J. Joosse, R.M. Buijs and F.J.H. Tilders (Eds.)

Progrers i n Brain Research, Val. 92

0 1992 Ekevier Science Publishers B.V. All rights reserved.

77

CHAPTER 7

Neuropeptide gene expression in transgenic animals David Murphy, Hwee-Luan Ang, Qi Zeng, Mei-Yin Ho, Judith Funkhouser and David Carter Neuropeptide Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511, Republic of Singapore

Introduction The regulation and function of the peptidergic neuron is best studied within the complexities of the whole organism. In vitro systems have their place in the study of neurosecretion, but can never mimic the entire gamut of developmental and physiological influences that govern the overall operation of those neurons that secrete biologically active peptides. In this context, it might be anticipated that technologies that allow for the manipulation of specific genes within whole intact mammal organisms, would be seized upon by neuroscientists. However, reports on the expression of neuropeptide genes in transgenic animals, or the expression of transgenes in peptidergic neurons, have been few. This is probably because transgenesis technologies remain unavailable to most neuroscientists. It is, however, to be hoped that, as transgenic technologies are developed and refined, and become more widely available, more studies will address problems in neurosecretion using whole-animal DNA-mediated gene transfer (Rosenfeld et al., 1988; Rossant, 1990). In this paper we will review the few published studies describing the application of transgenesis techniques to investigate neuropeptide gene regulation and function. Emphasis will be on strategies adopted and lessons learned rather than a detailed cataloguing of results. A description of some of the work of this laboratory on the expression of the

bovine vasopressin and oxytocin genes in transgenic mice and rats will be followed by a summary of the potential future of novel transgenesis approaches to furthering our understanding of the peptidergic neuron. Mammalian organisms that bear genetic changes as a consequence of direct and specific experimental genomic manipulation are termed “transgenic” (Jaenisch, 1988). Characteristically, transgenic animals carry the same genetic change (the transgene) in every cell. Further, if the genetic change is present in the cells of the germ-line it will be passed on to subsequent generations. Extensive breeding thus allows for the generation of a large number of genetically identical transformed organisms that bear a specific chromosomal alteration in every cell. Thus a large pool of identical experimental animals can be generated. Of fundamental importance has been the observation that transgenes are often expressed. The tissue-specific, developmental and physiological control of expression is dependent upon the regulatory elements contained within the transgene. If the entire repertoire of cis-acting elements associated with a particular gene have been included in the transgene, either through good luck or good planning, then the expression of the transgene will exactly parallel the pattern of activity of the endogenous gene. The techniques available for the generation of transgenic mice are well known (Hogan et al., 1986) and it is beyond the scope of this paper to describe

78

them in any detail. Suffice to say that three methodologies have been developed all of which depend upon intervention at the preimplantation stages of early embryo development. 1. The physical introduction, by microinjection, of cloned DNA fragments into the pronuclei of fertilised one-cell eggs (Hogan et al., 1986; Murphy and Hanson, 1987). 2. The infection of early embryos with recombinant retroviruses (Soriano et al., 1986; Hogan et al., 1986). 3. The isolation from blastocysts of totipotent “embryonal stem” (ES) cells (Robertson, 1987), their manipulation in vitro by DNA-mediated gene transfer (Lovell-Badge, 1987) and their reintroduction into mouse embryos by blastocyst colonisation (Bradley, 1987). The worth of other methods, such as DNAmediated gene transfer into embryos using sperm as a vector (Lavitrano et al., 1989; Maddox, 1989), or DNA ionotophoretic microinjection (Lo, 1983), has not been proven. Microinjection remains the most popular and successful method of transgenic animal generation (Murphy and Hanson, 1987). However, the technique is limited in that it inevitably results in the aquisition of new genetic information by the recipient organism. Whilst the mechanism of integration of the injected DNA is not understood, it appears to be a random or illegitimate process, with no recombinatorial specificity with respect to either host or target sequences. The endogenous gene remains physically unaltered. However, it is now possible to physically alter the endogenous genes of the mouse (Capecchi, 1989a,b). This is achieved by applying recently developed screening techniques that allow for the selection of rare homologous recombination events in ES cell cultures transformed with cloned DNA fragments. Clearly, technologies that result in specific endogenous mouse neuropeptide genes being inactivated or subtly altered will be extremely powerful (Rossant, 1990).

Neuropeptide gene expression in transgenic mice

The literature to date Experiments in which neuropeptide genes have been expressed in transgenic mice can be divided into two categories: those that seek to explore the mechanisms by which the regulation of these genes are controlled; and those that seek to explore the physiology and function of the mature products encoded by neuropeptide genes. Often, these two aims cannot be separated, or are dependent upon each other. Thus, a maximal array of regulatory elements, sufficient to direct the correct expression of a particular neuropeptide transgene, must be defined before physiological or functional studies, involving the appropriate expression of altered, mutated or even different peptides, can be performed. Initial strategies A number of studies have sought to determine the sequence requirements for the correct tissue-specific and physiological regulation of neuropeptide genes. These studies are outlined in Table I. Early workers were encouraged by the studies of Hanahan (1985) who was able to show correct pancreatic ,&cell specific expression of an oncogene reporter element, the SV40 large T-antigen, when controlled by only 600 bp of 5 ’ upstream sequences from the rat insulin I1 gene. Attempts to duplicate this approach using promoter sequences from neuropeptide genes proved to be somewhat disappointing. Thus, a hybrid oncogene made up of 1.6 kb of the human growthhormone releasing factor (GRF) promoter linked to SV40 large T-antigen, failed to elicit expression in the hypothalamus or brain (Botteri et al., 1987). Rather, expression was detected in the thymus, a tissue that does not express the GRF gene. Ectopic expression of the large T-antigen oncogene in the thymus of the transgenic mice resulted in a marked hyperplasia (Botteri et al., 1987). Similarly, expression of a hybrid transgene made up of 1.25 kb of the bovine vasopressin (VP) promoter linked to the

79 TABLE 1 Expression of neuropeptide genes in transgenic mice

5‘ end

(Kb)

3 ’ end

(Kb)

Peripheral

Neuronal _ _ _ ~

~-

SP

Ec

3.5

+

--

~~

M GnRH B VP B VP B OT R OT

5.0 1.25 9.0 0.6 0.36

M GnRH SV40 T-Ag B VP B OT R OT

4.5

R VP R OT

1.4

0.36

R VP R OT

2’15 1.27

R POMC R GRF R GRF R GRF H VIP

0.77 1.6 1.6 1.6 5.2

~-

-

2.6 1.27

+

1

~

~

~

_

___ _ _

-

~

+ + +

1

2 3

nd

nd

5

-

nd nd

nd nd

5 5

?

_

-

-

~

_

Ec

-

-

SV40 T-Ag NGF c-Ha-ras CAT

+

-

_

+ +/-

+

neo

SP

-

-

ML

_

~

? nd

+/-

? ?

nd

?

+

+ ~

-

+

+

~

+ ~

-

4

6 7 7 I

8

~

Abbreviations: B, bovine; CAT, chloramphenicol acetyl transferase; EC, ectopic; H, human; GnRH, gonadotrophin releasing hormone; GRF, growth hormone releasing factor; M , mouse; ML, mini-locus; neo, neomycin resistance gene; nd, not determined; NGF, nerve growth factor; OT, oxytocin; POMC, proopiomelanocortin; R, rat; SP, specific; VIP, vasoactive intestinal peptide; VP, vasopressin. References: 1 , Mason et al., 1986b; 2, Murphy et al., 1987; 3, Carter et al., 1989; 4,Ang et al., 1991; 5 , Young et al., 1990; 6, Tremblay et al., 1988; 7, Botteri et al., 1987; 8, Agoston et al., 1990.

SV40 large T-antigen reporter could not be detected in the hypothalamus of carrier mice. Expression, which resulted in a profound tumorigenesis, was detected in the anterior pituitary gland and the 0cells of the pancreatic islets (Murphy et al., 1987; see below). Again, neither of these tissues express the endogenous vasopressin gene at a detectable level (Murphy et al., 1989; D.M. and D.C., unpublished observations). These studies taught two important lessons. Firstly, the limited GRF or V P promoter segments included in these hybrid transgenes contained insufficient regulatory information to allow the expression of a reporter element to mimic that of the native, endogenous gene. Secondly, it became apparent that the removal of regulatory sequences from their normal genomic context, and/or their ar-

tificial association with new downstream sequences, could result in unexpected tissue specificities.

Ectopic expression of transgenes in peptidergic neurons The creation of novel expression patterns by the experimental combination of specific regulatory elements was first described in mice transgenic for a fusion gene made up the mouse metallothionein-I promoter linked to either the rat or the human growth hormone genomic coding and downstream sequences (Swanson et al., 1985). Whilst expression of the hybrid transgene generally mimicked that of the endogenous metallothionein gene (Palmiter et al., 1983), ectopic activity was detected in neurons of specific brain regions. Endogenous growth hor-

80

growth hormone coding and downstream sequences linked to a variety of promoter -enhancer elements (metallothionein; human vasopressin, 1.5 kb; calcitonin, 1.35 kb; growth hormone, 180 bp; Herpes simplexvirus (HSV) thymidine kinase, 209 bp; prolactin, both 3 kb and 450 bp; prolactin enhancer linked to HSV thymidine kinase promoter; growth hormone enhancer linked to the HSV thymidine kinase promoter). Two additional constructs assessed were a hybrid consisting of the metallothionein promoter and a rat calcitonin reporter, which was found to elicit cortical expression, and a fusion made up of the metallothionein promoter and a GRF reporter, which was not active in the brain. These data are summarised in Table 11. It was found that ectopic neuronal expression was not a unique feature of the metallothionein promoter, as three other promoter - enhancer sequences (vasopressin, calcitonin and HSV thymidine kinase) were able to

mone expression is confined exclusively to the mouse anterior pituitary gland (Frohman, 1987), whilst mouse metallothionein is only found in nonneuronal cells of the brain (Swanson et al., 1985). Expression of the metallothionein-growth hormone hybrid was, however, detected in neurons of the hypothalamus, hippocampus and cortex (Swanson et al., 1985). Interestingly, the predominant cell type expressing the metallothionein-growth hormone transgene in the hypothalamus were the magnocellular neurons (Russo et al., 1988; Table 11). Indeed, ectopic growth hormone immunoreactivity was shown to colocalise with vasopressin in the supraoptic nucleus and with oxytocin in the paraventricular nucleus (Russo et al., 1988). These observations led to a series of experiments in which the expression patterns of 14 fusion genes were tested in transgenic mice (Table 11). Twelve of the hybrids contained reporter sequences based on either the rat or human TABLE I1 Expression of chimeric genes in the brains of transgenic mice 5 ' end

Neuronal expression

3'end Hypothalamus SON

MT MT MT MT MT * MT hVP 1.5 kb rCal 1.35 kb TK rGH rGH-TK rPRL 3 kb rPRL 0.45 kb rPRL-TK

HIP PVN

NEO

OLF

SCN

rGH hGH rGH cDNA rCal hGRF rVP hGH hGH hGH hGH hGH hGH hGH hGH ~

Abbreviations: Cal, calcitonin; G H , growth hormone; h, human; HIP, hippocampus; CA3, pyramidal field and dentate gyrus; MT, metallothionein; NEO, neocortex, layers I1 and 1V; OLF, nucleus of the lateral olfactory tract and piriform cortex; PRL, prolactin; PVN, paraventricular nucleus; r, rat; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; TK; Herpessimplex thymidine kinase; VP, vasopressin. All data taken from Russo et al. (1988) except * taken from Habener et al., (1989).

elicit varying degrees of activity in overlapping sets of cortical and hypothalamic neurons. Thus a variety of 5 ’ promoter -enhancer sequences contain elements that, when juxtaposed to elements within the growth hormone coding and downstream sequences, are able direct expression to peptidergic neurons. Russo and colleagues (1988) then started the process of mapping the sequences within the growth hormone gene that are required for ectopic neuronal expression. A mini-transgene made up of the metallothionein promoter linked to the rat growth hormone cDNA was not expressed in brain neurons, indicating that the sequences required for ectopic neuronal activity must reside within the introns of the growth hormone gene, or lie downstream of the last exon. In a separate experiment, Habener and colleagues described the expression in mice of a hybrid gene made up of the mouse metallothionein promoter linked to the rat genomic sequences encoding vasopressin (1989). This transgene, like the metallothionein - growth hormone hybrids described above, was predominantly expressed in parallel with the endogenous metallothionein gene. However, expression was also detected in the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus - cells which normally express vasopressin (Table 11). Thus, both a transgene made up of a vasopressin promoter linked to growth hormone coding sequences (Russo et al., 1988), and a transgene consisting of the metallothionein and vasopressin coding sequences (Habener et al., 1989), are able to elicit expression in the same (appropriate) cell type. Whilst the vasopressin promoter in the former transgene is human derived, and the vasopressin coding sequences of the latter are from the rat, there is a considerable degree of homology between the two species in the overlapping sequences that are present in both transgenes. These common sequences, extending 7 bp upstream of the TATA box to 6 bp upstream of the methionine translation initiation codon (83 bp in human, Sausville et al., 1985; 74 bp in rat, Schmitz et al., 1991) contain 5 5 identical nucleotide residues, in terms of both position and identity, localised in a

number of homology blocks. However, the putative “neuron specific element” located just downstream of the rat TATA box, and highlighted by Habener and colleagues (1989) as a possible mediator of hypothalamus-specific expression is not present in the human sequence (Sausville et al., 1985). Other brain-specific cis-acting elements may reside in the common sequence. However, a more likely explanation for the expression o f these two transgenes in hypothalamic neurons is that the interaction of different elements from particular genes, contained in sequences both upstream and downstream of the start of transcription, brought together in a novel fusion gene, are able to elicit expression in a particular sub-set of hypothalamic neurons. These data are important in that they point to genomic processes that might mediate the expression of genes in normal pepidergic cells. Expression of some neuropeptide genes in peptidergic cells is probably mediated by an interaction between neuronal transcription factors (some of which may be unique to the brain, but not necessarily so) and two or more regulatory elements located both upstream and downstream of the start of transcription. The nature of these elements has yet to be determined, but may encompass “silencer”-type sequences (Wuenschell et al., 1990) as well as classical enhancer-type motifs. Further, these data suggest that the elements that elicit expression in specific sets of neurons are no different from those that regulate expression inother cellsof the organism. The “brain specific regulatory sequence” is as yet more an idea of mythology rather than an experimentally tested entity. The reproducible ectopic expression of a transgene in specific peptidergic cells has practical implications that have yet to be exploited. Although not mediated by “natural” elements, it may be possible to express novel proteins in specific peptidergic cells (for example, those of the vasopressin and oxytocin systems) using a combination of sequences derived from the growth hormone and metallothionein genes. Such artificial neuronal expression vectors may be useful in physiological studies.

82

Successful experiments A small number of workers have reported the production of animals that bear transgenes that are expressed in an appropriate manner. Transcriptional regulation A number of transgenes encoding neuropeptides are transcriptionally regulated in a manner that mimics that endogenous gene. However, few studies have examined the situation in the brain. Thus, although transgenes made u p of the vasoactiveintestinal peptide (VIP) gene (Agoston et al., 1990) and the pro-opiomelanocortin gene (Tremblay et al., 1988) have been shown to be appropriately expressed in intestine and anterior pituitary gland respectively, n o studies have reported a n examination of the expression of these transgenes in the brain. Similarly, although agrowth hormone releasing factor (GRF) - SV40 large T-antigen fusion was described as being expressed ectopically in the thymus (Botteri et al., 1987), the same paper reported that GRF-nerve growth factor (NGF) and G R F rus fusions were expressed in brain, but no description of the expressing cell types was presented. Thus it is not known whether expression of these transgenes in the brain is appropriate o r not. The best example of the successful mimicry by a transgene of the endogenous gene is the appropriate expression of a gonadotrophin releasing hormone (GnRH) transgene in mice (Mason et al., 1986b). A DNA fragment consisting of a 13.5 kb segment of murine genomic DNA containing the entire G n R H coding sequences and 5 kb of 5 ' flanking DNA and 3.5 kb of 3 ' flanking DNA was introduced into 2 independently derived transgenic lines. By selective breeding, the G n R H transgene was then introduced into a mutant strain of mouse (the hypogonadal strain, hpg) that lacks G n R H as a consequence of a genoniic deletion of 33.5 kb of DNA encompassing thedistal half of thegene (Mason et al., 1986a). The hpg mouse is infertile due to a postnatal block in gonadal development. However, the neurons that would normally express G n R H are present in these animals, implying that the mutant phenotype is en~

tirely due t o the absence of the G n R H gene product. 'The presence of the functional G n R H transgene in the hpg background restored fertility, as well as pituitary and serum luteinising hormone, folliclestimulating hormone and prolactin levels, to those of normal animals. Further, immunocytochemical and in situ hybridisation analysis revealed transgene expression in the appropriate hypothalamic cells. Expression was not, however, totally specific and low-level ectopic expression was also observed in the liver and in the paraventricular nucleus. Post t ranscript ional regulation Gene expression is regulated at many levels following the initial generation by polymerase I1 transcription of a primary RNA. In neurons, alternative pre-RNA splicing to produce different mature mRNAs (Leff et al., 1986; Maniatis, 1991) is a n important mode of post-transcriptional regulation, which provides for thegeneration of adiversity of peptide products from a single transcription unit. In the case of the calcitonin a n d calcitonin-generelated peptide (CGRP) gene, the two peptides, calcitonin and C G R P , are encoded by alternatively spliced mature transcripts derived from the same transcription unit and pre-mRNA (Amara et al., 1982). Calcitonin mRNAs are primarily observed in thyroid c-cells, whilst C G R P mRNAs are expressed i n the adrenal medulla and in specific neurons of the central a n d peripheral nervous systems (Rosenfeld et a!., 1983). The calcitonin-CGRP coding sequences have been expressed in transgenic mice under the control of the metallothionein promoter (Crenshaw et al., 1987). Expression of this transgene was found to be widespread throughout the animal, but the splicing choices observed were very tissue-specific. Thus, the splicing route leading to calcitonin m R N A was the choiceof the visceral and muscle tissues, whilst most brain neurons decided upon the C G R P route. Presumably tissue-specific splicing factors are able to recognise sequences in the pre-mRNA. Transgenic mice ought to be useful in the identification of these cis-acting elements.

83

Altering peptidergic neurons Having described a set of sequences able to elicit appropriate expression of a transgene to a defined cell-type, it ought to be possible to use that information to alter that cell type, and hence the whole organism, in a specific way by expression-targeting gene products that might evoke a particular novel physiologic consequence. A good example of this approach to understanding peptidergic neuron function has been provided by the recent derivation, using transgenic techniques, of immortal GnRH synthesising cell lines (Mellon et al., 1990). A transgene consisting of 2 kb of the GnRH promoter driving the expression of SV40 large T-antigen, was introduced into 9 independent founder mice. Two of these animals displayed anterior hypothalamic tumours, and cells from one of the lines have successfully been cultured in vitro. These immortal cells express GnRH and the neuronal markers neuron-specific enolase and neurofilament, but do not express a range of glial markers. The cells, which extend neurites, release GnRH in response to depolarisation and have neuron-type regulatable fast N a + channels. These cells are clearly a remarkable example of the ability of transgenesis techniques to provide powerful new tools to address difficult questions in neurobiology. However, the application of this technology to other neuropeptide genes has thus far proved disappointing(Botteriet al., 1988; Murphyet al., 1987). It has been suggested that the GnRH neurons may be unusually prone to perturbation of their growth control, being neural crest derived and exhibiting GnRH expression prior to the cessation of cell division (Mellon et al., 1990). It is considered that the majority of neurons, being post-mitotic, are resistant to the growth-stimulating effects of oncogenes. However, it is too early to be dogmatic about the general applicability of an approach which will be the subject of much future experimentation. Vasopressin and oxytocin gene expression in transgenic animals

The vasopressin and oxytocin systems The neuropeptides vasopressin (VP) and oxytocin

(OT) are encoded by linked genes that are very homologous both at the structural and the sequence level (Ivell, 1987; Richter, 1989; Schmitz et al., 1991). The structure of the V P - O T locus is diagrammed in Fig. 1. The V P and O T genes are transcribed towards each other from opposite strands of the DNA duplex (Mohr et al., 1988; Schmitz et al., 1991). Both V P and O T are derived from preprohormones that are processed into the mature peptide forms. The V P gene gives rise to three mature peptides (Ivell, 1987; Richter, 1989). Exon I encodes the signal peptide, the nonapeptide V P and the N terminus of neurophysin, a molecule that binds V P and which may act as a carrier. Exon I1 codes for the bulk of the neurophysin, the Cterminus of which is encoded by exon 111, along with a 39 amino acid glycopeptide of unknown function. Exon I of the OT gene also codes for the signal peptide, the neuropeptide and the N terminus of the corresponding neurophysin. Exon I1 encodes the bulk of the neurophysin molecule, the C terminus of which is found in exon 111. No glycopeptide-like molecule is encoded by the OT transcription unit. Both the V P and OT genes are expressed in anatomically and functionally discrete groups of hypothalamic neurons. It is beyond the scope of this review to delve too deeply into V P and O T neurobiology. Rather, it is hoped that a brief description of the regulation and function of the ex-

-

I

I

I

1

VP 1 25 Kb

CAT

VP 13.4 OT 4.2

1 kb

Fig. 1. Diagrammatic representation of the bovine VP-OT gene locus showing the extent of the four transgenes described in this study. Exons I , 11 a n d I I I of the two genes a r e illustrated. T h e arrows indicate the direction of transcription.

84

pression of the VP and OT genes in two hypothalamic cell groups, the supraoptic nucleus (SON) and the suprachiasmatic nucleus (SCN), will be sufficiently illuminating, not only of the complexity of these systems,,but also of their experimental value.

The SON

The axons of oxytocinergic and vasopressinergic SON neurons project to the posterior lobe of the pituitary. PreproVP and preproOT made in SON cell bodies are cleaved and processed during their passage to the posterior pituitary. Here, the mature peptides are stored in nerve terminals until released into the general circulation in response to physiological demand. Although it has been reported that VP and OT are expressed in mutually exclusive SON neurons, both genes appear to be coordinately regulated in response to physiological stimuli. OT released into the general circulation stimulates the contraction of smooth muscle cells that bear the appropriate receptors. OT is thus involved in the reproductive behaviors associated with parturition and lactation. VP and OT mRNA levels increase in the SON during pregnancy and lactation (Lightman and Young, 1987). The well-established role of the VP made in the SON and released from the posterior pituitary is to adapt the animal to conditions that increase plasma osmolality. Circulating VP interacts with specific receptors in the kidney collecting ducts and promotes the conservation of water. As a consequence of a stimulus that results in an increase in plasma osmolality -classic paradigms in the rat are dehydration (total fluid deprivation) for up to 3 days or replacement of normal drinking water with 2% (w/v) NaCl for up to 14 days - VP stores in the posterior pituitary become depleted and there is a need to increase synthesis of preproVP in the hypothalamus. As a consequence of dehydration, the pattern of V P gene expression in the SON changes in three ways: firstly, there is an increase in the rate of transcription of the V P gene (Murphy and Carter, 1990; the OT gene has not been investigated in this respect); secondly, there is an increase in the steady-state levels of the VP and OT RNAs (Murphy and Carter, 1990, and references

therein); and thirdly, the poly(A) tail length of the both RNAs specifically increases (Carazzana et al., 1988; Zingg et al., 1988; Carter and Murphy, 1989; Murphy and Carter, 1990). The neuronal and intracellular signalling pathways, and the VP and OT gene cis-acting elements with which they ultimately interact, have not been elucidated.

The SCN

In the SCN, the V P gene (but not the OT gene) is expressed in parvocellular cells of the dorsomedial region. This part of the SCN is thought to contain the central circadian generator of the mammalian brain (van der Pol, 1980; Moore, 1983) and is thus involved in anticipatory- or predictive-type behaviors. Vasopressinergic axons project from the SCN to other parts of the central nervous system. Although there is no evidence that VP is involved either in the generation of circadian signals, V P may be involved in the transmission of such signals to other parts of the CNS. As such, the VP system can be regarded as the hands of the clock. In the SCN, the VP gene is regulated according to the time of day, probably by the circadian clock. Again, like the SON, regulation is at both transcriptional and posttranscriptional levels. Firstly, at night, the rate of transcription of the V P gene is reduced compared to the rate of transcription during the day (DC and DM, unpublished results). Secondly, the steadystate level of VP RNA is reduced at night compared to the day (Uhl and Reppert, 1986; Burbach et al., 1988; D.C. and D.M. unpublished results), and thirdly, the poly(A) tail length of the VP RNA is shorter at night than during theday(Robinson et al., 1988; Carter and Murphy, 1989). The VP and OT systems offer a number of advantages for the study of the peptidergic neuron at the molecular level. 1 . There is a vast body of literature on the VP and OT systems on which to base molecular studies. 2. There are a number of well-established models for the physiological manipulation of the VP and OT systems in the rat which can be readily adapted to molecular studies in the mouse. 3. The VP and OT genes are expressed in well-

85

established, discrete tissue-specific sites, either in specific groups of neurons in the brain, or in defined peripheral sites. This is unlike many other neuropeptide genes which display a much broader spectrum of tissue specificities. 4. The VP and OT genes have been cloned and sequenced from many vertebrate species and at least four mammalian species (rat, Schmitz et al., 1991 and references therein; human, Sausville et al., 1985; mouse, Hara et al., 1990; cattle, Ruppert et al., 1984) thus facilitating comparative studies. The VP and OT genes are small and can be readily manipulated using recombinant DNA techniques. Further, the VP and OT transcription units together form an interesting example of a particularly small differentially regulated neuropeptide gene locus. 5. VP and OT epitomise the dual roles that neuropeptides can play, either as hormones in endocrine pathways, or as neurotransmitters following synaptic transmission. 6 . VP and OT are involved in both adaptive and predictive (the circadian clock) hypothalamic functions. For these reasons, we and others have sought to apply molecular techniques t o the VP and OT systems. However, the VP and OT systems, interesting as they are, present one major problem in that there are no appropriate cell lines available that express these genes, thus precluding gene transfer experiments. From the viewpoint of the molecular geneticist this is a powerful disincentive to their study. To overcome this difficulty we and others have resorted to the use of transgenic mice. Transgenic studies on the VP and OT systems We want to use transgenic animals in order to learn, firstly, about how the hypothalamus processes information leading to an adaptive response, and secondly, about how that response is transmitted, either to the rest of the animal through the pituitary or to other regions of the CNS. However, at this stage we are only in a position to be able to ask two much more basic, specific questions about the VP and OT systems. 1. What are the cis-acting sequences needed to

mediate the appropriate restriction of V P and OT gene expression to specific hypothalamic cell groups? 2. What are the cis-acting sequences required to mediate the physiological and circadian regulation of the VP and OT genes, at both a transcriptional and a post-transcriptional level? Having defined such sequences it will then be possible to use this information to ask precise questions about the physiology and neurobiology of the VP and OT genes, with a view to touching on some of the broader issues mentioned above. Experiments in this laboratory have thus far used bovine transgenes in transgenic mice. Bovine VP and OT genomic clones were the kind gift of Seigfreid Ruppert (Heidelberg; Ruppert et al., 1984). The rationale behind the use of bovine transgenes was that there is sufficient sequence divergence between the bovine and mouse V P and OT genes (Richter, 1989) to allow species-specific oligonucleotide probes to be designed. When used on Northern blots these probes have enabled us to distinguish between transcripts derived from the endogenous mouse VP and OT genes and RNAs transcribed from the bovine VP and OT transgenes. Fig. 2 shows the use of these probes on bovine and wildtype mouse tissues and demonstrates that the patterns of expression of the VP and OT RNAs in cattle and mice show distinct differences. Whilst VP and OT RNAs are present in both the bovine and murine hypothalamus, peripheral expression shows a marked species specificity. VP and OT transcripts are found at high levels in the mouse neurointermediate lobe (NIL; Murphy and Carter, 1990), but only a low level of the VP RNA can be detected in the bovine NIL. Murine ovaries (Fig. 2) and testes (Ang et al., 1991) are devoid of VP and OT RNAs, but bovine ovaries (Ivell, 1987; Fig. 2) and testes (Ang et al., 1991) contain high levels of OT RNA. These species differences have complicated the analyses of transgenic mice bearing bovine VP and OT transgenes. Tissue-specific expression of the vasopressin gene As described above, initial experiments designed

86

to elicit the expression of the SV40 large T-antigen in the vasopressinergic cells of the hypothalamus employing a 1.25 kb promoter derived from the bovine VP gene only resulted in ectopic transgene expression (Murphy et al., 1987; Fig. 1). Expression of immunoreactive large T-antigen, and consequent tumorigenesis, was noted in the anterior lobe of the pituitary and the endocrine pancreas (Murphy et al.,

Fig. 3. Northern analysis of the expression of SV40 large Tantigen RNAs in the VT-C transgenic mouse line bearing a bovine VP-SV40 hybrid oncogene. A. Animal bearing pancreatic and pituitary turnours. B . Animal with no Figns of tuniour development. Key: P , pancreas; AL, anterior lobe of the pituitary gland; T, testis; H , hypothalamus; C, cortex; N, neurointermediate lobe of the pituitary gland; 0, ovary. 50pg of RNA was loaded into each lane apart from hypothalamus (10

!-a).

as.

m

MURINE

BOVINE

b

' - ..

MURINE

Fig. 2. Expression of the VP and OT genes in selected bovine and murine tissues. Total cellular RNAs were isolated as described (Ang et al., 1991) and fractionated through 1% (w/v) agarose gels containing formaldehyde. Following transfer to Amersham Hybond-N, the RNA was hybridised to 32P-labelled oligonucleotideprobesspecific for the rodent or bovine VP and OT genes (Ang et al., 1991). RNA quantities used: cortex, 50 pg; cerebellum, 50 pg; hypothalamus (HYPOTHAL), mouse 5 pg; bovine 10 pg; neurointermediate lobe of the pituitary gland (NIL), mouse 10 pg, bovine, 80 pg; anterior lobe of the pituitary gland, mouse 50 pg, bovine 100 pg; ovary, mouse 100 pg, bovine 100 pg (VP probe) or 10 pg (OT probe).

1987). Subsequent RNA analysis revealed that the transgene was also transcriptionally active in the mouse ovary (Fig. 3). However, no pathological perturbation has ever been observed in this tissue. No large T-antigen RNA or immunoreactivity, nor any disruption of the normal anatomy, has ever been observed in the brains of mice carrying the VPSV40 transgene. From these results, we concluded, firstly, that sequences required for the correct expression of the VP gene were missing from the VPSV40 transgene and, secondly, that when removed from its normal genomic context, the bovine VP promoter directs expression to novel and unexpected cell types. We note that many transgenes consisting of the coding sequences for the SV40 large Tantigen linked to heterologous promoters are found to be expressed in the endocrine pancreas. This suggests that sequences of the large T-antigen reporter may carry with them some regulatory or functional specificities that direct expression to the pancreas. The same 1.25 kb of the bovine vasopressin gene promoter was then linked to the chloramphenicol acetyl transferase reporter (CAT, Gorman et al., 1982; Fig. 1). In two independently derived lines, expression at the level of RNA was detected in virtually

87

Fig. 4. Northern analysis of the expression of CAT RNAs in a transgenic mouse line bearing a bovine VP-CAT fusion gene. Key: L, lung (50 pg); P, pancreas (50 p g ) ; S, spleen (50 pg); H , heart (50 pg); Lv, liver (50pg); Th, thymus (50pg); K , kidney (50 pg); T , testis (50 pg); Ad, adrenal gland (20 pg); 0, ovary (20 pg); St, striatum (15 pg); MO, medulla oblongata and pons (15 p g ) ; Mb, midbrain (15 p ) ; Cb, cerebellum (15 p g ) ; Cx, cortex (15 p g ) ; Hip, hippocampus (15 pg); Hy, hypothalamus (15 p g ) ; N , neurointermediate lobe of the pituitary gland (1.6 p g ) ; AL, anterior lobe of the pituitary gland (15 p g ) .

all tissues examined. A representative Northern blot is shown in Fig. 4. Oddly, no C A T enzyme activity was detected in any expressing tissue, although this same construct directs the synthesis of functional protein in transfected tissue culture cells (Karen Pardy (IMCB), D.C. and D.M., unpublished results). These disappointing early results led us t o introduce a more extensive bovine transgene into mice. A map of this transgene, which consists of a 13.4-kb segment of bovine D N A encompassing the entire coding region and approximately 9 kb of 5 ’ upstream sequences, is shown in Fig. 1 . This transgene utilised bovine V P coding sequences, as detected by species-specific oligonucleotide probes, as reporters of expression, thus allowing us t o assay for possible cis-acting sequences that might reside within the coding sequences, introns, o r regions 3 ’ of the VP gene. Encouragingly, we find that expression in these lines is restricted t o a very few tissue types. Particularly, we find specific expression of the bovine vasopressin gene in the hypothalamus of three out of the four lines. Of the lines examined thus far (three out of four) show a high level transgene expression in mouse ovary. Additionally ex-

pression has been detected in anterior pituitary (two out of four lines), kidney (two out of four lines) and hippocampus (one out of four lines). Examination of transgenic mice by in situ hybridisation has demonstrated that specific expression of the bovine transgene in the hypothalamus is found in a few magnocellular neurons of the supraoptic nucleus (Fig. 5). T h e number of cells expressing the transgene in the SON is small compared to the number expressing the endogenous mouse gene (Fig. 5 ) . These data allow us t o reach several conclusions. 1 . T h e 1.25 kb of the bovine vasopressin gene immediately upstream of the start o f transcription is a ubiquitous promoter when linked to C A T coding sequences. This promoter is profoundly influenced by regulatory motifs in adjacent sequences. Thus, when juxtaposed with elements within the SV40 large T-antigen coding sequences, expression is confined t o ovary, anterior pituitary and the endocrine pancreas. T h e 1.25-kb bovine V P promoter is unable t o mediate the expression of a reporter element exclusively to the vasopressinergic cells of the hypothalamus. 2 . A 13.4-kb bovine genomic fragment containing the entire V P coding sequences along with 9 kb of 5 ’ upstream sequences carries at least some of the information needed to direct expression t o magnocellular (and possibly vasopressinergic) neurons of the hypothalamus. Expression of this transgene shows a restricted pattern of expression indicating that it contains silencer elements not present in the immediate 1.25 kb upstream of the start of transcription. Tissue-specific expression of the oxytocin gene We and others have also sought to analyse the expression of oxytocin transgenes in transgenic mice. T w o independently derived lines have been described that bear transgenes made up of a 4.2-kb segment of bovine genomic DNA encompassing the entire O T coding sequences and 600 b p upstream of the transcriptional start site (Ang et al., 1991; Fig. 1). This transgene was not expressed in the mouse hypothalamus; a result suggesting that sequences enabling appropriate expression to be mimicked

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A

B

were missing from the transgene. However, both lines displayed expression of bovine OT in the transgenic mouse testis. The endogenous murine O T gene is not expressed in the testis, but wild-type cattle have abundant OT RNA in their seminiferous tubules, probably in Sertoli cells (Ang et al., 1991). Examination of the testes of the transgenic mice also revealed bovine OT RNA in seminiferous tubules, indicating that a bovine specificity is being brought to the mouse by the transgene. In a similar experiment, Young et al. (1990) have reported that a rat OT transgene consisting of the entire coding sequences and 360 bp upstream of the transcriptional start site also fails to display expression in the brain, and in the hypothalamus in particular.

VP-OT gene interactions The VP and OT genes are closely linked in the rat genome, being separated by only 11 kb, and the two genes are transcribed towards each other from opposite strands of the DNA duplex (Mohr et al., 1988; Schmitz et al., 1991). This structural organisation has been maintained in all species examined (mouse, Hara et al., 1990; human, Sausville et al., 1985; cattle, H.-L.A., unpublished results). The organisation of the VP and OT genes into this conserved structure, and the failure to achieve entirely appropriate expression with a number of transgenes of both bovine and rodent origin, has led to the suggestion that there may be a regulatory interaction between elements residing in the two transcription units. Enhancer- or silencer -type elements within the OT gene may act on the VP gene over the short

Fig. 5 . Detection by in situ hybridisation of rodent and bovine VP transcripts in the SON of a mouse line bearing a 13.4 kb bovine VP transgene. Methods have been described (Ang et al, 1991). The bovine oligonucleotide probe fails to detect transcripts in the wild-type mouse SON (panel A), although some background hybridisation is seen in the optic nerve. The bovine probe detects bovine V P transcripts in a few scattered magnocellular SON neurons from control transgenic mice (panel B) and from mice salt-loaded for 7 days (panel C). A rodent probe detects high levels of the endogenous mouse V P RNA in the SON of transgenic mice (panel D).

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region between the genes, and vice versa. This hypothesis has been strengthened by the recent report of Young et al. (1990) that a VP-OT “minilocus” expresses the OT component at high level in the oxytocinergic neurons of the supraoptic

A

B

A

B

sv40

28 S

st -

LT -

AVP

GH

18 S

and paraventricular nuclei. The “minilocus” consisted of a rat OT component (consisting of the entire coding sequences and 360 pb upstream of the transcriptional start site, which on its own does not display appropriate expression; see above) linked to a V P component (consisting of the entire coding sequences and 1.4 kb upstream of the start site of transcription). The VP component of this transgene was not expressed. Note that the transgene was not constructed in the “native” orientation. In the transgene, VP and OT are transcribed from the same strand of the chromosomal duplex in the same direction, suggesting that the enhancer-like elements within this transgene operate in a position- and orientation-independent fashion. We note that whilst these data are supportive of the hypothesis that there is a regulatory interaction between the V P and OT genes, they are also consistent with the notion that the locus contains regulatory redundancy.

Physiological regulation Having achieved tissue-specific expression of VP and OT transgenes, investigators are now asking if these same transgenes respond to the same physiological stimuli as the corresponding endogenous gene. Although expressed ectopically in the anterior pituitary gland, we asked if the transgene made up of 1.25 kb of the bovine VP promoter and SV40 large T-antigen (Murphy et al., 1987) was regulated in a transgenic line (VT-C; Murphy et al., 1987) in response to salt-loading. VT-C transgenic mice were subjected to salt-loading with 2% (w/v) saline for 7 days (Fig. 6, track B; Fig. 7 , track 2) and Fig. 6 . Salt loading reduces the level of transgene RNA in VT-C anterior pituitary gland. RNA was extracted from total pituitary glands of VT-C mice subjected to 5 days of salt loading (B) and from control VT-C mice (A). 20 pg of total cellular RNA was analysed by Northern blotting. Probes were complementary to SV40 large T-antigen (a short and a long exposure is illustrated) growth hormone and vasopressin RNAs. The VP RNA, which increases in abundance salt-loaded animals (Murphy et al., 1989) represents material in the neurointermediate lobe. The doublet of RNAs revealed by the SV40 probe correspond to the differentially spliced large T-antigen and small t-antigen mRNAs.

90 1 2 3 4

Thus, these transgenes contain sufficient regulatory sequences to mediate an increase in abundance of the VP and OT mRNAs in magnocellular neurons as a consequence of dehydration and lactation. The precise cis-acting elements remain to be mapped. Further, it is not yet known if the transgenes are responding at the transcriptional level. It is interesting to note that the bovine transgene V P RNA in transgenic mice does not respond to dehydration by increasing its poly(A) tail length; a dramatic and well-documented response of the rat RNA (Carrazanna et al., 1988; Zingg et al., 1988; Carter and Murphy, 1989; Murphy and Carter, 1990). However, the endogenous mouse VP RNA poly(A)

Fig. 7 . A high-salt diet reduces the level of transgene RNA in VTC anterior pituitary gland. VT-C mice were subjected to salt loading with a 2% (w/v) drinking diet for 5 (track 2) or 10 days (track 4), or with 0.9% (w/v) NaCl for 10 days (track 3) and compared to control animals (track 1). Total pituitary gland RNA was extracted and 2.5 p g was subject to Northern blotting. Probes used were complementary to SV40 large T-antigen or growth hormone. The doublet of RNAs revealed by the SV40 probe correspond to the differentially spliced large T-antigen and small tantigen mRNAs.

for 10 days (Fig. 7, track 4), or with 0.9% (w/v) saline for 10 days (Fig. 7, track 3). Surprisingly, in all cases, large T-antigen message levels were reduced when compared to controls (Fig. 6, track A; Fig. 7, track 1). It is possible that this effect is being mediated by components of the renin - angiotensin - aldosterone system. However, this curiosity has little physiological relevance given that the vasopressin gene is not expressed in anterior pituitary gland of mice (Murphy et al., 1989), rats (Murphy et al., 1989), nor cattle (Fig. 1). In the case of the 13.4-kb bovine V P transgene, we have shown that the abundance of bovine RNA in the hypothalamus increases in parallel with the endogenous mouse VP RNA in animals subjected to 7 days of salt-loading (Fig. 8). Similarly, Young and colleagues found upregulation of rat OT RNA steady-state levels when mice bearing the VP-OT “mini-locus’’ are subject to dehydration (Young et al., 1990b) or are lactating (Young et al., 1990a).

Fig. 8. Dehydration increases the level of bovine VP RNA in the hypothalamus of a mouse line bearing a 13.4 kb bovine V P transgene. Transgenic mice subjected to salt loading (dehydration; 2% w/v NaCl drinking diet for 7 days) were compared to control animals. Total cellular RNA was extracted from the anterior lobe (AP) and the neurointermediate lobe (NIL) of the pituitary gland and from the hypothalamus (HYP). RNA was then subjected to Northern analysis (anterior lobe, 35 pg; NIL, 3 p g ; hypothalamus, 25 pg). Bovine hypothalamic RNA (25 pg) was included as a positive control. Oligonucleotide probes used were complementary t o bovine VP (BVP), rodent VP (RVP) and rat alpha-tubulin (TUB). All probes have been described (Ang et al., 1991).

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tail similarly fails to respond to dehydration (Murphy and Carter, 1990) and the equivalent experiment will probably never be performed on cattle. It thus seems possible that the poly(A) tail effect is unique to the rat. Functional studies and the ectopic expression of VP and OT in transgenic mice The physiological consequences of disrupting normal VP and O T metabolism in transgenic mice has yet to be fully explored. Habener and colleagues have examined the physiological consequences of VP over-expression in lines bearing a transgene made up of the mouse metallothionein promoter directing the expression of the rat V P coding sequences (Habener et al., 1989). This transgene is expressed in many peripheral tissues, as well as the magnocellular neurons of the hypothalamus (see above). The transgene RNA was translated, and the preproVP was processed into mature VP peptide in the brain, the pancreas, and, to a small extent, the kidney. In other tissues the predominant product was an unprocessed precursor. Plasma VP levels were elevated in these animals, but, surprisingly, plasma osmolality was also increased. It might be anticipated that animals which hypersecrete VP might over-conserve water and become hypoosmolar. This is not the case, and in fact the transgenic mice developed a mild diabetes insipidus of nephrogenic origin, attributable to an apparent homologous desensitisation of renal V, receptors (Habener et al., 1989). Such metabolic compensation in response to excessive circulating levels of an ectopically produced neuropeptide was also found in mice expressing sequences encoding somatostatin under the control of the metallothionein promoter (Low et al., 1985). Despite plasma somatostatin levels several orders of magnitude in excess of normal, the transgenic mice were free of any discernible physiological disorder. Similarly, there have been no reports of any physiological, neurological or pathological consequences of the central or peripheral ectopic expression of VP or OT in any of the transgenic mice described to date.

Conclusions The mapping of the cis-acting elements that mediate the specific expression of the peptides vasopressin and oxytocin in particular groups of neurons is proving to be a difficult task. At this stage it appears that tissue specificity is at least partly dependent on the selective repression (silencing) of a promoter that is otherwise ubiquitously, but weakly, expressed. It is possible that regulatory signals, possibly enhancers, in the V P gene interact with the OT transcription unit, and possibly vice versa. Transgenes that are, at least in part, subject to correct tissue-specific cues, are physiologically regulated. It is likely that physiological regulation is brought about by separate cis-acting elements and transcription factors’ other than those responsible for the tissue-specific expression. Physiological studies on the VP and OT genes using transgenic mice will have to await a much better definition of appropriate regulatory sequences.

Perspectives In recent years, a number of new developments and refinements have augmented the repertoire of techniques available that enable the investigator to intervene, by DNA-mediated gene transfer, in the operation of cells within the whole organism. The application of these techniques to problems on neurosecretion will contribute greatly to our knowledge of these systems. 1. Cell ablation

The ablation of specific groups of neurons has, for some time, been an important tool in neurobiology. Recently, the specificity of tissue and developmentally controlled gene expression has been exploited to enable the ablation of cells expressing a particular gene in the cell-type of interest (Evans, 1989). With this technique, the expression of a toxic compound or metabolite at a particular time or in a specific cell type induces cellular “suicide”. Three cell-killing systems are available: the Diptheria toxin A chain (DT-A) gene (Palmiter

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et al., 1987), the ricin gene (Landel et al., 1988) and the Herpes simplex thymidine kinase (HSV-TK) gene (Heyman et al., 1989). DT-A and ric h are extremely toxic protein molecules - a single molecule of either is sufficient to kill the host cell. HSV-TK is completely harmless when expressed in cells of the normal animal. However, if a nucleoside analogue such as Ganciclovir is administered to the host, cells expressing HSV-TK will incorporate the analogue into their DNA, leading to an inhibition of DNA synthesis and cell death. The HSV-TK cell-killing system is thus inducible, being subject to the administration of the nucleoside analogue, whereas both DT-A and r i c h will kill as soon as the genes expressing them are active. However, although both DT-A and ricin will kill quiescent cells, HSV-TK will only effectively kill cells that are actively dividing. Thus, the HSV-TK system is not suitable for postmitotic neurons. Once the cis-acting sequences needed to direct the expression of a particular peptide gene have been elucidated, it will be possible to incorporate a toxin gene into these sequences and then ablate expressing cells in transgenic carrier mice. Using this technology, dwarfism has been induced in transgenic mice expressing the DT-A gene (Behringer et al., 1988) or the HSV-TK gene (Borreli et al., 1989) under the control of the growth hormone promoter - enhancer sequences in somatotrophs. However, as yet there has been no report of this technology being applied to the ablation of peptidergic neurons.

I

2. Signal transduction molecules and transcription factors The inappropriate expression of wild-type or mutated molecules that disrupt normal intracellular signalling pathways or transcriptional regulation pathways might be used to disrupt the normal control of a gene of interest. This approach has been used to investigate the growth hormone system. The enzymatically active intracellular subunit of the cholera toxin gene was expressed in the anterior pituitary gland under the control of growth hormone regulatory sequences (Burton et al., 1991).

The intracellular subunit of the Cholera toxin is non-cytotoxic but induces cellular cAMP to extremely high levels through an irreversible activation of G,, a signal transduction molecule that mediates normal pathways of cAMP induction. Mice expressing the growth hormone-cholera toxin transgene demonstrated somatotroph proliferation, pituitary hyperplasia, elevated serum growth hormone levels and gigantism. These events were probably triggered by the chronic elevation of cAMP in somatotrophs, and vividly illustrate the utility of such transgenes in physiological engineering experiments. In a reciprocal experiment, Struthers et al. (1991) used growth hormone regulatory sequences to direct the expression of a non-phosphorylatable mutant cAMP responsive element binding protein (CREB) to the anterior pituitary. Some of the transcriptional effects of cAMP are mediated by CREB through an interaction with specific genomic sequences. CREB action is regulated by its phosphorylation state. The non-phosphorylatable CREB mutant fails to activate cAMP responsive genes and when overexpressed in the anterior pituitary gland it induced hypoplasia and dwarfism. The specific expression of these and other molecules (such as the c-fos gene) in neuronal cells has not yet been reported, but the approach offers a novel route to the disruption of the normal physiology of neurosecretory systems. 3. Cell-fate mapping and neuronal tracing The specific expression of markers such as pgalacosidase or fire fly luciferase in specific peptidergic neurons of transgenic animals offers a novel way to monitor cell fates. In some cases, the expression of the marker in the brain is a consequence of the chance integration of the transgene in a segment of the chromosome active in neurons (Allen et al., 1990). If such reporter molecules could be designed to be transported down axons, it may be possible to trace the projection routes of neuronal pathways from specific cells in the brain to the rest of the central nervous system.

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4. Homologous recombination and gene targeting

It is now possible t o physically alter the endogenous genes of the mouse (Capecchi, 1989a,b). This is achieved by applying recently developed screening techniques that allow for the selection of rare homologous recombination events in embryonal stem (ES) cell cultures transformed with cloned DNA fragments. Totipotent ES cells are isolated directly from blastocysts (Robertson, 1987) and manipulated in-vitro by DNA-mediated gene transfer (Lovell-Badge, 1987). Rare homologous recombination events are selected for (Capecchi, 1989a,b), then positive transformants are reintroduced into mouse embryos by blastocyst colonisation (Bradley, 1987). Technologies that result in the inactivation or alteration of specific endogenous mouse neuropeptide genes will be extremely powerful in the study of the nervous system (Rossant, 1990). The first results from the application of this technology to genes suspected of being important in development are now starting to emerge (Wagner, 1990; Wright and Hogan, 1991), but as yet, no experiments on neuropeptides have been reported. Clearly, it would be of great interest to engineer a “Brattleboro mouse” (a mouse deficient in vasopressin) or a mouse unable to synthesise oxytocin. Another technology that allows for the specific depletion of defined gene products is the expression of “anti-sense” RNAs. Anti-sense RNAs are molecules complementary to the gene of interest. When used in in-vitro systems they have been shown to effectively reduce the level of a particular RNA and the protein it encodes (Mains et al., 1991). However, there are very few reports of the successful application of anti-sense technology to transgenic animals. The validity of the technology has been demonstrated by the expression in mice of RNAs complementary to myelin basic protein (MBP) in Schwann cells, which resulted in a depletion of MBP and a shivering phenotype (Katsuki et al., 1988). 5. Transgenic rats

The rat has been for many years the animal model of choice for studies in physiology and neurobiology. The rat has a size ccmpatible with a

whole range of physiological and neurobiological manipulations that would be unthinkable in the mouse. However, the rat has a reproductive capacity equal to, if not greater, than the mouse; a gestation and maturation period of the same length as the mouse, and a size that allows a large number to be housed in a relatively small space. Further, the brain of the rat is extremely well mapped and understood compared to that of the mouse. Mullins et al. (1990) reported the construction of the first trangenic rats. Transgenic rats promise to be a much more accessible and appropriate system for the study of problems in neurosecretion.

Summary Transgenic animal techniques offer today’s neuroscientist the ability to experimentally manipulate neurosecretory systems with a precision undreamt of by our predecessors. The range of techniques now available, building as it does on our growing knowledge of physiological systems at the inter- and intracellular level, allows us to critically define molecular lesions and ask about their consequences to the whole organism. Neuroscients should grasp the opportunities afforded by these recent developments.

Acknowledgements We thank Ms. Sandra Jones and her staff for their care of the IMCB transgenic animal colony. Dr. Duncan Smith (IMCB) is thanked for discussions and for his critical reading of the manuscript.

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