(Received for publication, June 19, 1995; and in revised form, August 30, 1995)
From the
To gain insights into the molecular mechanisms that restrict the expression of the oxytocin gene to anatomically defined groups of neurons in the hypothalamus, we generated transgenic mice bearing bovine oxytocin genomic fragments. Appropriate neuron-specific and physiological regulation was observed in mice bearing transgene bOT3.5, which consists of the oxytocin structural gene flanked by 0.6 kilobase pair (kbp) of upstream and 1.9 kbp of downstream sequences. bOT3.5 is expressed in oxytocin magnocellular neurons in the mouse supraoptic nucleus and paraventricular nucleus, but transgene RNAs are excluded from vasopressin neurons. Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increased the abundance of bovine oxytocin transcripts in the supraoptic nucleus, but not in the paraventricular nucleus, in parallel with the endogenous mouse oxytocin RNA. Surprisingly, mimicry of the endogenous oxytocin gene expression pattern was lost with larger transgenes. Addition of 0.7 kbp of contiguous downstream sequences (transgene bOT) or linkage to the bovine vasopressin gene (transgene VP-B/bOT3.5) repressed hypothalamic expression. No mice were derived bearing transgene bOT6.4, which consists of the oxytocin structural gene flanked by 3 kbp of upstream and 2.6 kbp of downstream sequences, suggesting that the presence of this DNA is detrimental to normal embryonic development. These data suggest that while bOT3.5 contains sufficient cis-acting sequences to mediate expression to particular subsets of hypothalamic neurons, the overall regulation of the oxytocin gene is governed by multiple interacting enhancers and repressors.
Ever since pioneering studies demonstrated that the posterior
pituitary contains activities that stimulate uterine contraction (1) and milk ejection(2) , the oxytocin (OT) ()system has been a favorite model for the study of the
regulation and function of an identified class of central peptidergic
neurons. OT is synthesized as a prepropeptide in the cell bodies of
anatomically defined groups (nuclei) of hypothalamic magnocellular
neurons. This precursor is subject to cleavage and other modifications
as it is transported down the axon to terminals located in the
posterior pituitary(3) . The mature peptide products (the
nonapeptide OT and a putative carrier molecule termed neurophysin) are
stored until neural inputs governed by physiological stimuli elicit
their release into the general circulation (4) . Through an
interaction with OT receptors located in the uterus and mammary gland,
OT stimulates smooth muscle contraction, leading to parturition or milk
ejection.
The single-copy structural gene encoding the OT prepropeptide consists of three exons and encompasses <1 kbp(5) . The OT gene is highly homologous at both the structural and sequence level to the gene encoding the related neuropeptide vasopressin (VP). In mammals, the two genes are separated by a short intergenic region of 11 kbp in the rat (5) and of 3 kbp in the mouse (6) and are transcribed toward each other from opposite strands of the DNA duplex. An attractive feature of the OT system is that, within the hypothalamus, the expression of the OT gene is confined to anatomically defined groups of magnocellular neurons in the supraoptic nucleus (SON) and in the paraventricular nucleus (PVN). VP is also expressed in hypothalamic magnocellular neurons, but VP and OT are rarely found in the same cell(7, 8, 9) . The OT gene is also expressed in a number of peripheral tissues(10) .
Studies on the physiological regulation of OT gene expression in the hypothalamus have benefited from the exploitation of well established paradigms for the modulation of the activity of OT neurons. These experiments have revealed that OT gene expression in magnocellular cells is increased in response to functional demand. Thus, during pregnancy and lactation, when pituitary stores of OT are depleted, the abundance of the OT mRNA in the hypothalamus increases(11) . Surprisingly, despite being expressed in distinct magnocellular neurons, the VP mRNA also increases in abundance during pregnancy and lactation(11) . Conversely, osmotic stimuli (such as salt loading) that result in functional demand for VP and an increase in VP gene transcription (12, 13) and VP mRNA abundance (14, 15, 16, 17) also result in similar changes in OT expression(16, 18) .
Little is known about the regulation of OT gene expression in the hypothalamus in terms of either the synaptic regulation of phenotype and physiological status or their mediation by second messengers, transcription factors, and cognate OT gene cis-acting elements. In vitro studies have described cis-elements in the OT gene promoter that are able to mediate transcriptional regulation, but the physiological relevance of these findings remains to be determined. For example, Richard and Zingg (19) have shown that the human OT promoter is able to direct the high level expression of a reporter in the mouse neuroblastoma cell line Neuro 2A. The sequences mediating this effect have been shown to consist of at least three cooperating elements located within the 50 base pairs upstream of the mRNA cap site. Both rat and human OT promoters contain direct and inverted repeats of an AGGTCA nuclear hormone receptor-binding site. Transfections into heterologous cultured cells have shown that these sequences can mediate transcriptional up-regulation of the OT promoter in response to the ligand-dependent binding of the estrogen(20, 21) , thyroid hormone(22) , and retinoic acid (23) receptors. Although the presence of these receptors in oxytocinergic neurons has not been demonstrated (24) , the finding that OT gene expression in the rat uterus (25) is estrogen-responsive (26, 27) may provide a physiological context within which these elements function. The bovine OT promoter is not responsive to estrogen(28) .
To define broad regulatory regions required for appropriate expression, we have generated transgenic mice bearing the bovine OT gene. Here, we describe a bovine OT transgene that is expressed in a cell type-specific manner in the mouse hypothalamus, within which it is subject to physiological regulation. The hypothalamic expression of this transgene is repressed by downstream sequences and by sequences in the VP gene.
Figure 1: Structures of bovine VP and OT transgenes. Shown are the structures of the bovine VP and OT transgenes described in this study and their relationship to the rat VP-OT locus (5) and to previously described rat (37) and bovine (30, 31) transgenes. The horizontal arrows indicate the direction of transcription. Shaded boxes represent exons. Rat transgenes are shown above the locus map, and bovine transgenes are shown below. Expression patterns for either VP (left side) or OT (right side) are described. HYP, hypothalamus; TEST, testis.
Figure 2: Expression and physiological regulation of bovine OT RNA in the hypothalamus of bOT3.5. A, Northern analysis of hypothalamic RNA from the following: lane 1, bOT2 (30 µg)(30) ; lane 2, bOT3.5 line 1 (30 µg); lane 3, VP-B/bOT3.5 line 3 (30 µg); lane W, wild-type mouse (40 µg); lane B, wild-type cattle (20 µg). B, Northern analysis of hypothalamic RNA (extracted using Trizol) from hypothalami (six/group) of control (C1-C3; 50 µg/lane) and salt-loaded (S1-S3; 50 µg/lane) bOT3.5 line 1 and from wild-type mouse (50 µg) and wild-type cattle (50 µg). RNAs were subjected to Northern blotting and probed sequentially with oligonucleotides corresponding to bovine OT RNA (BOT), rodent OTRNA (ROT), rodent VPRNA (RVP), and glyceraldehyde-6-phosphate dehydrogenase RNA (GAPDH). Note that the rodent OT probe fails to detect the bovine OT transcript in the cattle hypothalamus, while the bovine OT probe does not hybridize to the mouse OT RNA in the wild-type mouse hypothalamus.
Figure 3:
Expression of bovine OT RNA in transgenic
mice bearing the bOT3.5 transgene. Total RNA was extracted from tissues
from two independently derived transgenic mouse lines (lines 2 and 4),
and 50 µg was analyzed by Northern blotting(31) . For line
2, the following tissues were examined: BR, brain minus
hypothalamus; P, pituitary gland; Hy, hypothalamus; Lv, liver; S, spleen; AD, adrenal gland; K, kidney; T, testis; L, lung; Th,
thymus; Ht, heart; Ov, ovary; Ut, uterus;
and BH, bovine hypothalamus (positive control). For line 4,
the following tissues were examined: St, striatum; MO, medulla oblongata and pons; MB, midbrain; Cb, cerebellum; C, cortex; H, hippocampus; AP, anterior pituitary gland; N, neurointermediate
lobe of the pituitary gland; Hy, hypothalamus; Lv,
liver; S, spleen; P, pancreas; AD, adrenal
gland; K, kidney; T, testis; L, lung; Th, thymus; Ht, heart; Ov, ovary; Ut, uterus; and BH, bovine hypothalamus (positive
control). Filters were probed first with an oligonucleotide specific
for the bovine OT RNA (BOT) and then with an oligonucleotide
corresponding to the rat -tubulin RNA (TUB) as a control
for intact total RNA and even transfer. Note that
-tubulin RNA is
expressed at different levels in different tissues, but the relative
pattern of expression observed is consistent between animals. Four
independently derived transgenic lines were examined. Representative
Northern analyses from two lines (lines 2 and 4) are shown. Line 3
showed no expression of the transgene (data not shown). Expression in
the testis, lung, and hypothalamus was seen in each of the three
remaining lines. Line 1 also showed some expression in the midbrain,
cortex, kidney, and liver (data not shown). Bovine OT RNAs in different
tissues do not migrate at the same rate due to poly(A) tail length
variations(40) .
Figure 4: Expression of the endogenous OT gene and of the bOT3.5 transgene in the murine hypothalamus is confined to the SON and PVN. Coronal brain sections from transgene bOT3.5 line 1 (BOT3.5) and wild-type (WT) mice were probed with labeled oligonucleotides corresponding to the rodent (ROT) or bovine (BOT) OT RNAs. Representative sections are shown in the vicinity of the PVN and SON. Note that the bovine OT probe does not give any signal in the wild-type mouse hypothalamus.
Figure 5:
The OT and VP genes are expressed in
mutually exclusive magnocellular neurons in the SON and PVN of the
mouse, and the bOT3.5 transgene is expressed only in oxytocinergic
neurons. Coronal brain sections from control (C) or
salt-loaded (S) transgene bOT3.5 line 1 (BOT3.5) and
wild-type (WT) mice were probed simultaneously with a S-labeled oligonucleotide (indicated by an asterisk) and a digoxygenin-labeled oligonucleotide. The
oligonucleotides used were rodent OT (ROT), bovine OT (BOT), and rodent VP (RVP). Representative areas of
the SON and PVN are shown. The digoxygenin probe is revealed by brown coloration of the cell body cytoplasm (in focus in the upper panel). The radioactive probe is revealed by grains that
are in a different focal plane to the nonradioactive probe (shown in
focus in the lower panel). A, rodent OT and rodent VP
are present in mutually exclusive sets of magnocellular neurons. B, bovine OT colocalizes with rodent OT. C, bovine OT
is excluded from neurons expressing rodent
VP.
We have previously shown that transgene VP-B is expressed throughout the nervous system(31) . However, when linked to bOT3.5 in transgene VP-B/bOT3.5, no expression of the VP component was detected (data not shown), and transcripts from the OT component were seen in the testis and lung (Fig. 6), but not in the hypothalamus (Fig. 2A and Fig. 6).
Figure 6:
Expression of bovine OT RNA in transgenic
mice bearing the VP-B/bOT3.5 transgene. Total RNA was extracted from
tissues from three independently derived transgenic mouse lines (lines
1-3), and 50 µg was analyzed by Northern
blotting(31) . For lines 1 and 2, the following tissues were
examined: BR, brain; Lv, liver; S, spleen; AD, adrenal gland; K, kidney; T, testis; L, lung; Th, thymus; Ht, heart; Ov,
ovary; Ut, uterus; and BH, bovine hypothalamus
(positive control). For line 3, the following tissues were examined: BR, brain minus hypothalamus; P, pituitary gland; Hy, hypothalamus; Lv, liver; S, spleen; K, kidney; T, testis; L, lung; Th,
thymus; Ht, heart; Ov, ovary; Ut, uterus;
and BH, bovine hypothalamus (positive control). Filters were
probed first with an oligonucleotide specific for the bovine OT RNA (BOT) and then with an oligonucleotide corresponding to the
rat -tubulin RNA (TUB) as a control for intact total RNA
and even transfer. Note that
-tubulin RNA is expressed at
different levels in different tissues, but the relative pattern of
expression observed is consistent between animals. Four independently
derived transgenic lines were examined. Representative Northern
analyses from three lines (lines 1-3) are shown. Expression in
the testis and lung was seen in each line. Bovine OT RNAs in different
tissues do not migrate at the same rate due to poly(A) tail length
variations(40) .
Figure 7: Salt loading increases the abundance of mouse and bovine RNAs in the SON of bOT3.5 transgenic mice, but has no effect on the level of these transcripts in the PVN. A, serial sections through the SON of control (C) or salt-loaded (S) bOT3.5 line 1 probed with oligonucleotides corresponding to the rodent (ROT) or bovine (BOT) OT RNA. B, Voxelview analysis of the level of expression of RNAs detected by the bovine OT probe (BOT) and the rodent OT probe (ROT) in the PVN and SON in control (open bars) and salt-loaded (closed bars) bOT3.5 line 1 transgenic mice. C, serial sections through the PVN of control or salt-loaded bOT3.5 line 1 probed with oligonucleotides corresponding to the rodent (ROT) or bovine (BOT) OT RNA. The base of the brain (ventral surface) is to the right. Serial sections run rostral to caudal from the left to the right.
While the molecular mechanisms underlying the cell-specific and physiological regulation of the OT gene are the subject of much interest(34) , mechanistic understanding has lagged behind descriptive studies because of the lack of appropriate cell culture systems. To circumvent this obstacle(35) , we have applied transgenesis to the OT system.
We have previously described the expression in mice of the 4.2-kbp transgene bOT(30, 36) . bOT RNAs were detected in the testis and lung, but not in the hypothalamus. Initially, we thought that bOT must be missing elements crucial for expression in OT neurons. However, we now show that bOT contains too much sequence information, rather than too little. Surprisingly, bOT3.5, which differs from bOT only in that it lacks 0.7 kbp of distal downstream flanking sequence, is expressed consistently in the hypothalamus, within which transgene expression is confined to magnocellular cells in the PVN and SON that also express the endogenous mouse OT gene. Transgene RNAs, like the endogenous OT transcripts, are excluded from neurons expressing mouse VP RNAs. We suggest that the downstream sequences present in bOT (but absent from bOT3.5) contain one or more repressor elements that prevent the detectable expression of the former transgene in hypothalamic oxytocinergic neurons. It follows that in the normal genomic context of the OT gene, the activity of the putative repressors must be repressed or overridden.
We were unable to generate any transgenic mice
bearing construct bOT6.4 (Table 1). bOT6.4 differed from bOT only
in that the sequence upstream of the start of transcription consisted
of 3.0 kbp, rather than the 0.6 kbp found in the latter transgene.
Young et al.(37) have described a similar situation
with a rat OT transgene. No transgenic mice were represented in the
pups resulting from the injection of fertilized one-cell mouse eggs
with ROT1.63, which consisted of the entire rat OT structural gene
flanked by 0.36 kbp of upstream and 0.4 kbp of downstream
sequences (Fig. 1). Both rat and cattle OT genes contain
sequences that are detrimental to normal development. In the bovine
gene, these sequences must reside in the 2.4 kbp of distal upstream
sequences, present in bOT6.4, but absent in bOT. Presumably, ectopic OT
expression, mediated by these sequences at an inappropriate time, is
incompatible with embryogenesis. In the context of the normal OT gene,
this toxic effect must be repressed.
Because of the close linkage
between the VP and OT genes, it has been suggested that interactions
between the two transcription units may be important in locus control.
Here, we show that the bovine VP and OT genes can affect the expression
of each other. The VP-B/bOT3.5 transgene consists of the VP-B and
bOT3.5 constructs linked 3` to 3` (Fig. 1). We have previously
shown that VP-B, consisting of the bovine VP structural gene flanked by
1.25 kbp of upstream and 0.2 kbp of downstream sequences, is expressed
in all neuronal tissues examined (31) . As described above,
bOT3.5 is expressed in OT neurons, testis, and lung. In mice containing
the VP-B/bOT3.5 transgene, expression of the VP component could not be
detected in any central or peripheral tissue, while expression of
transgene OT RNA was seen only in the testis and lung. Thus, sequences
within the OT component of the transgene prevented expression of the VP
portion in neuronal cells, while sequences within VP-B repressed the
hypothalamic expression of bOT3.5. Support for the concept of
cross-talk between the VP and OT genes has also come from the
experiments of Young et al.(37) . These authors
described mice bearing a minilocus transgene (V2) consisting of
construct ROT1.63 (see above and Fig. 1) linked to sequences
from the rat VP gene (transgene RVP3.55) (Fig. 1). No transgenic
mice were derived from the microinjection of fertilized one-cell mouse
eggs with ROT1.63 (see above). On its own, RVP3.55, consisting of the
structural gene flanked by 1.4 kbp of upstream and 0.4 kbp of
downstream sequences (Fig. 1), was not expressed. (
)In the V2 minilocus transgene, the two genes were arranged
in the opposite transcriptional orientation (VP 3` to OT 5`) to the
native genes (VP 3` to OT 3`). The VP component of the V2 minilocus was
again silent. However, the OT transcription unit present in V2 was
expressed in oxytocinergic magnocellular
neurons(37, 38, 39) . Thus, elements within
the VP component must repress the deleterious effects of the OT
portion, allowing transgenic mice to be generated. Thus, the close
linkage of the VP and OT genes may be important for overall locus
control, perhaps by coordinating responses of the two genes to
developmental or regulatory cues. However, it should be noted that our
data also suggest that there is redundancy in the regulatory elements
contained within the two transcription units as the bOT3.5 transgene,
consisting only of OT gene sequences and proximal flanks, is expressed
autonomously in oxytocinergic neurons. Similarly, we have described
bovine VP transgenes that are properly regulated in the
hypothalamus(31) .
Physiological stimuli are known to affect the pattern of OT gene expression in the hypothalamus, and we asked if these same stimuli altered the expression of the bOT3.5 transgene. We have shown that the level of bovine OT RNA increased in the hypothalamus during salt loading, in parallel with the endogenous mouse OT RNA. Thus, the sequences mediating the physiological up-regulation of bovine OT gene expression reside within the confines of the bOT3.5 transgene. Interestingly, while salt loading resulted in a significant increase in both the endogenous mouse and transgenic bovine OT RNAs in the SON, no change in either RNA could be seen in the PVN. These data suggest that in the mouse, unlike in the rat(16) , the two nuclei respond differentially to an osmotic stimulus.
In summary, transgene bOT3.5 contains sufficient sequence information to direct expression to murine oxytocinergic magnocellular neurons, within which it is subject to physiological regulation. However, this expression pattern is not found when the same bOT3.5 sequences are linked either to the contiguous 0.7 kbp of downstream sequence (in bOT) or to the VP gene (in VP-B/bOT3.5), which is closely linked to the OT gene in mammals(5, 6) . The expression patterns seen with bovine VP (31) and OT ( (30) and this report) transgenes in mice suggest that the cell type-specific expression of these genes is mediated by interactions between multiple enhancers and repressors located in both transcription units.