Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor.

In the anterior pituitary gland, there are five phenotypically distinct cell types, including cells that produce either prolactin (lactotrophs) or growth hormone (somatotrophs). Multiple, related cis-active elements that exhibit synergistic interactions appear to be the critical determinants of the transcriptional activation of the rat prolactin and growth hormone genes. A common positive tissue-specific transcription factor, referred to as Pit-1, appears to bind to all the cell-specific elements in each gene and to be required for the activation of both the prolactin and growth hormone genes. The data suggest that, in the course of development, a single tissue-specific factor activates sets of genes that ultimately exhibit restricted cell-specific expression and define cellular phenotype.

Two different cis-active elements transfer the transcriptional effects of both EGF and phorbol esters.

Short cis-active sequences of the rat prolactin or Moloney murine leukemia virus genes transfer transcriptional regulation by both epidermal growth factor and phorbol esters to fusion genes. These sequences act in a position- and orientation-independent manner. Competitive binding analyses with nuclear extracts from stimulated and unstimulated cells suggest that different trans-acting factors associate with the regulatory sequence of each gene. A model is proposed suggesting that both epidermal growth factor and phorbol esters stimulate the transcription of responsive genes via discrete classes of hormone-dependent, enhancer-like elements that bind different trans-acting factors, even in the absence of hormone stimulation.

Some neurons of the rat central nervous system contain aromatic-L-amino-acid decarboxylase but not monoamines.

Neurons containing the enzyme aromatic-L-amino-acid decarboxylase (AADC) but lacking either tyrosine hydroxylase or serotonin were found in the spinal cord of neonatal and adult rats by light and electron microscopic immunocytochemistry. The majority of these neurons localized to area X of Rexed contact ependyma. Thus, spinal AADC neurons have the enzymatic capacity to catalyze directly the conversion of the amino acids tyrosine, tryptophan, or phenylalanine to their respective amines tyramine, tryptamine, or phenylethylamine. These amines normally present in the central nervous system may be of potential clinical significance as endogenous psychotomimetics.

Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization in neurons of the brain stem.

Neurons containing the enzyme aromatic L-amino acid decarboxylase were immunocytochemically localized in the brain stem of the rat. The enzyme occurred as expected in previously well characterized monoaminergic cell groups, and in addition in some nuclei with unknown neurotransmitters. Major aggregates of neurons that were immunoreactive for aromatic L-amino acid decarboxylase but contained neither tyrosine hydroxylase nor serotonin, were found in the pretectal nuclei, the lateral parabrachial nucleus, and the dorsolateral subdivision of the nucleus tractus solitarius. Aromatic L-amino acid decarboxylase was also present in serotonin neurons and the majority of catecholamine cell groups. Dopamine, noradrenaline, and adrenaline cells exhibited characteristic staining intensities to anti-aromatic L-amino acid decarboxylase reflective of relative enzyme levels in the different groups. Some cells in the dorsal motor nucleus of the vagus that were previously classified as dopaminergic lacked immunoreactivity to aromatic L-amino acid decarboxylase.

Positive and negative elements contribute to the cell-specific expression of the rat dopamine beta-hydroxylase gene.

Dopamine beta-hydroxylase catalyzes the final step in noradrenaline synthesis and is expressed exclusively in noradrenergic and adrenergic cells. In order to identify elements within the dopamine beta-hydroxylase (DBH) gene which contribute to the regulation of tissue-specific expression, we have analyzed the expression of the rat DBH promoter by transient transfection in both DBH-expressing and non-expressing cell lines. We have found that 1 kilobase of the DBH promoter can direct expression of the luciferase reporter gene in the DBH-expressing PC12, CATH.a, and SK-N-SH cell lines, but not in the non-DBH-expressing C6 glioma or CA77 cell lines. This activity was localized to a region between -133 and -173 upstream of the transcription start site. This element, however, also directed expression in non-DBH-expressing cell lines, but was inhibited when sequences between -212 and -388 were included. This inhibitory region contains sequences homologous to a silencer element recently identified in the human DBH gene, and shares homology with other previously identified silencer elements. Gel retardation experiments demonstrate that the rat DBH inhibitory region and the silencer elements found in the rat sodium type II channel and SCG10 genes bind a similar factor. The region between -133 and -173, which contains a consensus cyclic AMP response element (CRE), was also found to be responsive to cAMP in both DBH-expressing and non-expressing cells. Inclusion of sequences between -173 and -190 diminished the cAMP induction in PC12 cells, and nearly abolished the induction in C6 and CA77 cells, suggesting the presence of an additional negative element which inhibits cAMP induction in non-DBH expressing cells. DNA binding assays using antibodies to CRE binding protein-related transcription factors identified ATF-1 binding to the rat DBH-CRE, and further suggest that inhibition of cAMP regulation may be due to inhibition of ATF-1 binding by an additional factor, which binds to the DBH promoter immediately upstream of the CRE. These results demonstrate the importance of both positive and negative regulatory elements in the regulation of tissue-specific expression of the rat DBH gene.

Aromatic L-amino acid decarboxylase in the rat brain: coexistence with vasopressin in small neurons of the suprachiasmatic nucleus.

We demonstrated the coexistence of aromatic L-amino acid decarboxylase (AADC) and arginine-vasopressin in neurons of the hypothalamic suprachiasmatic nucleus of Sprague-Dawley rats. Neurons that lacked monoamines but expressed immunoreactivity to the enzyme AADC occupied the rostral and caudal poles of the suprachiasmatic nucleus and mediodorsal and dorsolateral positions along the entire extent of the nucleus. AADC was also localized in similar neurons of the suprachiasmatic nucleus of rats from other strains including the homozygous Brattleboro rat.

Tissue-specific expression of the nonneuronal promoter of the aromatic L-amino acid decarboxylase gene is regulated by hepatocyte nuclear factor 1.

The rat aromatic l-amino acid decarboxylase (AADC) gene contains alternative promoters which direct expression of neuronal and nonneuronal mRNAs that differ only in their 5'-untranslated regions (UTRs). We have analyzed the expression of the nonneuronal promoter of the rat AADC gene in the kidney epithelial cell line LLC-PK1 and in cells which do not express the nonneuronal form of AADC by transient transfection. These studies revealed that the first 1.1 kilobases of the nonneuronal promoter, including the nonneuronal-specific 5'-UTR (Exon 1), contains sufficient information to direct tissue-specific expression. Serial deletions of this promoter localized the cis-active element to a region between -52 and -28 base pairs upstream of the nonneuronal transcription start site. An A/T-rich sequence, within this region which we have termed KL-1, was found to bind a kidney and liver-specific factor by DNase footprint analysis and was capable of directing tissue-specific expression from a heterologous promoter. Moreover, when the KL-1 sequence was mutated in the context of the entire promoter sequence, all transcriptional activity was abolished. DNA sequence comparison revealed that the KL-1 fragment is highly homologous to the binding site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not express the nonneuronal form of AADC resulted in activation of transfected AADC nonneuronal promoter constructs. These results strongly suggest that the transcription factor which regulates the tissue-specific expression of the nonneuronal form of AADC mRNA is HNF-1.

A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues.

We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.

Distinct promoters direct neuronal and nonneuronal expression of rat aromatic L-amino acid decarboxylase.

Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28) catalyzes the decarboxylation of L-dopa to dopamine in catecholamine cells and 5-hydroxytryptophan to serotonin in serotonin-producing neurons. This enzyme is also expressed in relatively large quantities in nonneuronal tissues such as liver and kidney, where its function is unknown. Neuronal and nonneuronal tissues express AADC mRNAs with distinct 5' untranslated regions. To understand how this is accomplished at the genomic level, we have isolated rat genomic DNA encoding AADC. The organization of the AADC gene suggests that there are two separate promoters specific for the transcription of neuronal and nonneuronal forms of the AADC message. A small exon containing 68 bases of the neuronal-specific 5' end is located approximately 9.5 kilobases upstream of the translation start site, which is contained in the third exon. Approximately 7 kilobases upstream from the neuron-specific promoter is another small exon containing 71 bases of the 5' end of the nonneuronal AADC message. These data suggest that transcription initiating at distinct promoters, followed by alternative splicing, is responsible for the expression of the neuronal and nonneuronal forms of the AADC message.

A two-base change in a POU factor-binding site switches pituitary-specific to lymphoid-specific gene expression.

The structurally related POU homeo domain proteins Pit-1 and Oct-2 activate pituitary- and lymphoid-specific transcription, respectively, by binding to similar AT-rich motifs in their target genes. In this study we identify bases critical for recognition and activation by Pit-1 and examine how small differences in Pit-1 and Oct-2-binding sites can impart differential transcriptional responses in pituitary and B-lymphoid cells. Scanning mutagenesis of Pit-1 response elements in both the rat prolactin and growth hormone genes reveals a critical binding motif recognized in an identical manner by the native Pit-1 protein and cloned Pit-1 gene product. This motif, ATTATTCCAT, differs by only two bases from the octamer element, ATTTGCAT, required for Oct-2-dependent activation of immunoglobulin genes. Cross recognition of Pit-1 and Oct-2 sites by both factors can be demonstrated in competitive binding assays, in which an oligometric Pit-1 site from the prolactin gene is converted to an Oct-2 site by a double point mutation. In contrast to the binding data, no cross activation of transcription is detectable in cultured cell lines. When inserted immediately 5' to a prolactin TATA box, the wild-type prolactin element enhances transcription strongly in pituitary cells but is inactive in B cells, whereas the octamer variant of the prolactin site activates expression in B cells but is silent in pituitary lines. Both elements are nonfunctional in heterologous cell lines that lack Pit-1 and Oct-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the neuronal promoter of the rat aromatic L-amino acid decarboxylase gene.

The rat aromatic L-amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5' untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non-AADC-expressing cells. We demonstrate the use of the neuronal-specific UTR in individual dopamine-, norepinephrine-, and serotonin-containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68-bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis-active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue-specific expression of the neuronal promoter may not be controlled by cis-active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.

Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia: studies in vivo and in vitro.

We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.

Different forms of adrenal phenylethanolamine N-methyltransferase: species-specific posttranslational modification.

Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.

Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements.

The pit-1 gene is a member of a large family of genes that encode proteins which are involved in development and which contain a highly homologous region, referred to as the POU domain. Pit-1, a pituitary-specific transcription factor, can activate the transcription of the growth hormone and prolactin promoters. It is expressed in mature thyrotroph, somatotroph and lactotroph cell types of the anterior pituitary which arise sequentially during development; somatotrophs and lactotrophs, which secrete growth hormone and prolactin, respectively, are the last to arise. Intriguingly, during ontogeny, pit-1 transcripts are observed in the rat neural tube and neural plate (embryonic day 10-11) and disappear thereafter (day 13), only to reappear exclusively in the anterior lobe of the pituitary gland (day 15) just before activation of prolactin and growth hormone. This biphasic pattern suggests a complex mechanism of initial activation of pit-1 gene expression. Transcription and transfection analyses in vitro using wild-type and mutated promoters indicate that Pit-1 can positively autoregulate the expression of the pit-1 promoter as a consequence of binding to two Pit-1-binding elements. Mutation of the 5' Pit-1-binding site abolished positive autoregulation, whereas mutation of the element located immediately 3' of the cap site markedly increased expression of the pit-1 promoter. These data are consistent with a positive, attenuated autoregulatory loop that seems to function in maintaining pit-1 gene expression.

A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone.

The substance 3,5,3-triiodothyronine (T3) stimulates growth hormone gene transcription in rat pituitary tumour cells. This stimulation is thought to be mediated by the binding of nuclear T3 receptors to regulatory elements 5' to the transcriptional start site. Understanding of the mechanism by which thyroid hormone activates gene transcription has been limited by failure to purify nuclear T3 receptors because of their low abundance, and by the absence of defined T3 receptor-DNA binding sites affecting T3 regulation. Recently, human and avian c-erb-A gene products have been shown to bind thyroid hormone with high affinity and to have a molecular weight and nuclear association characteristic of the thyroid hormone receptor. In the present report, we describe the development of an avidin-biotin complex DNA-binding assay which can detect specific, high-affinity binding of rat pituitary cell T3 receptors to the sequence 5'CAGGGACGTGACCGCA3', located 164 base pairs 5' to the transcriptional start site of the rat growth hormone gene. An oligonucleotide containing this sequence transferred T3 regulation to the herpes simplex virus thymidine kinase promoter in transfected rat pituitary GC2 cells, and specifically bound an in vitro translation product of the human placental c-erb-A gene. The data provide supporting evidence that the human c-erb-A gene product mediates the transcriptional effects of T3 and also that GC2 cell nuclear extracts contain additional factors that modify the binding of pituitary T3 receptors to the rat growth hormone gene T3 response element.

Discrete cis-active genomic sequences dictate the pituitary cell type-specific expression of rat prolactin and growth hormone genes.

The anterior pituitary gland, which is derived from a common primordium originating in Rathke's pouch, contains phenotypically distinct cell types, each of which express discrete trophic hormones: adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), prolactin, growth hormone, and follicle stimulating hormone (FSH)/luteinizing hormone (LH). The structurally related prolactin and growth hormone genes, which are evolutionarily derived from a single primordial gene, are expressed in discrete cell types--lactotrophs and somatotrophs, respectively--with their expression virtually limited to the pituitary gland. The pituitary hormones exhibit a temporal pattern of developmental expression with rat growth hormone and prolactin characteristically being the last hormones expressed. The reported co-expression of these two structurally related neuroendocrine genes within single cells prior to the appearance of mature lactotrophs, in a subpopulation of mature anterior pituitary cells, and in many pituitary adenomas raises the possibility that the prolactin and growth hormone genes are developmentally controlled by a common factor(s). We now report the identification and characterization of nucleotide sequences in the 5'-flanking regions of the rat prolactin and growth hormone genes, respectively, which act in a position- and orientation-independent fashion to transfer cell-specific expression to heterologous genes. At least one putative trans-acting factor required for the growth hormone genomic sequence to exert its effects is apparently different from those modulating the corresponding enhancer element(s) of the prolactin gene because a pituitary 'lactotroph' cell line producing prolactin but not growth hormone selectively fails to express fusion genes containing the growth hormone enhancer sequence.

The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions.

Pit-1 is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (POUS) domain and a more divergent homeodomain (POUHD). Analysis of mutant Pit-1 proteins suggests that, while the POUHD is required and sufficient for low affinity DNA binding, the POUS domain is necessary for high affinity binding and accurate recognition of natural Pit-1 response elements. Pit-1 is monomeric in solution but associates as a dimer on its DNA response element, exhibiting DNA-dependent protein-protein interactions requiring the POUS domain. Analysis of alpha-helical domains and conserved structures in Pit-1 suggests that POU domain proteins interact with their DNA recognition sites differently than classic homeodomain proteins, with both the POUHD and the POUS domain contacting DNA. Transcriptional activity of Pit-1 on enhancer elements is conferred primarily by a Ser- and Thr-rich N-terminal region unrelated to other known transcription-activating motifs.

A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally.

The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types within an organ, a central question in development. The rat prolactin and growth hormone genes are expressed selectively in distinct cell types (lactotrophs and somatotrophs, respectively) of the anterior pituitary gland, reflecting differential mechanisms of gene activation or restriction, as a result of the interactions of multiple factors binding to these genes. We find that when the pituitary-specific 33-kD transcription factor Pit-1, expressed normally in both lactotrophs and somatotrophs, is expressed in either the heterologous HeLa cell line or in bacteria, it binds to and activates transcription from both growth hormone and prolactin promoters in vitro at levels even 10-fold lower than those normally present in pituitary cells. This suggests that a single factor, Pit-1, may be capable of activating the expression of two genes that define different anterior pituitary cell phenotypes. Because a putative lactotroph cell line (235-1) that does not express the growth hormone gene, but only the prolactin gene, appears to contain high levels of functional Pit-1, a mechanism selectively preventing growth hormone gene expression may, in part, account for the lactotroph phenotype.

Phenylethanolamine N-methyltransferase-containing neurons in rat retina: immunohistochemistry, immunochemistry, and molecular biology.

We sought to characterize in detail neurons in rat retina that contain phenylethanolamine N-methyltransferase (PNMT), the epinephrine biosynthetic enzyme. Cell bodies and processes of PNMT-containing neurons in retina were identified by immunohistochemistry. The coexistence of other catecholamine biosynthetic enzymes in the same cells was also investigated. Biochemical, molecular biological and immunochemical methods were applied to determine whether retinal PNMT is similar to the adrenal enzyme, since regulation of PNMT in retina and adrenal appears to be different. The results show that there are two types of PNMT-containing cells: those containing PNMT exclusively and those containing PNMT with two other catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), but not dopamine beta-hydroxylase (DBH). PNMT-only cell bodies are localized in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Their processes are observed in outer and inner strata of the inner plexiform layer (IPL). Only a small fraction of PNMT neurons in INL also contain TH and AADC. These cells send their processes to the adjacent stratum of the IPL. Antibodies to bovine adrenal DBH, however, fail to localize DBH in any rat retinal cells. Immunochemical titration shows that PNMT from both retina and adrenal gland has the same immunoreactivity. Furthermore, a PNMT-cDNA probe hybridizes equally with PNMT-mRNA isolated from both the retina and the adrenal gland. These results indicate that PNMT is identical in these tissues.

A family of POU-domain and Pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development.

The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types, within an organ, a central question in development. The rat prolactin and growth hormone genes are selectively expressed in distinct cell types (lactotrophs and somatotrophs) of the anterior pituitary gland, which reflect differential mechanisms of gene activation or restriction because of interactions of multiple factors binding to these genes. We find that the pituitary-specific 33,000 dalton transcription factor, Pit-1, normally expressed in somatotrophs, lactotrophs, and thyrotrophs, can bind to and activate both growth hormone and prolactin promoters in vitro at levels even tenfold lower than those normally present in pituitary cells. In the case of the prolactin gene, high levels of expression in transgenic animals required two cis-active regions; a distal enhancer (-1.8 to -1.5 kb) and a proximal region (-422 to +33 bp). Each of these regions alone can direct low levels of fusion gene expression to prolactin-producing cell types in transgenic mice, but a synergistic interaction between these regions is necessary for high levels of expression. The initial appearance of the prolactin transgene expression closely follows the appearance of high levels of Pit-1, but later increases in expression coincident with appearance of mature lactotrophs suggest the operation of additional, critical positive factor(s). Unexpectedly, transgenes containing the distal enhancer removed from its normal context are expressed in both the prolactin-producing lactotrophs and the TSH-producing thyrotrophs, thereby suggesting that sequences flanking this enhancer are necessary to restrict expression to the correct cell type within the pituitary. These data indicate that distinct processes of gene activation and restriction are necessary for the fidelity of cell-type specific expression within an organ. Consistent with this model, we find that lactotroph cell lines that cannot express the growth hormone gene contain high levels of functional Pit-1. We suggest a large, highly related POU-domain gene family, potentially exceeding 100 members, has been conserved and expanded in evolution to meet the increasing requirements for more intricate patterns of cell phenotypes. The POU-domain subgroup of the homeodomain gene family, in concert with other homeodomain proteins and with other classes of transcription factors, is likely to contribute to the establishment of the mammalian neuroendocrine system.