
octamer sequence (ATTTGCAT) that binds different factors in lymphoid and non-lymphoid cells. This binding site is sufficient to impart lymphoid-specific promoter activity (58, 62, 53). While Pit-l can bind at tenfold lower affinity to the octamer sequence, Pit-l fails to activate transcription units containing the octamer, rather than Pit-l elements. The binding sites for Drosophila homeodomain gene products are AIT-rich (33), and the consensus yeast MATa2 product binding site (37) is remarkably similar to the Pit-l recognition element. Therefore, it is possible that the members of the homeodomain and POU-domain families responsible for developmental activation of gene transcription bind to highly related A/T-rich elements. This would be analo gous to the steroid hormone T3 receptor gene family that exhibits remarkable similarity of their binding sites (reviewed in 3; 20, 24, 25). The presence of multiple required elements, many of which bind Pit-l in the prolactin gene, appears to be an important aspect of its developmental activation because a single element is insufficient to produce marked in creases in gene expression. Therefore, combinatorial effects of multiple Pit-l elements and sites for other transcription factors must constitute the code responsible for the temporal and quantitative patterns of prolactin gene ex pression. In lactotrophs, additional factors may also bind to Pit-l sites and functionally effect prolactin gene transcription.
Ontogeny of Pit-l Expression in Somatotrophs, Lactotrophs, and Thyrotrophs Because the distal enhancer contains Pit-l binding sites, at least two of which are necessary for expression in prolactin-producing cells (46), possibly Pit-l was responsible for the activation of the distal enhancer-containing fusion genes in thyrotrophs (16). Hybridization histochemistry of sections im munostained for the TSH peptide revealed that Pit-l mRNA colocalized with TSH immunostaining in some thyrotrophs (Figure 7). The ontogeny of Pit-l expression correlates with the appearance of pituitary cell types during de velopment. Expression of Pit-l appears to be restricted to the developing anterior pituitary on embryonic day (16) (e16) with hybridization for Pit- l more difficult to detect in el 5 embryos. Pit-l gene expression is, therefore, found early in the ontogeny of the pituitary prior to the reported appearance of TSH, growth hormone, and prolactin. Many reports suggest that prolactin is expressed postnatally many days after the initial expression of Pit-1, but reports vary widely in the initial appearance of prolactin from e21 to just after birth in the rat (59, 10) and 8 days postpartum in the mouse (54), This apparent discrepancy between the appearance of Pit-l and the initial expres sion of prolactin might indicate that low levels of prolactin expression could be activated by Pit-l very early in development, but that other factors either cooperate with or replace Pit-l to generate detectable levels of expression at
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
POU-DOMAIN DEVELOPMENTAL REGULATION
781
RL
TSH Figure 7
Expression of Pit-l in lactotrophs
and thyrotrophs based on colocalization of Pit-I, by
hybridization histochemistry with immunostaining for PRL and TSH.
later times coincident with birth of the lactotroph. Consistent with this explanation, we found the expression of prolactin transgenes on mouse day e16 (rat day el7) (16). These analyses indicated that prolactin gene expression was activated at low levels in the rodent fetus, coordinate with or closely following the expression of Pit-l during ontogeny. The progressive increase in expression of two orders of magnitude, however, suggests that additional factors must be involved in establishing the patterns of cell-specific restriction and physiologic levels of gene expression. Because Pit-l at levels tenfold lower than those in pituitary cells
(42) is
capable of activating the expression of both the growth hormone and prolactin fusion genes and because the Pit-l gene is expressed in somatotrophs, Iacto trophs
(35, 6), and thyrotrophs (16), there are likely to be restrictive mech
anisms that prevent the expression of growth hormone and prolactin genes in
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
782
INGRAHAM ET AL
lactotrophs and thyrotrophs, respectively. Sequences flanking the distal en hancer are likely to be required for restricting prolactin gene expression from thyrotrophs. Restrictive mechanisms could represent either negative regula tion or the lack of complementing factors that interact with or displace Pit-l to give physiologic levels of prolactin expression. On this basis, it is expected that there are additional factors that bind to the prolactin and growth hormone 5' flanking regions and play pivotal roles in regulating the physiologic levels of prolactin and growth hormone gene expression. The role of Pit-l during the organogenesis of the pituitary may be to activate initial transcription of specific genes in a pituitary-specific manner, while additional activation and restrictive mechanisms are responsible for delineating specific cell type within the pituitary gland. These additional mechanisms that regulate the differentia tion into lactotrophs and somatotrophs should provide an important paradigm for differentiation during mammalian development.
A Large Family of POU-Domain Regulatory Genes in Mammalian Brain Development Brain development involves an intricate program of gene expression that leads to the establishment of many neuronal phenotypes and a precise complex pattern of connections between them. The nuclear events underlying neuronal development and the accompanying diverse patterns of specific gene expres sion remain largely unknown. It is likely, however, that the precise temporal and spatial patterns characteristic of mammalian brain development reflect sequential activation of a complex network of regulatory factors similar to those presumed to account for establishing structural patterns in Drosophila (51,23). The specification of a variety of neuronal phenotypes derived from an apparently homogeneous population of neuroepithelial cells are pre requisites for brain development. The possibility that novel POD-domain proteins were expressed during establishment of the nervous system with distinct spatial and temporal pat terns, potentially exerting some roles in specifying neuronal phenotypes, was investigated using a strategy based on the structure of the initial four members of the POU-domain gene family. Degenerate oligonucleotides based on two highly conserved amino sequences in the A region of the POU-specific domain and in the C-terminal portion of the POU homeodomain were used as primers for the polymerase chain reaction employing DNA complementary to brain and testes mRNAs as templates. Four new members of the POU-domain gene family were identified; three from brain cDNA (Brn-l, Brn-2, and Bm-3) and one from rat testes cDNA (Tst-l), although this gene is pre dominantly expressed in the brain. Predicted amino acid residues for each new member are shown in Figure 6b. Structural comparisons of the four new POU-domain proteins revealed that
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
POU-DOMAIN DEVELOPMENTAL REGULATION
783
they are all highly related to Pit-I, Oct-I, Oct-2, and unc-86. Recently another POD-protein has also been identified in C. elegans (ceh-6) (6a). These nine proteins constitute a distinct POD-domain protein family. Three of the new proteins (Brn- l , Brn-2, and Tst- l ) are highly homologous, with> 94% of the amino acid residues being identical among them throughout the entire POD-domain. Even the variable region between the POD-specific domain and the POD-homeodomain is well conserved among these proteins. Brn-3 is highly related to unc-86, including three characteristic additional amino acid residues in the region between the A and B portions of the POD-specific domain. Pit-l is distinct from the other eight proteins in the POD-family domain at several amino acid positions that otherwise would be totally conserved, and it is thus far the most divergent member of the family. The known POD-domain proteins appear to segregate into four classes arbi trarily referred to as POD-I (Pit-I), POD-2 (Oct-I, Oct-2), POD-3 (Brn-l, Brn-2, Tst-I), and POU-4 (Brn-3, unc-86). The N' terminal basic part of the POD-homeodomain is identical for the first 11 or 12 amino acids, and 15-18 amino acids in the C-terminal WFC region (see Figure 5) are particularly well-conserved between all members of each class. A consensus sequence for the POD-domain emphasizes the degree of primary amino acid sequence conservation among all nine family members. It is predicted that the POD III gene family will contain the most members. Each gene is expressed in at least one tissue other than brain and generates transcripts of different size. Thus Bm-l and Bm-2 exhibit virtually identical patterns of expression in the central nervous system, but Brn-l is clearly expressed in the medullary zone of the kidney, while Brn-2 is not (30). Hybridization for Brn-l and Brn-2 is found in at least some classes of neurons at all levels of the neuraxis. Almost all regions of the cerebral (layers II-V) and cerebellar (Purkinje cells) cortices are labeled, as are the basal forebrain cholinergic system, ventral midbrain dopamine system, paraventricular and supraoptic neuroendocrine system, somatic motoneurons in the cranial nerve nuclei and ventral hom, and tectum (Figure 8). In contrast, the Brn-3 gene transcript exhibits a considerably more restricted pattem with relatively dense hybridization limited to the habenula, posterior hypothalamic area, infer ior olive, inferior colliculus, and nucleus ambiguus. Brn-3 transcripts are uniquely present in sensory ganglion cells (in the trigeminal and dorsal root ganglia), which are derived from the neural crest. The Tst-I gene, while expressed in testes, is particularly well-expressed in brain and displays a characteristic pattern of expression in the cerebral (layers V-VI) and cere bellar (granule cells) cortices, as well as in the striatum. medial habenula, su perior colliculus and parabigeminal nucleus, and dorsal motor nucleus of the vagus nerve. Thus each POD-domain gene exhibits a unique, restricted pattern of expression, though not obviously correlated with known de-
INGRAHAM ET AL
784
Table 1 Distribution of POU-domain mANA expression in the adult rat neNOUS system determined by in situ hybridization
SENSORY GANGLIA
Pit·,
Brn-'
Brn· 2
•
•
•
+ +
CORTEX
isoeortox. layers 2-5 layers 5-6
olfactory bUlb. mitral poriglomorular
n. lateral olfactory tract
+
+
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+
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+ +
+
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+++ + + +
+
+
++
+ ++
+ +++
+
+ +
+ ++
+ +
... +
presubiculum. layer 2 CA1/Subiculum dentate gyrus subependymal zone AMYGDAlA
SEPTUM
BASAL GANGLIA striatum substantia innominata
s. nigraJyentral tegmental a.
THALAMUS medial habenula lateral habenula parafaseicular n. reticular n. zona incerta
HYPOTHALAMUS suprachiasmatiC n.
supraoptic/parallenltieular n. medial preoptic n.
tuberomammiUa'Y n. lateral mammillary n. medial rnammill9.ry n.
posterior hypothalamic a.
B�NSTEM-SENSORY
superior eolliculus, inter. gray parabigeminal (I. superior olive inferior COIliCUJu5. dorsal n.
area postrema
B�NSTEM - MOTOR motor
n. trigeminal
facial n.
hypoglossal n. n. ambiguuA
+
+
n. incertus
... ...-+
+ ... +
deep nuclei Purldnje
... ++
... ++
...
+
+
•
periaqueductal gray
interpeduncular n. CEREBELLUM & RELATED
granule redo.
inferior olive
SPINALCORO ventral horn
+ +++
+ ... +.+ ++
++
d
+.
++ t
+ ++
.+
+
++
+ ...++
+
++
... +
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Qct·2
++
dorsal motor n. vagus
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Oct·,
•
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piriform
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•
+++ + ++ ++ + ++
islands of Galleja
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
Brn·3
++1-+
+++
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Distributors of POU-domain mRNA expression in the adult rat nervous system
detennined by
in situ hybridization. The hypothalmic pattern of expression is noted.
POU-DOMAIN DEVELOPMENTAL REGULATION
785
Annu. Rev. Physiol. 1990.52:773-791. Downloaded from www.annualreviews.org Access provided by WIB6080 - Universitat Zu Kiel on 12/26/14. For personal use only.
velopmental, functional, or neurotransmitter-related systems in the nervous system. All four new POU-domain proteins are widely expressed in all levels of the neural tube during at least some part of this period. The anatomical restriction for each gene product in the developing neural tube is distinct however, and reflects the adult loci of expression. Expression is observed in the ventricular (proliferative) zone of the neuroepithelium, the mantle layer, the early cortical plate (Bm-l, Bm-2), and external granular layer of the cerebellum (Tst-l) (Figure 9). These data suggest that the genes are expressed
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