Expression and regulation of sex determining in the mouse.

Veronica Mercedes Narvaez Padilla

A thesis submitted for the Degree of Doctor of Philosophy 1996

Department of Developmental Genetics Department of Biology National Institute for Medical Research, University College London The Ridgeway, Mill Hill, Gower Street, London, NW7 lAA London, WCIE GET ProQuest Number: 10016705

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 In memory of Pepe and Arturo. ...Many times Fve been alone and many times Fve cried. Anyway you’ll never know the many ways Fve tried...

(Lennon & M cC artney)

Little darling, it’s being a long cold lonely winter. Little darling, it feels like years since it’s been here. Here comes the sun, here comes the sun and I say It’s alright!

(G. Harrison) Contents Contents ...... 4 List of figures and tables ...... 6 Acknowledgements ...... 8 A b str a c t ...... 11 Chapter 1: Introduction ...... 12 1.1 Sex ...... 12 1.2 Sex Determination vs Differentiation ...... 12 1.3 Environmental sex determination ...... 12 1.4 Genetic Sex Determination ...... 13 1.4.1 Drosophila ...... 14 1.4.2 C. elegans...... 18 1.4.3 Mammals ...... 21 1.4.4 More variations in genetic sex determination ...... 21 1.5 Organogenesis of the genital system ...... 21 1.5.1 The urogenital system ...... 21 1.5.2 The indifferent gonad ...... 25 1.5.3 Origin, migration and fate of the primordial germ cells ...... 26 1.5.4 Testis Differentiation ...... 26 1.5.5 Ovary differentiation ...... 27 1.6 Testis Determining Factor ...... 28 1.7 Genes involved in sex determination or differentiation ...... 29 1.7.1 S ry...... 29 1.7.2 Sox-9 ...... 36 1.7.3 Dax-1...... 37 1.7.4 AMH ...... 40 1.7.5 S F -i ...... 41 1.7.6WT-J...... 41 1.8 Aims of this thesis ...... 41 Chapter 2: Materials and Methods ...... 43 2.1 Commonly used solutions and enzymes ...... 43 2.2 Bacterial strains, plasmids ...... 43 2.3 Purification of DNA fragments from agarose gels ...... 44 2.4 Spectrophotometric quantitation of nucleic acids ...... 44 2.5 Recombinant DNA techniques...... 44 2.5.1 Preparation of competent bacteria ...... 44 2.5.2 Transformation ...... 44 2.5.3 Blunt End Ligations ...... 44 2.5.4 Cohesive End Ligations ...... 45 2.5.5 Small scale plasmid isolation (minipreps): ...... 45 2.5.6 Large Scale Plasmid Purification (maxipreps) ...... 45 2.5.7 Sequencing ...... 45 2.6 PCR techniques...... 46 2.6.1 Isolation of genomic DNA for PCR ...... 46 2.6.2 Oligonucleotides used: ...... 47 2.6.3 Primer combinations and annealing temperatures ...... 48 2.6.4 Oligonucleotide Purification ...... 49 2.6.5 PCR procedure ...... 49 2.6.6 RNA Purification for RT-PCR ...... 49 2.6.7 Reverse Transcription of RNA ...... 50 2.7 Probe Labelling and Southern hybridization ...... 50 2.7.1 DNA labelling ...... 50 2.7.2 Oligonucleotide labelling ...... 50 2.7.3 DNA Transfer and Hybridization ...... 50 2.8 detection ...... 51 2.8.1 Sample preparation ...... 51 2.8.2 Electroblotting ...... 51 2.8.3 Detection of the protein with antibodies ...... 51 2.9 Production of transgenic mice ...... 51 2.9.1 Mouse Stocks ...... 51 2.9.2 Microinjections of fertilized mouse eggs ...... 52 2.9.3 X-Gal Staining ...... 52 2.9.4 Paraffin sections ...... 53 2.10 RNAse Protection ...... 53 2.10.1 Probe Synthesis...... 53 2.10.2 Hybridization ...... 53 2.10.3 Digestion ...... 54 2.10.4 Probes ...... 54 Chapter 3: Siy regulation ...... 55 3.1 Introduction ...... 55 3.2: DNAse Hypersensitive Site Mapping ...... 55 3.2.1 Introduction ...... 55 3.2.2 Results...... 57 3.2.3 Discussion ...... 57 3.3 Transgenic Studies ...... 61 3.3.1 Introduction ...... 61 3.3.2 Results...... 62 3.3.3 Discussion ...... 81 Chapter 4: Sry mis-expression ...... 85 4.1 Introduction ...... 85 4.2 Results ...... 85 4.2.1pFLAG...... 85 4.2.2 pVACS...... 91 4.3 Discussion ...... 94 Chapter 5: D ax-1 regulation ...... 96 5.1 Introduction ...... 96 5.2 Results ...... 97 5.2.1 D-12/Z...... 99 5.2.2 D -3/Z ...... 105 5.2.3 D-4.5/Z...... 105 5.2.4 D -6/Z ...... 108 5.2.5 F(-18^-4) + D-12/Z ...... 108 5.2.6 D-12/Z + F(+0.7-^+13) ...... I ll 5.3 Discussion ...... I ll Chapter 6: Dax-1 as a transgene ...... 118 6.1 Introduction ...... 118 6.1 Results...... 118 6.1.1 Mouse Dax-1...... 118 6.1.2 Human DAX-1...... 121 6.2 Discussion ...... 121 Chapter 7 : Concluding Remarks ...... 125 Chapter 8: References ...... 129 List of figures and tables.

Figure 1.1 Sex determination in Drosophila ...... 17 Figure 1.2 Sex determination in C. elegans...... 20 Figure 1.3 Action of sex determining genes in C. elegans...... 20 Figure 1.4 Development of the mammalian urogenital system ...... 23 Figure 1.5 Sexual differentiation in mammals ...... 24 Figure 1.6 Mouse and human Y ...... 30 Figure 1.7 Inverted repeat structure of the genomic locus of S r y ...... 31 Figure 1.8 Model for the formation of the adult circular transcript of S r y ...... 33 Figure 1.9 Schematic representation of Sry embryonic transcript ...... 34 Figure 1.10 Schematic representation of the Sry open reading frame ...... 35 Figure 1.11 Schematic representation of the conservation between human and mouse DAX-1 /Dax-1...... 39 Figure 3.1 Mapping of DNAse hypersensitive sites in the Thy-1 ...... 58 Figure 3.2 Mapping of DNAse hypersensitive sites in the Sry gene ...... 59 Figure 3.3 Schematic representation of constructs pZlO, pZ ll and pLUG l ...... 63 Figure 3.4 Ectopic expression of lacZ in line pZlO-C ...... 64 Figure 3.5 Expression of transgene pZll in embryonic tissues ...... 66 Figure 3.6 Expression of transgene pZll in adult tissues ...... 67 Figure 3. 7 Analysis of the transcript expressed by pZll transgene ...... 68 Figure 3.8 Expression of pLUGl transgene in VPL54 line ...... 70 Figure 3.9 Expression of pLUGl transgene in VPL44 line ...... 71 Figure 3.10 Analysis of expression of pLUGl transgene in 11.5 dpc genital ridges ...... 72 Figure 3.11 Ectopic expression of lacZ in line VPL43 ...... 73 Figure 3.12 Schematic representation of pHOXY-8 ...... 75 Figure 3.13 Region of homology between Mus musculus testis-specific somatic cytochrome C gene and S ry ...... 78 Figure 3.14 Schematic representation of p741.mutB ...... 79 Figure 3. 15 RT-PCR on adult testes of transgenic line 741.mutB-1467 ...... 80 Figure 4.1 Schematic representation of FLAG and VACS constructs ...... 86 Figure 4.2 Analysis of expression of the Flag transgene in whole embryos ...... 88 Figure 4.3 Analysis of expression of Flag transgene ...... 89 Figure 4.4 Analysis of protein expression in Flag4 and Flag5 lines ...... 90 Figure 4.5 Transgenic male VACS-27, carrying construct pVACS ...... 92 Figure 4,6 Expression of VACS and Flag transgene ...... 93 Figure 5.1 Schematic representation of Dax-1 constructs used in transgenic m ice ...... 98 Figure 5.2 Expression of lacZ in D-12/Z-tl and D-12/Z-t2 embryos at 11.5 dpc ...... 100 Figure 5.3 Expression of lacZ in line D-12/Z-1317 ...... 101 Figure 5.4 Expression of lacZ in line D-12/Z-1322 ...... 102 Figure 5.5 Expression of lacZ in line D-12/Z-1581 ...... 103 Figure 5.6 Sections through the genital ridges of transgenic embryos from line D-12/Z-1322...... 104 Figure 5.7 Different levels of expression of lacZ in line D-12/Z-1322 ...... 106 Figure 5.8 Expression of lacZ in D-12/Z-tl to -t5 11.5 dpc embryos ...... 107 Figure 5.9 Expression of lacZ in 11.5 dpc genital ridges of D-6/Z-1634 ...... 109 Figure 5.10 Expression of lacZ in F(-18->-4)\D-12/Z-tl 14.5 dpc transgenic embryo ...... 110 Figure 5.11 Expression of lacZ in line D-12/Z\F(+0.7->+13)-1378 ...... 112 Figure 5.12 Expression of lacZ in line D-12/Z\F(+0.7->+13)-1372 ...... 113 Table 1 Summary of lacZ expression obtained with the different Dax-1 constructs used ...... 114 Figure 5.13 Ectopic expression of lacZ in different transgenic lines ...... 117 Figure 6.1 Expression of D-12/cDNA transgene in 14.5 dpc embryos ...... 119 Figure 6.2 Expression of D-12/cDNA transgene in genital ridges of 11.5 dpc em bryos...... 120 Figure 6.3 RT-PCR of 11.5 dpc embryos carrying human DAX-1 as a transgene ...... 122 Figure 7.1 Expression pattern of Dax-1, Sry and Sox-9...... 128 Acknowledgements

Everybody knows that a thesis cannot be written in isolation. In order to accomplish this difficult task, help and support from all over is needed; so I want to thank a lot of people which without them this thesis just hadn’t existed. First, all the people that in some way were directly related with the work:

Robin, first of all, for letting me work in your lab and in this way giving me the opportunity to live in a wonderful city of a beautiful country. Thanks for feeding me (and everybody else) all Thursdays, and for the delicious dinners you cooked for us. Thank you for your kindness and helpfulness each time problems arose. But mainly, thanks for all the trouble you had gone through with the correction of this thesis. I think you have done a marvellous job! I'm sure that if it makes any sense at all, it is all because of you. ...ps: I'll still wait for a real ride in your car!..and a squash game....

Amanda, because thanks to you I finally got blue gonads! (that is, thanks to you, this thesis doubled its size). Thank you for rescuing my trust and fondness for science, for being an excellent psychologist, reading my mind and cheering me up before I decided to commit suicide, and for being patient with me in my “lack of concentration” moments. It’s being a pleasure working with you.

Nigel, for taking care of me since the first day I stepped into this side of the Atlantic, for introducing me into squash and for the pleasant after-game drinks. You've been an excellent friend. Also, thanks for all the help in the lab and with the computers, and for trying to get me the printer on time....

Sara, thanks for enabling me to understand Portuguese. Thanks for being such a caring and understanding friend. Sorry for my finishing of your tequila. I'll send you a replacement!

Mike, Cheers! For teaching me to appreciate Oasis and Pulp, to pronounce correctly York, for greeting me in such nice ways as “senorita muy bonita”, to help me understand the meaning of "not too bad", and for all your attempts to learn Spanish.

Ariel, thanks for bringing a drop of fun to the lab. I have really enjoyed your madness!... and your cakes! Thanks for my coloured pencils. And thanks for the patience you had in correcting this thesis, and for the encouragement you gave me to finish it. Thanks for the "spotty" mouse. Isabelle, thanks for converting me into a Guinness addict. Thank you for the many puh lunches you paid for me . Thanks for taking care of me when I overdosed myself with those pints. Thanks for reminding me what it meant to be a beginner in doing transgenics, and merci for the banana beers.

Larysa, for being a very friendly E.T. and for being so quiet, a lesson maybe I should learn.

Lidia, for giving me an example of somebody organized and with common sense. It was chaos after you left. For being so patient in explaining all the protocols and having everything ready. How could you change us for a bunch of Germans?

Adam, for teaching me to do the RNAse protections, and for your good pronunciation of E nglish.

Katarina, thanks for the Swedish lessons and for sharing with me the colour of your rabbit.

Daniel, for sharing with me the lab in the night with nice music.

Blanche, thanks for the biscuits and carrot cakes.

Shan, thanks for the help in the writing of this thesis.

Sophie, thanks for my French lessons.

Silvia, thanks for my Italian lessons.

Charlotte, for being my hot companion in the RNAse protections.

Dalia, for sharing the beginning with me.

Tristan, thanks for organizing the basketball, ...and for all the dead mice.

Paul, Shanthi, Aine, Teresa and Vladimir for teaching me about spermatogenesis, deformed heads and arrests.

Elizabeth, Zoe, Mandy and Rosemary for taking so good care of my babies and for the thousands (literally) of tails.

Thanks to all the people in the Graphics department for all the figures of this thesis.

To all the people in the library, for all the references they had to get for me and for keeping such a nice and organized library. And now to all the people that although not directly involved with this work, still helped a lot in other ways:

To the Fir Island “neighbourhood” (Davindo, Davinda, Nazy, Mohamed and Reza) for the excellent Indian and Persian food, and for all the fun. Nazy, thanks for all your prayers for me. Reza, thanks for taking care of my fish when I had to fly away.

To Martha, for the rock&roll lessons, and Samuel for the basketball ones.

Thanks to Gaby and Martin for giving me asylum in Cambridge each time London became too pathetic, for being always at hand to help and for having shared the traumatic experience of a first "real" winter.

To all my e-mail friends from Mexico that although far away, kept me in touch with reality, specially Nina, Susana, Diana, Ale and Mario which were the more faithful.

Many thanks to my mother for the weekly letter I received during all the PhD, and my sister for the not so weekly, but constant letters.

To my cousin Adriana for sharing and understanding the tension of the writing process.

Thanks to the British Council for paying for my life the first year in this country and the University of Mexico for paying for my life the last four years.

And finally, Alejandro, special thanks to you because, you more than anybody else, knows what all this has meant, and has suffered the consequences. Thank you very much for all your love, care, tenderness and support, and for the patience in waiting for me. Pm going!

10 Abstract

The aims of my thesis were to characterize the regulatory elements of the mouse gene Sry, the testes determining factor, and of the mouse gene Dax-1, a candidate for an ovary determining gene; and to analyze their function during development. The first strategy used was to map DNAse I hypersensitive sites (DHSs) in the regions flanking the open reading frame (ORF) of Sry, as it has been found that DHSs often correspond to DNA sequences that function as regulatory regions. No DHSs were found in the first four kb surrounding the ORF.

Another strategy for finding regulatory regions was to use transgenic mice harbouring lac-Z reporter constructs with different amounts of flanking genomic sequence. None of the transgenic reporter constructs with Sry sequences were found to be expressed at a high enough level for the detection of B-galactosidase expression by staining with X-gal. The endogenous low levels of expression and the complex genomic structure of Sry might account for this result.

Since Sry expression is tightly regulated during development, we attempted to misexpress it to see if it produced an abnormal phenotype. Three lines carrying Sry under the control of a constitutive promoter were produced. No obvious phenotype has been observed to date.

Dax-1 has at least two important sites of expression: the developing gonads and the adrenal glands. Reporter constructs with Dax-1 genomic sequences allowed us to define upstream regions responsible for expression in these two tissues. Within 18 kb 5' of the ORF we have found a gonad-specific and an adrenal-specific expression elem ent.

In order to study the function of Dax-1 in the sex determining pathway, transgenic animals with extra copies of this gene have been produced. Male to female sex-reversal was expected. Up to now, no sex-reversal has been found in these animals, however the level of expression may be too low.

11 Chapter 1; Introduction

1.1 Sex

In the context of evolution, the sole goal of every organism in this planet is to reproduce. This can be achieved sexually or asexually. Most animal species use sexual reproduction; but sexual reproduction has an evolutionary problem. If a sexual population is made to compete against an asexual one in the same conditions, the asexual population will soon have an advantage and displace the sexual one (Maynard Smith, 1978;Williams, 1975). So why sex hasn’t disappeared? Sex mixes together the genes of two organisms. What advantages does this mixing give to the organisms? Many different hypotheses have been postulated. Some claim that sex exists to repair damaged genes or to get rid of accumulating mutations (Bernstein, et al., 1985;Kirkpatrick and Jenkins, 1989;Kondrashov, 1988). Others claim that sex is needed to combat parasites (Hamilton, et al., 1990;Hamilton and Zuk, 1982;Lively, et al., 1990). A parasite has to find a way of getting into its host, so parasites are always evolving mechanisms to "fool" the defence barriers of the host. In long-lived species, parasites have time to overcome the defence barriers. By the time the host has to reproduce, is better if the offspring are given different defences by mixing their own defences with someone else's. There is a better chance that parasites already adapted to their host individual, won't be adapted to the new combination of genes. In any case, sex is the most widely used way of reproduction, and having two sexes is the most typical form. Therefore, understanding how an organism becomes one sex or the other is a question that has fascinated humanity since a long time ago.

1.2 Sex Determination vs Differentiation

Sex determination is the process which selects if an organism will be male or female. Sexual differentiation is the process by which an organism develops as a male or female individual.

Considering the immense variety of ways in which sex is determined in nature, only some examples of sex determination in vertebrates and invertebrates will be reviewed here. In some species, sex is determined by the genetic components of the fertilized egg. In others, it is a matter of environmental conditions.

1.3 Environmental sex determination.

Environment can determine sex in many different ways. In most turtles and crocodilians, sex is determined by the temperature at which the egg is incubated. In

12 turtle species, females are generally produced when the eggs are incubated at high temperatures (30®C and above), and males when eggs are incubated at low temperatures (22-27®C); whereas the converse is true for most crocodilian species (Ferguson and Joanen, 1982;Head, et ah, 1987;Janzen, 1992). In many of these cases, administration of estradiol will counteract the masculinizing effects of the male-producing temperature. So it has been proposed that temperature and estradiol act in a common pathway in temperature-dependent sex determination (Johnston, et ah, 1995;Wibbles, et ah, 1994). In other organisms, sex can be determined by the location of the developing embryo. One example is the case of the worm Bonellia viridis. The females of this worm live in small burrows and have a long proboscis which protrudes for food collection. Male worms are considerably smaller than the females. If a larva comes into contact with the proboscis of an adult female, it will move on to the uterus of the female and differentiate into a male, living there as a parasite, fertilizing the eggs of the female. Larvae that fail to reach a female develop into females themselves (Bacci, 1965). Experiments in which larvae are cultured with and without an exogenous proboscis indicate that this effect may be mediated by a substance secreted by the proboscis. However the finding that some males are produced even in cultures without a proboscis and some females in cultures with a proboscis, suggests that some intrinsic factors may exist such as a genetic predisposition to one sex or the other (Leutert, 1975). Another example of position-dependent sex determination can be found in Crepidula fornicata, a snail. Here, individuals pile on top of each other to form a mound. Young individuals are always male, but soon after, their male reproductive system degenerates. Their future sex is determined depending on the position in the mound. If it is attached to a female, it will become male. If it is removed from its attachment, it will become a female. The presence of large number of males will cause some of them to become females. However, once an individual is a female, it can never become a male (Gilbert, 1994).

It is known that daylength, crowding and nutritional status can also affect sex determination in a variety of species, as in aphids (Richards and Davis, 1977) or some species of fish (Bacci, 1965;Mittwoch, 1973). Overall, not much is known of the molecular mechanisms by which environment decides the sex of any organism.

1.4 Genetic Sex Determination. When sex is determined by genetic components, the sex of the organism is set from the moment of conception. In this way, each individual will develop into a predestined sexual fate. Amongst the organisms that have their sex determined genetically, there are some very well studied examples, specifically. Drosophila melanogaster and Caenorhabditis elegans. In both of these organisms somatic sex determination.

13 germline sex determination, and dosage compensation (a mechanism that equalizes X expression between the two sexes despite their disparity in X chromosome dosage) are controlled by means of a chromosomal signal known as the X:A ratio (X chromosomes to autosomes ratio). A variety of mechanisms are used for establishing and implementing the chromosomal signal, and these do not appear to be similar in the two species.

1.4.1 Drosophila. An X:A ratio of 1:1 gives a female, while a ratio 1:2 gives a male (Bridges, 1921). The first step in the sex determining cascade is to assess this ratio. There are genes linked to the X chromosome that act as numerators and genes linked to autosomes that act as denominators (Fig. 1.1). Two of the numerator genes are sisterless a (sis-a) and sisterless b (sïs-6)(Cline, 1988). When two copies of these genes are present, the feminizing switch gene Sex-lethal (S%Z)(Cline, 1986) is turned on. In XX embryos Sxl is activated during the first two hours after fertilization producing a specific female transcript (Bopp, et al., 1991). Early Sxl function is necessary for both the female development pathway and for dosage compensation. When Sxl is non functional in an XX organism, the male phenotype emerges and the X chromosomes are transcribed more rapidly (to the same degree as the single X in XY embryos) (Cline, 1983). This gives an overproduction of X gene products that is lethal to the embryo. In order to be activated, in addition to the sis genes, Sxl needs the products of the daughterless (da) (Cline, 1989), hermaphrodite (her) (Pultz and Baker, 1995) and runt (Duffy and Gergen, 1991) genes. Maternal expression of da is essential for the activation of Sxl at early stages (Salz, et al., 1989). The her gene has both a maternal and a zygotic function required for normal female development. Maternal her function is needed early in the hierarchy as a positive regulator of Sxl. Zygotic her function is needed later for female sexual differentiation (Pultz and Baker, 1995). Runt acts as a position-specific element necessary for the uniform expression of Sxl. Loss of this gene results in a failure to activate Sxl in the central region of female embryos (Duffy and Gergen, 1991). This gene is located in the X chromosome, so it might also be one of the numerator elements of the X:A signal.

One of the major denominator elements appears to be the deadpan (dpn) gene (Younger- Shepherd, et al., 1992). Males with too high ratios of sis-b to deadpan activate Sxl and die, while females with too low ratios fail to activate Sxl and also die. The gene products of da, sis-a, sis-b, and deadpan are all transcription factors of the basic helix-loop-helix (bHLH) family. bHLH form homo- or heterodimers via the HLH domain (Murre, et al., 1989). By dimerizing, they bring together their basic domain, enabling them to bind to DNA in a sequence-specific manner (Ferre-d'Amare, et al., 1993). Therefore, it is possible that da, sis-a, sis-b and deadpan form heterodimers with one

14 another. It has been shown that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently adjust the level of active transcription from this promoter (Cabrera and Alonso, 1991). The dpn product represses this da/sis-b dependent activation by specifically binding to a unique site within the promoter (Hoshijima, et al., 1995). Another gene that has also been implicated in the regulation of the activation of Sxl is extramacrochaetae (emc). This gene is also an HLH protein, but lacks a basic domain so it is unable to bind to DNA (Ellis, et al., 1990).lt represses activation of Sxl by inhibiting the binding of the da/sis-b heterodimers to DNA (Van Doren, et al., 1991). In this complex of activation/repression of Sxl, at least two other genes are known to participate: Groucho (gro), whose protein binds specifically to dpn and seems to act as a corepressor (Paroush, et al., 1994); and snf {sans-fille, although it is not true that mutants lacking this gene just produce no daughters; they are completely sterile), which is a positive regulator of Sxl in both the germline and the soma (Salz, 1992). When Sxl is activated from the early promoter by all these genes, the transcript produced is automatically spliced into a female specific mRNA that codes for a functional protein. This protein functions as a splicing factor that removes the male exon from the transcript that is produced later, from the late constitutive promoter (Sakamoto, et al., 1992). The male exon includes a stop codon in the RNA transcript, so no protein is produced when this exon is not removed, as is the case in normal XY embryos where no early Sxl transcript is produced.

The next gene in the pathway is transformer {tra) (McKeown, et al., 1988). Sxl regulates somatic sex determination by controlling the processing of tra mRNA. tra mRNA is alternatively spliced in males and females. There is a female-specific mRNA and a non-specific mRNA found in males and females. This non-specific transcript contains a termination codon early in the message so the protein is non functional. Sxl protein is thought to activate a female specific 3’ splice site in the tra pre- mRNA causing it to be processed in a way that removes the exon which codes for the stop codon (Nagoshi, et al., 1988). To achieve this, Sxl protein blocks the binding of the splicing factor U2AF to the non-specific splice site by specifically binding to the polypyrimidine tract adjacent to it. This causes U2AF to bind to the lower affinity 3’ splice site generating the female specific transcript (Varcarcel, et al., 1993). The protein produced by this message acts together with the product of the transformer 2 {tra- 2) gene to control the sex-specific alternative splicing of transcripts from the douhlesex {dsx) gene. The tra-2 gene uses alternative promoters and splicing patterns to generate four different mRNAs that together encode three putative RNA-binding polypeptides. Only the products expressed in somatic tissues function to regulate the RNA splicing of doublesex. The products expressed in the male germ line play an unknown, but essential, role in spermatogenesis (Mattox and Baker, 1991). It is proposed that tra-2

15 binds to the transcript of the doublesex {dsx) gene only in the presence of the female specific tra protein (Mattox and Baker, 1991).

Doublesex is active in both males and females, but its primary transcript is processed in a sex-specific manner by tra and tra-2. The transcript produced when tra is present encodes an active protein that inhibits male development. On the other hand, when tra is not produced, dsx is spliced in the default male specific manner. This transcript encodes an active protein that inhibits female traits and promotes male ones (Ryner and Bruce, 1991). When dsx is absent, both the male and female primordia develop and intersexual genitalia are produced. Thus, the main role of dsx is to actively inhibit the development of the inappropriate genitalia. Once this cascade of genes has been established, the complete male or female phenotype depends on the further interactions of dsx with its target downstream sex differentiation genes, many of which are known (Bownes, 1994;Chase and Baker, 1995;McKeowm and Madigan, 1992).

16 XX XY

sis-a dpn sis-b emc runt gro

da snf her

Activate Sxl from early No Sxl activation from promoterI early promoter. Sxl mRNA from early promoter No Sxl protein splices to give protein I Splices late promoter RNA in Late promoter RNA splices in a way a protein is produced a way no protein is produced I tra is spliced in the default Splices tra mRNA so it gives manner, coding for a non an active protein functional protein

Splices dsx mRNA to give the dsx is spliced to give the female specific transcript. male specific protein. 1

Female specific protein Male specific protein directs female somatic diracts male somatic differentiation pathway. differentiation pathaway

Figure 1.1: Sex determination in Drosophila Schematic representation of the interaction of genes that lead to Drosophila sex determination.

17 1.4.2 C. elegans

As in Drosophila, sex in Caenorhabditis elegans is determined by a regulatory cascade of several interacting autosomal and X-linked genes in response to the X/A ratio (Madl and Herman, 1979). XX animals (high X/A) develop as self-fertile hermaphrodites, and XO animals (low X/A) develop as males. Three regions of the X chromosome containing sex determining signals have been defined (Akerib and Meyer, 1994). In one of these regions, a gene, encoding an RNA binding protein was found. Analysis of transgenic animals carrying extra copies of this gene led to the proposal of this gene as a candidate for a primary sex-determining signal. This gene has been called fox-1 (for feminizing locus on X) (Hodgkin, et al., 1994). xol-1 (for XO-lethal) is the earliest-acting gene in the known hierarchy that controls sex determination and dosage compensation (Miller, et al., 1988). The primary sex- determining signal (the X/A ratio) directs the choice of sexual fate by regulating xol-1 transcript levels: high xol-1 expression during gastrulation triggers male development, whereas low expression at that time permits hermaphrodite development (Fig. 1.2). Inappropriately high xol-1 expression causes hermaphrodites to activate the male program of development and die from a disruption in dosage compensation (Rhind, et al., 1995). Xol-1 represses the function of the sdc genes. The sdc-1, sdc-2 and sdc-3 genes may be analogous to the Sxl gene in Drosophila in that they affect both sex determination and X chromosome dosage compensation. The sdc genes are repressors of the function of the her-1 gene (DeLong, et al., 1993;Nusbaum and Meyer, 1989;Villeneuve and Meyer, 1987). her-1 activity is required for male development. It appears to be a secreted product functioning non-cell autonomously (Perry, et al., 1993). When sdc genes are inactive because of the high expression oï xol-1, the activity of her-1 is high. With high activity of her-1, the activity of tra-2 and tra-3 genes is repressed. tra-2 and tra-3 seem to be repressors of the activity of the fern genes, so in their absence, (being repressed by her-1 activity) the activity of the fern genes is high. The activity of the fern genes deactivate tra-1. The tra-1 gene is the terminal regulator in the sex determination pathway, directing all aspects of somatic sexual differentiation (Hunter and Wood, 1990). In this case, where tra-1 is inactive, the individual develops as a male. Conversely, when xol-1 is expressed at low levels (XX individuals), the activity of the sdc genes is high and the whole cascade is changed, her-1 activity is now low; tra- 2 and tra-3 activities are high; the fern genes are inactive; and tra-1 is active so the individual develops as an hermaphrodite.

A model proposed by Kuwabara, et. al. (1992) integrating the genetic pathway with the cellular mechanisms states that tra-2, being an integral membrane protein, binds the fem proteins when her-1 is not bound to it. tra-1 would be free to enter the nucleus and

18 bind to DNA to initiate transcription. When her-1 is present, it binds to tra-2, preventing it from binding to the fem proteins. The fem proteins would then bind to tra- 1 preventing its translocation to the nucleus, and therefore preventing its activation of downstream genes (Fig. 1.3).

19 sdc-1 fem-l xol-1 sdc-2 her-1 tra-2 fem-2 tra-1 sdc-3 tra-3 fem-3

XO HIGH HIGH HIGH MALE

XX HIGH HIGH HIGH HERMAPHRODITE

Figure 1.2; Sex determ ination in C. elegans

Schematic representation of somatic sex determination in C. elegans. The high/low designation reflects functional gene activity. (Taken from Gilbert, 1994)

tra-2 tra-2 her-1

fem fem

tra-1 tra-1

XX HERMAPHRODITES XO MALES

Figure 1.3: Action of sex determining genes in C. elegans

Scheme of model proposed by Kuwabara et al for the action of sex-determining genes in C. elegans. In XX individuals, the fem proteins are sequestered by the tra-2 protein, tra- 1 can then enter the nucleus and activate transcription. In XO individuals, her-1 binds to tra-2, thus preventing the binding of the fem proteins to tra-2. The fem proteins then bind to tra-1, preventing it to enter the nucleus (taken from Gilbert, 1994).

20 1.4.3 Mammals

In mammals, even though they also have an X and a Y chromosome, the mechanism used to determine sex is quite different from the examples mentioned above. In the late 1950s, three groups (Ford, et al., 1959;Jacobs and Strong, 1959;Welshons and Russell, 1959) showed that male development is determined by the presence of the Y chromosome, rather than the number of X chromosomes. Thus, XO individuals develop as females while XXY develop as males. In the late 1940s, Jost and colleagues (dost, 1947) had shown that the testis is needed to develop secondary sexual characteristics in the male. By removing the developing gonads of rabbit foetuses in utero he obtained female development of the reproductive tract and external genitalia whether the castrated organ was a testis or an ovary. Therefore, female development seemed to be the default pathway. Combining these two findings, it became apparent that a dominant-acting factor on the Y chromosome was directing the development of the gonads into testes, and hence, male development. This factor has been called “testis determining factor” {TDF in humans, Tdy in mouse). It was proposed that TDF would switch the development of the indifferent gonad from the default ovarian pathway into the male one, and the subsequent production of hormones by the developing testis would lead to the further development of secondary male sexual characteristics.

1.4.4 More variations in genetic sex determination

As in the case of environmental sex determination, where many different mechanisms have evolved, genetic sex differentiation has also been very creative. In some birds, reptiles and fish, the presence of a chromosome also determines the sex but in this case the heterogametic sex is the female. Females have a Z and a W chromosome, while males have two Z chromosomes. It is believed that in some cases there could be a feminizing signal in the W chromosome (Lacroix, et al., 1990). In other cases it could be the number of Z chromosomes which determined sex. Another interesting example is the wood lemming, which posses three sex chromosomes, X, X'" and Y. XY are males, while X*Y, X*X* and XX are females. It is believed that the X='= chromosome is a mutant X chromosome which overrules the masculinizing effect of the Y chromosome (Lau, et al., 1992).

1.5 Organogenesis of the genital system

1.5.1 The urogenital system

A unique feature of the developing gonad is that it is a bipotential organ, capable of differentiating either into an ovary in females or a testis in males. The gonads develop

21 together with the kidney system and a series of interconnecting ducts and excretory pathways known as the urogenital system.

The urogenital system starts its development by the differentiation of the intermediate mesoderm and the formation of the nephric ducts on both sides of the dorsal aorta. These ducts arise in the mesoderm ventral to the anterior somites at around 8 days post coitum (dpc) in mouse. The cells migrate caudally inducing the adjacent mesenchyme to form the pronephric tubules in the anterior region. This anterior region degenerates, but the more caudal regions of the pronephric duct persist and continue to grow caudally. This growing duct is referred to as the Wolffian duct. Two sequential sets of tubules differentiate adjacent to the Wolffian duct both spatially and temporally, the mesonephric tubules in the middle region and the metanephric tubules in its most caudal part. These tubules seem to be induced by the growth of the Wolffian duct itself. The gonads will develop in the inner region of the mesonephros at about 10.5 dpc in the mouse. The mesonephros degenerates later in development. The metanephros will give rise to the definitive kidney, Fig 1.4 (Herzlinger, 1994).

A second duct system, the Müllerian duct, starts its development parallel to the Wolffian duct at around 11 dpc. It is believed that its development somehow depends on the integrity of the Wolffian duct. Later in development, the Wolffian ducts will give rise to the male reproductive tract: the vas deferens, epididymis and seminal vesicle. The Müllerian ducts will give rise to the female reproductive tract: oviducts, uterus and upper vagina. Therefore, in each organism, according to its sex, one set of these ducts must be eliminated. In males, the developing testes produce Anti-Müllerian Hormone (AMH) and testosterone. AMH induces the regression of the Müllerian ducts while testosterone induces the differentiation of the Wolffian ducts. In females, in the absence of both hormones, the Wolffian duct degenerates and the Müllerian duct differentiates. In this way, only one reproductive tract system develops in each individual. Fig. 1.5 (Behringer, 1995).

22 Figure 1.4: Development of the mammalian urogenital system

23 Metanephric Kidney

Pronephros Müllerian Duct

"Indifferent" Genital ------^ Gonad Ridge

Mesonephros

Metanephros U Wolffian Duct Nephric (Wolffian) Duct

(a) (b) (c) (d)

(a) Differentiation of the Wolffian ducts,(b) The Wolffian ducts grow caudally, controlling the appearance of the mesonephros, metanephros and Müllerian ducts. The pronephros degenerates.(c> The genital ridges form, medial to the mesonephros,id) "Indifferent" gonads form on the genital ridges. The metanephros gives rise to the definitive mammalian kidney. Figure 1.5: Sexual differentiation in mammals.

24 Indifferent

Metanephric Metanephric Kidney n Kidney

MALE

Mullerian Degenerating Ducts Müllerian Ducts

U reter------p ^ Degenerating Wolffian Ducts

Urinary Bladder

Uterus Urethra Urethra Vagina

Fem ale the Wolffian ducts degenerate, ovaries form from the “indifferent” gonads, the Müllerian ducts give rise to the female reproductive tract. Male: the Müllerian ducts degenerate, testes form from the “indifferent” gonads, the Wolffian ducts give rise to the male reproductive tract. 1.5.2 The indifferent gonad

During its early stages, the developing gonad appears identical in both male and female individuals. It is composed of several cell types: germ cells, which will give rise to sperm in testes and oocytes in ovaries; supporting cells, which will give rise to Sertoli cells in males or follicle cells in females; steroidogenic cells, differentiating into either Leydig or Theca cells in male and female respectively; and connective tissue, which constitutes the blood vessels and tunica in both testes and ovaries, and gives rise to myoid cells in males and stromal cells in females. Apart from the germ cells, whose origin and migration to the gonads is well described (see below), it is not yet clear how the other cell types of the gonad originate.

Witschi (1951) (Witschi, 1951) claimed that the major constituent cells of the testes were of mesenchymal origin, while the ovary was predominantly composed of cells coming from the coelomic epithelium. He even thought that sex differentiation depended on the interaction of the cortical (female) region versus the medullary (male) region. He based this model on studies in amphibian gonads. Now there is evidence that this may not apply to mammals (see below).

On the other hand, many investigators suggest that the cells giving rise to the gonads of both sexes come from the mesonephros.(Kanai, et al., 1989;Merchant-Larios, 1979;Satoh, 1985;Wartenberg, 1982). It has been shown by whole mount staining against laminin that there are cellular bridges extending from the mesonephric tubules to the gonad primordium (Karl and Capel, 1995).

Others think that they come from the coelomic epithelium (Allen, 1904;Smith and MacKay, 1991;Torry, 1945;Whitehead, 1904;Yoshinaga, et al., 1988). Pores going from the surface epithelium to the interior of the gonad through which cells could be invaginating have been found (Capel and Lovell-Badge, 1993).

In vitro organ cultures have shown that the mesonephros needs to be present, adjacent to the gonad at 11.5 dpc in order for the testicular cords to develop (Buehr, et al., 1993;Merchant Larios, et al., 1993). When marked cells from the mesonephros were followed at 11.5 dpc through development it was found that myoid and endothelial cells migrate from the mesonephros to the gonad at this time(Moreno-Mendpza, et al., 1995). By 11.5 dpc, the gonad consists of small compact clusters of primordial germ cells surrounded by somatic cells. There is a fine meshwork of laminin localized at the surface of the cells throughout the gonad.

25 1.5.3 Origin, migration and fate of the primordial germ cells

In all vertebrates the primordial germ cells (PGCs) are migratory. They appear extraembryonically, distant from the gonad (Nieuwkoop and Satasurya, 1979) to which they migrate via passive and active migration mechanisms. In the mouse, PGCs can first be located (at about 7.5 dpc) at the base of the allantois, posterior to the definitive primitive streak in the extraembryonic mesoderm (Ginsburg, et al., 1990;Lawson and Hage, 1994;Ozdzenski, 1967). They can be identified by their ultrastructural morphology and surface alkaline phosphatase activity. By the end of gastrulation, PGCs can be seen in the endoderm that will give rise to the hindgut. Later they are found in the hindgut endoderm (8 dpc). This region invaginates, introducing the germ cells into the embryo, where they become embedded in the hindgut wall (9 dpc). From 9 dpc to 11 dpc, PGCs migrate out of the hindgut endoderm and through the dorsal mesentery, around the dorsal aorta and the coelomic angles towards the genital ridges. By 12.5 dpc most of the PGCs are found aggregated in tight clumps in the gonad (De Felici, et al., 1992;Wylie and Heasman, 1993).

Signals inducing the start of migration, its direction and the time and place to stop it are largely unknown. However, the participation of growth factors released by the genital ridges and the modulation of interactions with different cell types and extracellular molecules are thought to be important (Donovan, 1994). It has recently been shown that PGCs actively extend long processes, establishing nets which link them to each other. These processes appear as soon as PGCs leave the hindgut and it is possible that they play an important role in the aggregation of PGCs in the gonad (Gomperts, et al., 1994). PGCs proliferate from 50 per embryo at 8 dpc to about 25 000 per embryo at 13.5 dpc. The appearance and behaviour of PGCs seem much the same in males and females until 13 dpc, when they proceed through sexually different pathways. In testes, PGCs stop mitotic proliferation and enter a quiescent period which is resumed only after birth. In females, PGCs stop proliferating and enter the prophase of the first meiotic division at 13.5 dpc. PGCs of both sexes which do not reach the gonads, enter the first meiotic prophase simultaneously with those that reached the ovary (McLaren, 1995;Upadhyay and Zamboni, 1982).

1.5.4 Testis Differentiation

At around 11.5 dpc the male indifferent gonad starts differentiating. By 12.5 dpc it can clearly be recognized as a testes. Two main characteristics mark the appearance of the testes: the differentiation of the supporting cell lineage into Sertoli cells, and the formation of the seminiferous cords. The current model of sex determination proposes that the testis determining gene induces somatic cells to develop into Sertoli cells

26 (Lovell-Badge and Hacker, 1995), which produce AMH. The Sertoli cells are then thought to induce the differentiation of the testosterone producing cells (Leydig cells). Leydig cells can be seen as interstitial cells outside the seminiferous cords. Intimate contact between the pre-Sertoli and the germ cells is achieved through long pre-Sertoli cytoplasmic processes. The primitive sex cords aggregate into larger seminiferous cords, and become completely surrounded by basal lamina components to which early myoid-like cells are attached. A thick cellular layer rich in extracellular matrix, the tunica albuginea, separates the seminiferous cords from the surface epithelium. Blood vessels and mesenchymal cells in the stromal compartment make the seminiferous cords very conspicuous, so that at 12.5 dpc the testis is clearly distinguishable from the ovary. At 13.5 dpc the seminiferous cords are tightly packed and occupy most of the gonad except for a small region of disorganized cells lying between the mesonephric tubules and the newly formed cords. These cells are assumed to form later the rete testis, the ducts that will provide the link from the seminiferous cords to the excretory channels.

Evidence indicates that germ cells are not necessary for the development of the testes since both mutants and chemically treated animals lacking germ cells show normal testis cord formation(Merchant-Larios and Coello, 1979;Mintz and Russell, 1957).

1.5.5 Ovary differentiation

If the trigger that leads to male development does not occur during the receptive period of the indifferent gonad, the default ovarian pathway of development proceeds. While testis differentiation can be clearly seen at 12.5 dpc, at this stage the ovary still resembles an indifferent gonad. It contains a loosely organized system of cords(Byskov and Lintern-Moore, 1973) referred to as the intraovarian rete. A common basal lamina surrounds the branching system of intraovarian rete cords and on the inside surface the cells of the intraovarian rete make direct contact with small groups of oogonia. Some authors believe that the intraovarian rete cells are the precursors of the follicle cells (Byskov, 1978). At the hilus of the ovary, which is towards the cranial end, the cords make contact with a loosely defined mass of epithelial cells, the interconnecting rete, which joins to the mesonephric tubules (extra ovarian rete)(Byskov and Lintern-Moore, 1973). Between 15.5 dpc and birth, the intraovarian rete cords become more clearly defined and increase in length, creating a series of strands that connect at one end to the surface epithelium and at the other to the extra ovarian rete (Byskov, 1978). At birth, the intraovarian rete cords have a rosary-like appearance. The oocytes have expanded, particularly in the central region of the ovary. During the first week after birth, the intraovarian cords individualize forming follicles which only contain one germ cell in them (Odor and Blandau, 1969). The

27 remnants of the extra ovarian rete lose contact with the Wolffian duct at one end and the intraovarian rete on the other, becoming isolated as a mass of epithelial tissue, the rete ovarii (Byskov, 1978). By two weeks after birth, the ovary consists of many isolated follicles surrounded by a single layer of follicle cells and a thick basal lamina. The supporting cell lineage becomes the granulosa cells, while the mesenchyme becomes the theca cells. The granulosa cells are equivalent to the supporting Sertoli cells of the testis, and they too are capable of producing AMH, although they only produce it after birth. The theca cells are the steroid producing cells. Theca and granulosa cells form the follicles of the ovary which surround each oocyte. The initial development of the somatic components of the ovary does not seem to be dependent upon the presence of germ cells. However, without the presence of oocytes, follicles fail to form. The epithelial cells remain as intraovarian rete cords, and no theca cells are formed.

1.6 Testis Determining Factor

The search for the “testis determining factor” in mammals took a long time. The first candidate proposed for TDF was the H-Y antigen (Wachtel, et al., 1975). This was discovered by studying skin transplants between genetically identical mouse strains. The males accepted the female transplants, but not the other way around. Therefore it was proposed that a gene on the Y chromosome, Hya, produced a surface antigen that was being rejected by the females. At this time the Y chromosome was thought to carry very few genes and perhaps only one, TDF. Hya was put forward as a candidate essentially only because it was the first Y-linked gene for which there was an assay. Later, it was demonstrated that Hya was not necessary for testis development because mice without it developed normal testes (McLaren, et al., 1984).

From examining certain cases of human gonadal dysgenesis, another candidate for TDF was proposed. Page and his colleagues found an XX male with only 280 kb of Y specific DNA, and an XY female with a Y deletion that overlapped this region. The analysis of these patients suggested the position of TDF to be in a 140 kb region and the ZFY gene was found there (Page, et al., 1987). This gene was strongly conserved and was present on the Y chromosomes of all eutherian mammals examined. Nevertheless, embryos carrying a Y chromosome known to be mutant in Tdy (Ytdyml) (Lovell-Badge and Robertson, 1990) have normal Zfy expression in genital ridges, despite them developing as females (Gubbay, et al., 1990b). ZFY was definitively excluded as a candidate for TDF when four XX individuals with testicular development were found to have inherited a fragment of a Y chromosome that did not include ZFY (Palmer, et al., 1989). Analysis of these patients defined a region of 35 kb that must contain TDF. In this region a gene called SRY (sex determining region Y, Sry in mice) was identified (Gubbay, et al., 1990a;Sinclair, et al., 1990).

28 The evidence equating SRY/ Sry with TDF is very strong. It is expressed between 10.5 dpc and 12.5 dpc in the developing mouse testis, just prior to its differentiation. (Koopman, et al., 1990). It encodes a DNA binding protein consistent with its presumed regulatory role (Gubbay, et al., 1990a). Mutations within the DNA binding domain of SRY were associated with male to female sex reversal (Berta, et al., 1990;Harley, et al., 1992). And the strongest evidence is that female to male sex reversal was found when a genomic fragment of 14 kb carrying the mouse Sry gene, and no other, was introduced as a transgene in XX embryos (Koopman, et al., 1991). This latter experiment showed that Sry is not only involved in testis determination, but that it is the only gene from the Y chromosome required for this process.

1.7 Genes involved in sex determination or differentiation.

With the finding of Sry, the major switch of the male determining pathway was found. Nevertheless, as in the organisms described earlier, this gene is only the beginning of a regulatory cascade that must act in order to form a male individual. A number of genes critical for the development and differentiation of the gonads have been recently described. Among them, Dax-1 and Sox-9 have been implicated in the sex determining mechanism. AMH is known to be a sex differentiation gene. WT-1 and SF-1 have been shown to be necessary for the development of the gonad, and SF-1 could be a sex differentiation gene as well. WT-1 might even be involved in regulating Sry. Nevertheless, it is still not clear how they work or at what point on the pathway towards sexual differentiation they act. A brief description of what is known about these genes follows.

1.7.1 Sry Both human and mouse SRY/Sry genes reside on the short arm of their respective Y chromosomes, although they differ in their position, and the Y chromosomes are very different (Fig. 1.6). Human SRY is located 5 kb away from the pseudoautosomal boundary (Palmer M.S. et al., 1989), while mouse Sry is located at the opposite end of the chromosome from the pseudoautosomal region (McLaren, et al., 1988;Roberts, et al., 1988). The mouse Sry ORF is contained within 2.8 kb of unique sequence flanked by inverted repeats of at least 15.5 kb (Gubbay, et al., 1992) (Fig. 1.7).

29 Genes Genetic Functions ] GCY2 CSF2RA Mouse chromosome 19 IL3RA Mouse chromosome 14 ANT3 Possibly deleted In mouse? ASMT M1C2R Yp Tel Yp Tel MIC2 Breakpoints yjv! ly PAR Srncy { Hya) } - r S R Y

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Figure 1.6: Mouse and human Y chromosomes Summary of the known genes mapping to the human and mouse Y chromosomes. Taken from the Report of the Second International Workshop on Human Y Chromosome Mapping(Lau and Affara, 1996)

30 s M _ SM (M) S (S) -J—'-----'------'------"-----'— ^—•------"------'------'— '—'------'— Sry genomic locus 9.0 2.3 2.8 3.5 1.5 2.8 2.3 9.0

Figure 1.7 Inverted repeat structure of the genomic locus of Sry

S = Spe I sites; M = Msp sites; other vertical lines are EcoRI sites I = HMG Box It is known that mouse Sry is expressed in embryonic genital ridges (between 10.5 and 12.5 dpc) and in adult testes. Its precise timing of expression seems crucial as some experiments suggest that if SRY action is delayed by more than 14 hours, testis differentiation fails to occur (Palmer and Burgoyne, 1991). This suggests that during normal development, the gonad might only be receptive to the male trigger for a short period.

In the mouse, it has been found that the embryonic and adult transcripts differ (Hacker, et al., 1995). They have different start sites, the adult transcript initiating somewhere upstream of the embryonic one. The adult transcript was found to give a circular RNA (Capel, et al., 1993); presumably arising from normal splicing processes as a consequence of the unusual inverted repeat structure surrounding the Sry locus in the mouse (Fig. 1.8). It has been shown in vitro that the inverted repeats are indeed necessary for the formation of the circular transcript (Dubin, et al., 1995). It has been found that circular transcripts can be translated if they have internal ribonucleosome entry sites (IRES)(Chen and Sarnow, 1995). Nevertheless, no such sites have been found in the circular transcript of Sry and experiments to show attachment of this transcript to ribosomes failed (Capel, et al., 1993), Therefore it is not clear whether this RNA has any function at all. There is no evidence for circular transcripts from SRY in humans or other non-mouse species.

The embryonic transcript comprises a single exon of 4942 bp (Fig. 1.9)(Hacker, et al., 1995). It starts within the unique region of the locus, 283 bp 5’ of the HMG box. The transcript extends into the 3’ arm of the inverted repeat, and possesses an unusually long 3’ untranslated region (UTR) (3481 nucleotides). In this UTR, putative RNA destabilizing signals and multiple polyadenylation signals have been found. Three ATGs and a TCTG microsatellite have been found in the 5' UTR. All these signals in the UTR may account for the very low level of expression of this gene. The protein encoded by Sry is a member of a superfamily of DNA-binding proteins characterized by having a domain referred to as a High Mobility Group (HMG) box (Gubbay, et al., 1990a) (Fig. 1.10). This box is present in chromatin proteins, in general transcription factors for nucleolar and mitochondrial RNA polymerases and in gene- and tissue-specific transcriptional regulators (Jantzen, et al., 1990). Some HMG box proteins show little DNA sequence specificity, but it has been shown that human SRY binds DNA in a sequence specific manner having high affinity in its interaction with DNA (Harley, et al., 1992). A consensus DNA binding sequence has been found for it

32 Figure 1.8: Model for the formation of the adult circular transcript of Sry

Transcription from the spermatid-specific promoter within the inverted repeat leads to a pre-RNA that extends from the 5' repeat, through the unique region, and into the 3' repeat. Homology between the 5' and S' ends of this RNA molecule would allow a stem loop structure to form. The circle is form by the splicing between the splice acceptor (SA) and splice donor (SD) sites (from Capel, et. al., 1993)

33 ATG ATG

SA 8201 9432 SD 8201 9432 TGA

7480 10219 Figure 1.9 Schematic representation of Sry embryonic transcript.

The long ORF of the gene is shown as a large box with the initiatior methionine (M); red bands mark glutamine repeats; the HMG box is highlighted in magenta; the 5' and 3' untranslated regions are shown in blue boxes. Features of interest are shown: the TATTA degenerate promoter element, three transcription initiation points, two short ORF (m); SA, splice aceptor and SD splice donor sites used by the transcript expressed in the adult testis; transcript instability sequences (ATTTA) and polyadenylatiuon signals (AATAAA). The inverted repeat is shown by arrows. Also marked is the TAG stop codon identified in domesticus type Sry sequences (taken from Hacker, A. et. al. 1995).

34 SA SD V M (TAG) T ig A T T l m m tatta I I n h 1 234 395

-277 1185

TGA AATAAA AATAAA AATAAA

395 * t * 4666 ATTTA 1185 1656 2527 4650 Figure 1.10 Schematic representation of the Sry open reading frame.

The diagram shows the Sry ORF emphasizing the nature of the large glutamine/histidine repeat region at the carboxy terminal. The HMG box is highlighted in red.

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J0S djj |BA leuj eqd B|b use jeiu ojd 6jb sA| |ba siq A|6 n|6 p|/\| (AACAAT). Outside the HMG box there is little homology between SRY in different species. The mouse SRY protein has a glutamine-histidine rich repeat C-terminal to the HMG box, which is not present in human SRY. This repeat is polymorphic in different mouse strains. It has been suggested that this could account for the sex reversal found when the Y chromosome from certain Mus musculus domesticus strains are bred into a strain which normally has a Mus musculus musculus Y, C57BL6 (Coward, et al., 1994). However, recent evidence suggests that this is not the case (Carlisle, et al., 1996)

It is still not known if SRY acts as a transcriptional activator or repressor. There have been claims of it being both. Some genetic evidence discussed by McElreavey et al suggests it could be a repressor (McElreavey, et al., 1993). On the other hand, regions of glutamine-histidine rich repeats have been found to be activator domains in some proteins (Gerber, et al., 1994). Consistent with this, biochemical evidence presented by Dubin and Ostrer show that SRY can function as an activator (Dubin and Ostrer, 1994).

Sry exists as a single copy gene in most mammalian species including the mouse species Mus musculus, M. spicilegus, M. macedonicus, and M. spretus, which are closely related european species. Nevertheless, it has been found that in the more distant asian species, M. caroli, M. cervicolor, and M. cookii, up to six copies of Sry exist. In some related murid species Pyromys saxicola, Coelomys pahari, Nannomys minutoides, Mastomys natalensis, and Rattus norvegicus, up to 13 copies of Sry can be found (Nagamine, 1994). These data, together with the finding of great divergence at the DNA sequence and amino acid level shows that Sry is a very rapidly evolving gene. This could contribute to the reproductive isolation needed for speciation(Tucker and Lundrigan, 1993;Whitfield, et al., 1993).

1 .7 .2 S O X -9

Patients with a skeletal disorder known as campomelic dysplasia (CD) show XY sex- reversed female phenotypes in approximately 75% of cases. Cytogenetic analyses of five XY sex-reversed translocation patients revealed that a region of chromosome 17 (q23-25) was involved. Cloning of a translocation chromosome breakpoint from a sex- reversed patient, followed by mutation analysis of an adjacent gene, indicated that SOX-9, an STZY-related gene, was involved in both bone formation and control of testis development. (Foster, et al., 1994;Wagner, et al., 1994). Mutations in SOX-9 were found to be present in only one allele of the gene suggesting that sex reversal and CD could be due to haploinsufficiency of the SOX-9 gene product. In contrast with SRY, mutations in SOX-9 are not concentrated within the HMG box, but distributed throughout the gene.

36 There is no obvious difference between mutations that cause sex reversal and those that not. Two patients with typical features of CD were found in which the same mutation, a single insertion causing a frameshift, caused sex reversal in one but not in the other. This could be explained by the haploinsufficiency giving rise to a certain level of protein. This level could be just on the threshold, affected by other factors like genetic background, in which sometimes it would cause sex reversal and sometimes not.

The pattern of expression of Sox-9 in developing gonads has recently been studied in mice. It was found to be expressed in a low level in indifferent gonads of both sexes at 10.5 dpc. At 11.5 dpc, when Sry is being expressed, the expression of Sox 9 in testes increases, while in ovaries it disappears. It is interesting that this same pattern of expression was found in developing gonads of chick embryos, where no Sry is present. It seems that Sox-9 is an important gene for determining Sertoli cell differentiation which has been conserved during evolution(Morais da Silva, et al., 1996).

1.7.3 Dax-1

In humans, male to female sex reversal has been observed in individuals with duplications of the short arm of the X chromosome(Bardoni, et al., 1994;Fechner and al, 1993;German, et al., 1978). It has been shown that sex reversal results from the presence of two active copies of an Xp locus. That locus has been named DSS (Dosage Sensitive Sex reversal). It has been proposed that DSS, when present in two active copies, could be overriding the testis-determining mechanism. By mapping the duplication breakpoints in sex-reversed, Xp-duplicated patients and by localizing submicroscopic duplications of an XY sex reversed female, DSS was localized to a 160 kilobase region of chromosome Xp21. Some cases of male individuals which have DSS deleted have been found, so it seems that this locus is not required for testis differentiation. It has been proposed that DSS could have a role in ovarian development and could be involved in the sex determining pathway (Bardoni, et al., 1994).

Adrenal Hypoplasia Congenita (AHC) is an inherited disorder of adrenal gland development. Two forms of it can be found, an autosomal recessive form, and an X- linked form. The X-linked AHC is characterized by the absence of the permanent zone of the adrenal cortex and by structural disorganization of the glands.

Hypogonadotropic hypogonadism (HHG) is commonly associated with the X-linked AHC. AHC was mapped to the Xp21 region, the same region where DSS maps. A gene was cloned from this region by searching for expressed sequences. Analysis of this gene indicated that functional deficiency for this gene was responsible for AHC as

37 AHC patients lacked or had mutations in this gene. This gene has therefore been called DAX-1 (for DSS-AHC critical region on the X chromosome, gene l)(Zanaria, et al., 1994).

The DAX-1 open reading frame encodes a protein of 470 amino acids (Fig. 1.9). 225 amino acids in its C-terminal domain show similarity to the ligand-binding domain of the nuclear hormone superfamily. This domain has been shown for other members of the family to be important for hormone binding, dimerization and transactivation. There is some evidence that DAX-1 could be binding DNA as a homodimer (Zanaria, et al., 1994). On the other hand, the amino-terminal domain of DAX-1 shows no similarity to the DNA-binding domains of the steroid receptor superfamily. The DNA-binding domain of this family is highly conserved among its different members and is composed of two zinc fingers. In contrast, the DNA-binding domain of DAX-1 contains a novel N-terminal repetitive structure, organized into four incomplete repetitions of alanine- and glycine-rich repeats.

38 Putative DNA binding domain Putative ligand binding domain

470

► I— ►! S DAX/Dax

86.4 46.1 87.5 54.8 96.0 48.4 90.0 40.8 85.0 10 81.4 % aa identity

95.4 61.5 95.8 71.4 57.9 93.3 59.2 90.0 20 90.7 % aa similarity 87.9 65.0 77.0 65.1 90.7 86.7 64.6 82.2 57 86.0 % nt identity

121 187 247 449 1 1 A/B - ► c - > D RXRO/RxrO

87 100 95 99

Figure 1.11: Schematic representation of the conservation between human and mouse DAX-1/Dax-1. Comparison between human and mouse DAX-1 /Dax-1 and human and mouse RXR/3/ Rxr/3 . The percentage in amino acid and nucleotide identity or similarity is given below each region. Numbers refer to the human amino acid sequence, (from Swain, A. et. al., 1996).

39 The common embryological origin of the steroidogenic components of the gonads and adrenal glands, make it is possible for DAX-1 to be involved in the development of both organs. The localization oï DAX-1 within the DSS critical region, suggested that DAX-1 might correspond to DSS.

Mouse Dax-1 has been recently isolated and its pattern of its expression has been analyzed during embryonic development (Swain, et al., 1996) see Chapter 4. It was shown to be expressed in the developing adrenals and the hypothalamus. Furthermore, consistent with the proposal of Dax-1 as a candidate gene for DSS, it was shown to be expressed in the developing gonads with a sex-specific pattern. It starts its expression at around 10.5 dpc both in males and females. In males it is then repressed by 12.5 dpc in almost all the testes, except in cells which may give rise to the rete-testis. This pattern of expression coincides with that of Sry (although Sry turns off completely). In females, Dax-1 expression takes longer to be repressed, as it is seen strongly up to 13.5 dpc. From then onwards, it is repressed, only detectable in the possible future cells of the rete- ovarii and possibly in the external the rete-ovarii. As both SF-1 and DAX-1 are members of the nuclear superfamily, it has been proposed that they might interact, either at the level of or at protein function. It is interesting that in the 5’ flanking region of both the mouse and human Dax-1 genes, an SF-1 binding site has been found.

1.7.4 AMH Anti Müllerian Hormone (AMH), also known as the Müllerian Inhibiting Substance (MIS), is a member of the transforming growth factor-B (TGF-B) family (Cate, et al., 1986;Massague, 1990). AMH is an homodimeric glycoprotein expressed in Sertoli cells of embryonic testes, and by granulosa cells post-nataly in the ovary (Münsterberg and Lovell-Badge, 1991). Secreted AMH protein is responsible for the regression of the Müllerian ducts. AMH has been overexpressed in transgenic mice causing the elimination of the Müllerian ducts in female embryos (Behringer, et al., 1990). In mice lacking the AMH gene, females were found to be normal and fertile, but males developed as pseudo hermaphrodites with both male and female reproductive tracts (Behringer, et al., 1994). Testis development was not affected in these animals. The putative receptor of AMH has been cloned in rabbit, rat and mouse (Baarends, et al., 1994;Behringer, 1995;di Clemente, et al., 1994). Its pattern of expression (in the mesenchymal cells adjacent to the Müllerian ducts) suggest that AMH mediates the regression of the Müllerian ducts indirectly through mesenchyme tissue. Mice lacking the AMH receptor are currently being produced (Behringer, 1995), so insights

40 into the function of the AMH receptor during Müllerian duct regression will soon arrive.

1.7.5 SF-1

Steroidogenic factor 1 (SF-1), also known as AD4BP (AD4 element binding protein) is a regulating the steroidogenic P-450 genes in steroidogenic tissues. It is a member of the nuclear hormone receptor superfamily. This gene is homologous to the that regulates the fushi tarazu gene in Drosophila (FZT-Fl) (Lala, et al., 1992).

SF-1 transcripts are present in the gonads of both sexes early in mouse development. The levels decline in females at about 12 dpc (Ikeda, et al., 1994) when morphological differences are occurring, suggesting a role in sex differentiation. Studies in vitro have shown that the SF-1 protein can bind to the upstream region of the gene encoding AMH and activate transcription in co-transfection assays (Shen, et al., 1994). However, because it is expressed at earlier stages in both sexes, SF-1 alone cannot account for the testis- specific activation oiAmh expression at 11.5 dpc. In SF-1 deficient mice(Luo, et al., 1994) the earliest stages of gonadogenesis are relatively intact. The genital ridge forms, but from about 11.5 dpc, apoptosis is seen in the somatic cells of the gonad and adrenal glands. The animals are born with a complete absence of gonads and adrenals.

1.7.6W T -I

The Wilms’ Tumor gene (WT-1) plays a critical role in kidney and gonadal development. In WT-1 deficient mice, no kidneys or gonads are formed(Kreidberg, et al., 1993). It is observed, as in the case of SF-1, that the early formation of the genital ridge is not disrupted, but the gonads don’t develop and degenerate. However, it has been shown that the SF-1 and WT-1 genes are not dependent on each other for their expression, so they must be needed in different pathways in the developing gonad(Luo, et al., 1995).

1.8 Aims of this thesis

In order to obtain development of a complete organism with its hundreds of different cell types, it is fundamental to achieve a precise regulation and timing of the expression of many genes. Each cell type expresses a different set of genes at any given time. This regulation can be accomplished at several levels. First, by regulating the levels at which the gene is transcribed into RNA. Then, by selecting the RNAs for nuclear processing, in this way regulating which of the RNAs

41 get into the cytoplasm to become mRNA. Another step involves the selection of mRNA for translation; and finally, selection of some proteins for modification to allow them to be functional. All of these mechanisms can be used by a single gene.

In order to understand how a gene is regulated at the level of transcription, it is important to find regulatory elements flanking the gene. Promoters and enhancers are necessary for controlling the tissue specificity and temporal pattern of expression of the genes. A promoter is typically located immediately upstream from the site where transcription starts. The promoter site is required for the binding of RNA polymerase II and the accurate initiation of transcription. An enhancer activates the utilization of the promoter, controlling its efficiency and rate of transcription. They can activate the promoter at great distances (even at 50 kb away from the promoter). Their activating function is position independent because they can be either upstream or downstream of the promoter and still be functional. They can even be in the introns or the complementary DNA strand. It is thought that the way enhancers activate promoters is by the interaction they have with transcription factors binding to them. Because enhancers can be many kilobases away from the promoter, many different enhancers can collaborate in determining whether the gene is activated or not. The aims of this thesis were to find the regulatory regions of two crucial genes for the sex determination pathway: Sry and Dax-1; and to study the possible involvement of Dax-1 in the sex determining mechanism of the mouse. For these ends, several approaches, both in vivo and in vitro were attempted.

42 Chapter 2; Materials and Methods

2.1 Commonly used solutions and enzymes.

Ix Denhardt's: 0.02% (w/v) Ficoll (Type 400, Pharmacia), 0.02% (w/v) polyvinylpyrrolidone and 0.02% (w/v) BSA (Fraction V, Sigma) LB: 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract and 170 mM NaCl Ix MOPS: 20 mM 3-[N-Morpholino]propane-sulfonic acid, 5 mM Na Acetate and 1 mM EDTA PBS: 140 mM NaCl, 3 mM KCl, 10 mM Na2HP0 4 and 2 mM KH2PO4 Ix SSC: 150 mM NaCl, 15 mM Na Citrate, pH 7.0 Ix TAE: 40 mM Tris-acetate, pH 8.0, 1 mM EDTA Ix TBE: 89 mM Tris-borate, 89 mM boric acid and 2 mM EDTA 20X NET: 3M NaCl; 20 mM EDTA; 0.3 M Tris pH 7.5. STET: 8% sucrose, 5% Triton X-100, 50 mM Tris, pH 8 and 50 mM EDTA pH 8 TE : 10 mM TrisHCl, pH 7.5, 1 mM EDTA TER: 10 mM TrisHCl, pH 7.5, 1 mM EDTA, 30 |ig / ml RNAse

Restriction enzymes, Boehringer Mannheim and NBL. T3/T7 RNA polymerases, Promega T7 DNA polymerase, Boehringer T4 DNA polymerase, Boehringer Klenow (large fragment of E. coli DNA polymerase I), Boehringer DNAsel (RNAse free), Boehringer Ribonuclease A, Sigma Ribonuclease A/Tl mixture (Ambion) RNasin, Promega Calf intestinal phosphatase, Boehringer T4 Ligase, NBL Polynucleotide kinase, Pharmacia Reverse transcriptase from Moloney murine leukaemia virus, BRL Thermus aquations (Taq) DNA polymerase, Amplitaq, Perkin Elmer.

2.2 Bacterial strains, plasmids

DH5 F': supE44Alac\J169{0SOlacZAM15) hsdRnrecAlendAlgyrA96thi-lrelAl F'[traD36proAB'^lacl9lacZAM15] (Hanahan, 1983) pBluescript KS+ or KS-, Stratagene pUC18 or 19, NBL pOK12 (Vieira and Messing, 1991)

43 2.3 Purification of DNA fragments from agarose gels

DNA was run in agarose-TBE gels and fragments purified with the QUIAEX DNA gel extraction kit (QUIAGEN), or GeneClean II kit (Bio 101) using the manufacturer’s protocol.

2.4 Spectrophotometric quantitation of nucleic acids Nucleic acid concentrations were determined by measuring the UV absorption at 260 nm of diluted samples on a spectrophotometer (Kontron, Uvkon 860). An OD26O absorption unit of 1 corresponds to approximately 50 mg/ml for double-stranded DNA, 40 mg/ml for RNA or single-stranded DNA and 20 mg/ml for oligonucleotides of twenty bases.

2.5 Recombinant DNA techniques.

2.5.1 Preparation of competent bacteria. 50 ml of LB medium were inoculated with an overnight culture of E. coli and grown to an OD600=0.5-0.6 at 37 ®C. The culture was chilled on ice for 5 to 10 minutes. The cells were pelleted by centrifugation for 15 minutes at 4®C and then gently resuspended in 1/3 volume of RFl (100 mM RbCl, 50 mM MnC12, 30 mM potassium acetate, 10 mM CaC12, 15% (w/v) glycerol, pH 5.8) and incubated on ice for 25 minutes. The cells were re-pelleted for 30 minutes at 3000 rpm and resuspended in 1/12.5 of the original volume in RF2 (10 mM MOPS, 10 mM RbCl, 75 mM CaC12, 15% (w/v) glycerol, to pH 6.8). The cells were incubated on ice for 15 minutes, and 200 |il aliquots were quickly frozen in dry ice/ethanol and stored at -70 OC .

2.5.2 Transformation

Competent cells were thawed at 37®C and placed on ice. 50 ng of plasmid DNA (1-5 |ll1) or 10 |il of a ligation reaction were added to 50 pi of competent cells. Cells were incubated in ice for 5 minutes, then at 42 °C for 45 seconds and then returned to ice for 10 minutes. Bacteria were plated on LB agar plates (0.7%w/v agar in LB) containing the appropriate antibiotic for selection. When B-galactosidase was used as a genetic marker, plates contained 0.5 mM IPTG (isopropyl-B-D galactosidase) and 40 mg/pl X-gal. After o/n incubation at 37oC, colonies were selected for analysis.

2.5.3 B lunt End L igations

Vector and fragment (1:3 ratio) DNA were added to 10 pi of ligation mix (2 pi of NEB 1 buffer (New England Biolabs), 4 pi 1 mM ATP, 1 pi 20 mM Hexamminecobalt (III) chloride (HCC), 1 pi ligase (T4 DNA ligase, NEB) and 2 pi 0.3 M KCl) in a total volume of 20 pi. The reaction was incubated at RT for 20 minutes.

44 2.5.4 Cohesive End Ligations

Vector and fragment DNA (1:3 ratio) was added to ligation mix (1 |ll1 o f lOX T4 ligase buffer (NEB), 1 |il of T4 ligase (NEB)) in a total reaction volume of 20 |il, and incubated o /n at 150c.

2.5.5 Small scale plasmid isolation (minipreps):

2 ml cultures were grown overnight in LB media with the appropriate antibiotic. 1.5 ml of each were spun down and most of the supernatant, except for about 100 pi, was poured off. The pellet was then completely resuspended by vortexing and 0.3 ml of STET containing 1 mg/ml of freshly added lysozyme was “squirted” into it. This mixture was immediately boiled for exactly 90 seconds, and microfuged at 4 °C for 10 minutes. The supernatant was transferred to a new tube, an equal volume of isopropanol was added, vortexed to mix and microfuged for 10 minutes at 4 °C. All the supernatant was aspirated off and the pellet was washed with 200 pi of 70% ethanol. The pellet was resuspended in 40 pi of TE if the plasmid was used for sequencing or in 100 pi of TER if it was used for enzyme digestions.

2.5.6 Large Scale Plasmid Purification (maxipreps).

Maxipreps were prepared with the QUIAGEN Plasmid/Cosmid Purification Kit following the manufacturer’s instructions.

2.5.7 Sequencing.

A modification of the Sequenase Version 2.0 t& DNA Polymerase Sequencing Protocol was used (USB, Pharmacia). 1 pi of primer (10-50 ng) and 1 pi of 1 N NaOH and 1 pi of primer were added to 8 pi of plasmid DNA from a miniprep and incubated for 10 minutes at 68 °C. 4 pi of TDMN (280 mM TES (Sigma, free acid), 80 mM MgCl2, 200 mM NaCl, 10 pi / ml of concentrated HCl and fresh 52 mM DTT) were added, mixed by pipetting up and down, and incubated at room temperature for 10 minutes. 3.5 pi of labelling mix (1 pi of 0.1 M DTT, 0.4 pi of 5X dGTP labelling buffer, 1.6 pi of H2O and 0.5 pi of 35 S-dATP were added together with 5.5 pi of a 1:4 dilution of Sequenase in enzyme dilution buffer. This was incubated for 3 minutes at room temperature. 2.5 pi of A, C, G, and T termination mixes were added to four wells of a sequencing plate, and 3.5 pi of the template/primer/labelling reaction were added, mixed up and down with a pipet and incubated for 5 minutes at 37 °C. 4 pi of stop buffer were added to each sample, and they were heated at 80 °C for 3 minutes just before loading into the gel. The samples were run in a 6% (w/v) polyacrylamide, 8 M urea, IX TBE gel.

45 2.6 PCR techniques

2.6.1 Isolation of genomic DNA for PCR.

Genomic DNA for PCR was isolated from tails of 2-3 week born mice or from limbs of embryos. Tissue was incubated for 5 to 16 hours at 50 in 100 |il of Tris (pH 7.5) 100 mM, NaCl 100 mM, EDTA 1 mM, SDS 1% and proteinase K 100 pl/ml. An equal volume of phenol:chloroform (1:1) was added, mixed for 20 minutes and microfuged for 10 minutes. 1 pi of the upper phase was used for a 25 pi PCR reaction. PCR was used to detect the presence of the transgenes in the genome and reverse transcription PCR (RT-PCR) for detection of m RNA .

46 2.6.2 Oligonucleotides used:

oligo name: sequence: position in gene:

5’ oligos

BABBV5’: TGC TGC ACT GOT GGC GAA GC (347-366 in /3-actin) BAHHA5’: TGC GCC GTT CCG AAA GTT GC (398-417 in /S-actin) BACTIN5’: AGC ACA GAG CCT CGC CTT TG (505-524 in ft-actin) ACSRY5’: CCG ATC CTC TAG AGA GCA TG (junction fragment: (525-531 in ft-actin + 8294-8306 in Sry) pZ5A: TAG TGT TCA GCC CTA CAG CC (8220-8239 in Sry) pll94.1: TA GTT GGG CTG GAC TAG GG (7946-7965 in Sry) la c5 ’: GGA AAA CCC TGG CGT TAG CC (51-70 in lacZ) HPRT.IA: CCT GCT GGA TTA CAT TAA AGC ACT G (exon 3) m yol: TTA CGT CCA TCG TGG ACA GCA T lA: TCA ACT GCT GGA GTC TGA ACA TTG (37-60 in Dax-1 ) 6A: TGC TGA GAT TCA TCA ATA GC (244-260 in Dax 1 ) HDR'; CTC CGC CTC CAA GGT CCA AG (1292-1312 in DAX-1 cDNA) HDDS': CTC CCT CCA GAC GTC CCG GG (10 b in intron + 1402-1411 in DAX-1 cDNA) Y11A2: GTC TAG AGA GCA TGG AGG GC (8293-8313 in Sry) Y41A: CTA ACA GCT GAC ATC ACT (9414-9431 in Sry) Zfy 1.8: GAC TAG ACA TGT CTT AAC ACT TGT CC

3' oligos

SRYRV3’: CAG GTG TGC AGC TCT ACT CC (8672-8653 in Sry) SRYPST3’: GTT GAG GCA ACT GCA GGC TG (8590-8571 in Sry) pZ3’: AGA TGG GCG CAT CGT AAC CG (282-301 in lacZ) HPRT.2A: GTC AAG GGC ATA TCC AAC AAC AAA C (exon 8) myo2: TGG GCT GGG TGT TAG TCT TAT G (in m yogenin) Mut 3 : TAC AAA TAT ACT ATG CGA GC (466-475 in Dax 7-mut) 7B: CCT ACT ACC ACA CTC TTA GA (580-602 in Dax 1) 3B: GCA CAG AGC ATC TCC AGC ATC AT (321-343 in Dax-1) HD3': GCA CTT GTG TGG CCC ACA TG (1651-1670 in DAY-7) RGB: CAA GGG GCT TCA TGA TGT CC (1655-1684 in /3-globin) Y llB : CCA CTC CTC TGT GAC ACT (8546-8563 in Sry) Y11B2: TGG CAT GTG GGT TCC TGT CC (8614-8633 in Sry) YNLS-5’ CCT ATT GCA TGG ACA GCA GCT TAT G (in Zfy)

47 2.6.3 Primer combinations and annealing temperatures

Gene Prim er Fragment A n nealing screened: combination: size (bp): temperature:

ACSRY5’ + SRYRV3’ 384 @60 °C pFLAG+ ACSRY5’ + YllB 275 @58 OC pVACS BACTIN5’+ SRYRV3’ 404 @60 °C BACTIN5’+ YllB 295 @58 OQ pZ10+ pZ5A + pZ3’ 461 @58 °C pZll pll94.1+ pZ3' 703 @58 OC pLUGl pll94.1+ pZ3’ 555 @58 °C lacZ lac 5’ + pZ3’ 249 @58 °C

Hprt HPRT.IA + HPRT.2A 352 (in cDNA) @58-62 °C

Myogenin myo 1 + myo 2 249 @58-62 00

6A+7B 358* @62 oc 6a + mut 3 235 ** @62 oc Dax-1 1A + 3B 305 @62 OC 6A + RGB 310 (in cDNA) @62 oc 6A + RGB 890 (in genomic) @62 oc

DAX-1 HDR5'+ HD3' 378 (in cDNA) @62 oc HDD5'+ HD3' 260 (in genomic) @62 oc

Sry Y11A2 + Y11B2 340 @58 oc (embryonic)

Sry Y41A + Y11B2 397 @60 oc (adult)

Z/y Zfyl.S + YNLS-5’ 180 @58-62 oc

* These oligos detect the endogenous Dax 1 gene. The transgene carries a mutation engineered at position 465 (Amanda Swain, unpublished), so a Sac I site is created. After the PCR amplification, the product can be cut with this enzyme in order to distinguish between the transgene and the endogenous gene. After the digestion two fragments appear of the following sizes: 137 and 221 bp.

** 6A and mut 3 only detect the transgene.

48 2.6.4 Oligonucleotide Purification

Oligonucleotide primers were prepared by the service at NIMR. Typically, the oligos were received in 2 ml of ammonia. These were vacuum dried (Speedvac) and washed three

times in 100% ethanol. The pellet was resuspended in 400 | li1 of water and precipitated with 40 fil of 3 M sodium acetate and 800 |il of 100% ethanol and spun for 10 minutes in a m icrofuge.

2.6.5 PCR procedure

PCRs were carried out in a 25 pi volume containing 50 mM KCl, 10 mM Tris pH 8.3, 1.75 mM MgCl2, 0.1 mg/ml Gelatin, 0.375 mM dNTP, 500-700 ng of each primer and 2 units of Taq polymerase (Cetus). 1 pi of the DNA extracted from tails or limbs, or of the product of a reverse transcription reaction (see below) was used as template. Reactions were overlaid with mineral oil (Sigma) to prevent evaporation. The amplification was done in a Hybaid thermocycler either in 0.5 pi eppendorf tubes or in 96 multiwell plates. A first step of 3 minutes of dénaturation at 94 was used with all the programs. Then, 30 cycles of 45 s at 94 ®C, 45 s at different annealing temperatures depending of the primers used, and 50 s at 72 °C were done. All programs ended with 10 minutes at 72 °C. 15 pi of the reaction plus 10 pi of stop mix (TBE IX, Urea 9 M, bromophenol blue) were run in 2% agarose, 0.5X TBE gels to confirm the amplification of PCR products of predicted size.

2.6.6 RNA Purification for RT-PCR.

RNA was purified basically as described by (Chomczynski and Sacchi, 1987). Embryonic tissues were dissociated in 500 pi of solution D (4 M Guanidinum thiocyanate, 25 mM Sodium citrate, pH 7, 0.5% Sarcosyl and 0.1 M fi-mercaptoethanol). 50 pi of sodium acetate 3 M, pH 4 were added and extracted with 500 pi of phenol (saturated in water) : chloroform :iso amyl alcohol (25:25:1). Samples were precipitated with 500 pi of propanol for 3 hours at -20 and spun for 15 minutes at 4 °C. Pellets were resuspended in 20 pi of TE. 5pi of these were used for a control PCR reaction to check for DNA contamination. If DNA was present, 2.5 pi of lOX DNAse buffer (IM Sodium acetate, 100 mM MnCl2) and 40 u of RNAse-free DNAse (Boehringer) were added to the RNA samples and incubated at 37°C for half an hour. Another phenol : chloroform :iso amyl alcohol extraction and propanol precipitation was carried out and the pellet resuspended in 15 pi of TE.

49 2.6.7 Reverse Transcription of RNA

10 |i.l of isolated RNA for RT-PCR were incubated for 5 minutes at 65®C. 20 |il of cDNA synthesis buffer {50 mM Tris pH 8.3, 75 mM KCl, 5 mM MgCl2, 0.5 mM dNTP, 10 mM DTT, 2 p,g of oligo dT primer (Boehringer) and 100 units of reverse transcriptase (ERL)} were added and incubated for one hour at 37®C. 10 |ll1 of this reaction were used for PCR.

2.7 Probe Labelling and Southern hybridization

2.7.1 DNA labelling

DNA was labelled, by random primer, with the Megaprime DNA labelling kit (Amersham) following the instructions of the manufacturer. The reaction was done in 20 |il. If the probe was bigger than 400 bp, it was spun through a G50 Sephadex column for one minute to eliminate the unincorporated nucleotides. When the probe was less than 400 bp, a precipitation was done. 200 p,l of precipitation mix (Tris 10 mM, pH 7.5; sonicated herring sperm DNA, 0.5 mg/ml, EDTA 14.28 mM and ammonium acetate 2.57 M) and 400 |il of propanol were added. The sample was vortexed vigorously and spun for 15 minutes. The pellet was resuspended in 19 |l i 1 of water. Percentage of incorporation was measured in a scintillation counter by Cherenkov counting. The probe was boiled for 3 minutes before adding to the filters.

2.7.2 Ohgonucleotide labelling

Two complementary oligonucleotides (PI: 5’-GGC GTA ATA GCG AAG AGG CCC GCA CCG, and P2: 5’-AAG GGC GAT CGG TGC GGG CCT CTT GCG) were used for the detection of lacZ after RT-PCR. 15 ng of each oligonucleotide were mixed with 1 |il of a mixture of 5 mM each dATP, dGTP and dTTP, 2 |l i 1 of Megaprime Labelling Kit Buffer, 5 |il of p32dCTP, 1 |il of Klenow enzyme and volume reaction adjusted to 20 p.1. Polymerization was done at

RT for 20 minutes. 1 |l l 1 of dCTP (5 mM) was added and the reaction left for another 5 minutes. The reaction was stopped with l|il of EDTA 500 mM. Precipitation was performed as above to remove unincorporated nucleotides.

2.7.3 DNA Transfer and Hybridization

After electrophoresis, DNA was blotted to Hybond N+ membranes with 0.4 M NaOH, o/n. When DNA fragments bigger than 100 bp were used as a probe, the filters were prehybridized at 65°C for 3-6 hours in hybridization mix A (6X SSC, 5X Denhardt; 0.5% SDS and sonicated herring sperm DNA 100|ig/ml). The labelled probe was added, and left to hybridize at 65°C, o/n. The filters were washed 3 times with 2X SSC; 0.1% SDS for 15 minutes at 65 ®C and then two times with O.IX SSC; 0.1% SDS for 15 minutes at 65°C. When

50 DNA fragments of less than 100 bp were used as probes, prehybridization was carried out in hybridization mix B (6X NET; 0.1% SDS and lOX Denhardt) for 3-6 hours at 37°C. The labelled probe was added and hybridization was carried out at 50°C, o/n. The filters were washed with 6X SSC; 0.1% SDS once at RT for 15 minutes and twice at 50°C, 15 minutes. The filters were exposed to radiographic films or to the phosphoimager.

2.8 Protein detection

2.8.1 Sample preparation Basically the protocol described in Harlow and Lane (1988). have been followed. The sample was dissolved thoroughly in 10 volumes of sample buffer (2% SDS, 100 mM DTT, 60 mM Tris, pH 6.8, 0.01% bromophenol blue), boiled for 5 minutes and spun for 10 minutes. The supernatant was recovered and 10 pi of it were loaded to a 6% acrylamide, 10% SDS, Tris pH 8.8 gel.

2.8.2 Electroblotting The transfer of the proteins to nitrocellulose membranes was done by electroblotting in a semi-dry camera. The nitrocellulose filter was soaked in IX semi-dry buffer (48 mM Tris base, 39 mM glycine, 0.037% SDS, 20% methanol) and the proteins were transferred for one hour.

2.8.3 Detection of the protein with antibodies The nitrocellulose filters were washed in milk solution (5% powder skimmed milk, 0.05 % Tween-20 dissolved in PBS) for 20 minutes. The antibody was added and left at room temperature for one hour or overnight at 4®C. The filters were washed three times for ten minutes with milk solution, and the secondary antibody was added and left for one hour at room temperature. The filters were washed again three times for ten minutes at room temperature. Detection of the antibody was done with the ECL kit (Amersham).

2.9 Production of transgenic mice

2.9.1 Mouse Stocks

All mice were from stocks maintained at the animal facilities at NIMR. They were kept in a 12 hours light/12 hours dark cycle. Midday on the day of vaginal plug was taken to be 0.5 dpc. The stage of embryos was confirmed by examination of limb morphology and comparison to the schedule outlined in Hogan, et al. (1986).

51 2.9.2 Microinjections of fertilized mouse eggs.

The procedures used were essentially those described by Hogan et al. (1986). Ten 3-4 week

old (CBA X C57BL/10) FI female mice were superovulated by injecting intraperitoneally with 5 units (0.1 ml) of pregnant mares serum (Folligon, Intervet Laboratories) 48 hours before and 5 units (0.1 ml) of Human Chorionic Gonadotropin (Choluron, Intervet Laboratories) at 2 pm on the day of mating with (CBA x C57BL/10) FI male mice. The next day, oviducts from plugged females were removed and the fertilized eggs released by dissection under M2 medium (Hogan et ah, 1986). Hyaluronidase was added to a final concentration of 300 mg/ml. The eggs were washed through six drops of M2 medium to remove all cumulus cells and other debris and kept in a drop of M16 medium (Hogan et al., 1986) equilibrated in a humidified incubator at 37®C with 5% of COg, under liquid paraffin (Boots), until injection. M2 and M16 media were made fresh, weekly from stock solutions kept at -20oC. Media were filter sterilized (0.22 mm) and stored at 4°C.

For the injection of the DNA, one cell embryos were transferred to a drop of M2 medium under liquid paraffin in a depression slide and the purified DNA fragment was injected into either pronucleus. Embryos were injected in batches of 30-50, and returned to M16 medium. The microinjection setup consisted of a Nikon Diaphot-TMD inverted microscope or a Leitz DM IRB/E microscope fitted with Nomarski optics, combined with Leitz-E micromanipulators. Fertilized eggs that survived the microinjection procedure were reimplanted immediately or after o/n culture into the oviducts of pseudopregnant females of 6-8 weeks, plugged on the same day of the reimplantation. Mice were anaesthetised by intraperitoneal injection of 0.3 ml of 0.5 mg/ml midazolam (Hypnovel, Roche), 2.5 mg/ml fluanisone and 0.079 mg/ml fentanyl citrate (Hypnorm, Crown Chemical Company). The time was considered as 0.5 dpc if the eggs were reimplanted the same day and as 1.5 dpc if reimplanted the next day. The embryos that developed were either allowed to be born for the production of transgenic lines or harvested earlier for staining with X-gal or for RT-PCR as "transients". Genomic DNA obtained from tail tips or limbs (from embryos) was used in PCR reactions to detect the presence of the transgene.

2.9.3 X-Gal Staining.

Embryos injected with lacZ constructs were analyzed for B-galactosidase expression by staining with X-gal (5-bromo, 4-chloro, 3-indolyl, B-D-galactosidase, Sigma). They were dissected out of uteri and rinsed in phosphate buffered saline (PBS), opened so to expose the gonads, and fixed with paraformaldehyde 2%; glutaraldehyde 0.1% in PBS 30 minutes at 4®C. Embryos were washed 3 times with PBS at room temperature and stained with 0.5 ml per embryo of staining solution (1 mg/ml X-gal, 5 mM K3Fe(CN)6, 6.5 mM K4Fe(CN)6, 20 mM MgCl2) during 24 hours at 37®C in the dark.

52 2.9.4 Paraffîn sections

After X-gal staining, samples were imbedded in paraffin for sectioning. Samples were washed three times with PBS and desiccated by passages through rising concentrations (50, 70. 85. 95 and 100%) of ethanol. Afterwards, the samples were changed to histo-clear and then imbedded in paraffin.

2.10 RNAse Protection.

2.10.1 Probe Synthesis The linearized DNA template was gel purified. Then a phenol:choloroform:isoamyl alcohol extraction was performed and the DNA precipitated with sodium acetate and ethanol. DNA was washed with 75% ethanol and resuspended in TE at 0.5 mg/ml. 1 pi of this DNA was added to a 20 pi labelling reaction 40 mM Tris-HCl pH 7.9, 6 mM Mg C12 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 2.5 mM ATP/CTP/GTP, 800 Ci/mmole ^^UTP (Amersham) and 20 units of the correct RNA polymerase. The reaction was incubated for one hour at 37 °C. The DNA template was digested from the riboprobe by adding 1 pi of RQl DNAse (Pharmacia) and incubating for a further 15 minutes at 37 °C. 20 pi of formamide dye (100% formamide, 10 mM EDTA, 0.1% Bromophenol blue/ Xylene cyanol) were added, the sample heated at 90 for 3 minutes and loaded in a 6% polyacrylamide, 8 M urea gel, run for about 1.5 hours at 50 Watts. The probe was then cut out from the gel and eluted in 400 pi of elution buffer (0.5 M Ammonium acetate, 1 mM EDTA, 0.1% SDS, 10 pg E. Coli tRNA) for at least 3 hours at 37 ®C. The elution buffer was transferred to a new tube and precipitated with 2 volumes of 100% Ethanol and 0.3 M Sodium acetate for one hour on dry ice. The pellet was resuspended in 20 pi of water and 1 pi was used for counting incorporation of radioactivity.

2.10.2 Hybridization.

The RNA probe synthesized above was combined with the RNA samples using around 400,000 cpm of the probe per sample. They were vacuum dried and resuspended in 20 pi of hybridization buffer (80% formamide, 40 mM PIPES pH 6.7, 0.4 M NaCl and 1 mM EDTA) Samples were incubated for 5 minutes at 80 °C and transferred to 50 °C and left to hybridize o/n.

53 2.10.3 Digestion

400 )ll1 of digestion mix (NaCl 0.5 M, 10 mM Tris, 1 mM EDTA, 20 |Lig/ml RNAse A and 20

U/ml TI were added to each sample and incubated for 30 minutes at 37 ®C. 20 | l l 1 of 10% SDS and 50 pg of proteinase K were added and incubated for another 15 minutes at 37 °C. A phenol:chloroform; isoamyl alcohol extraction was carried out, and the RNA precipitated with ethanol on dry ice. Samples were dried under vacuum and resuspended in 4 pi of formamide dye, boiled 3 minutes and loaded in a 6% polyacrylamide gel.

2.10.4 Probes Probe 2: Nhe I (6937) to Xba I (8298) genomic fragment of Sry Probe D: Sap 62 (Dresser, et al., 1995) Probe Hha: PCR product of pFLAG construct from bp 300 in the fi-actin promoter to bp 8592 in Sry.

54 Chapter 3: iSrv regulation

3.1 Introduction

Sry is an extremely important gene (especially for women!) as it is responsible for initiating male development. One big question is how does Sry carry out this function? It is known that Sry is a transcription factor, so presumably the way it is eliciting male development is by turning on genes and starting a cascade of gene activity leading to the formation of a testis. It is also important to understand how this first trigger of the cascade is being regulated. Therefore, we were interested in finding the regulatory elements which convey on Sry its precise timing and place of expression. Sry must be turned on at about 10.5 dpc by a factor present in both XX and XY genital ridges, although its down regulation around 12.0 dpc could be male specific (Lee and Taketo, 1994). We tried to find regulatory elements in vitro, by mapping the DNAse I hypersensitive sites in the Sry locus; and in vivo by using transgenic mice with reporter genes attached to Sry sequences. We were also interested in understanding why such precise timing of expression is needed. To address this question we tried mis expressing Sry under the control of a constitutive promoter. Apart from being expressed in the developing gonads, Sry is expressed in adult testes, so the regulation of the activation of Sry at this stage was also studied. In addition, we searched the Sry flanking DNA sequences conserved with other genes with the hope of finding specific regulatory elements.

3.2: DNAse Hypersensitive Site Mapping

3.2.1 Introduction

Sry expression is tightly regulated during development. Its transcript is found only in the male genital ridges of 10.5 to 12.0 dpc mouse embryos (Hacker, et al., 1995;Jeske, et al., 1995;Koopman, et al., 1990). We were interested in finding the regulatory elements responsible for this pattern of expression. The initial approach attempted was to map DNAse Hypersensitive Sites (DHSs) in the Sry genomic locus.

It is known that the organization of the chromatin with respect to transcriptionally active and inactive genes seems to play an important role in transcriptional regulation, not only at a structural, but at a functional level. Weintraub and Groudine (Weintraub and Groudine, 1976) showed that chicken globin gene DNA in actively transcribing cells is preferentially sensitive towards digestion with DNAse I and that it is relatively insensitive to this enzyme in non transcribing cells. It was found later

55 that the general sensitivity to DNAse I is not restricted to the coding region of active genes, but extends to their flanking chromatin (Lawson, et al., 1980;Stalder, et al., 1980). Within these regions of general DNAse I sensitivity, some sites hypersensitive to DNAse I digestion have been found. These DHSs were mapped, originally in viral regulatory regions (Scott and Wigmore, 1979;Varshavsky, et al., 1978), and then in cellular promoters (Wu, et al., 1979). In every case the DHSs were found to map to sequences that control the transcriptional activity of neighbouring genes.

Two classes of DHSs have been found, ubiquitous and cell-type specific. It has been proposed that ubiquitous DHSs mark the boundary of an active chromatin segment by isolating other transcription units from the effect of enhancer elements present in this segment (Chung, et al., 1993). Examples of this type of constitutive DHSs have been found in the ends of the Drosophila 87A7 heat shock locus, and in the 5' region of the human, mouse and chicken B-globin gene. The chicken 5' constitutive DHS was found to isolate a reporter gene from the activating effects of a nearby B-globin control region (Chung, et al., 1993). This supported the idea that certain chromatin elements serve to block in one direction the interactions between activators and promoters.

The appearance of cell-type specific DHSs correlate with gene expression. They mark short regions of nucleosome free chromatin 50-400 bp in length, which will be accessible to specific non-histone DNA binding proteins (Emerson and Felsenfeld, 1984;McGhee, et al., 1981). Generally these regions behave as cell-type specific enhancers or locus control regions. An example of this type of DHS can be found in the Thy-1 gene, which is normally expressed in the brain and in the thymus. This gene shows three DHSs, two constitutive ones, and one only present in tissues where the gene is expressed (Spanopoulou, et al., 1988). Mapping of DHSs is therefore frequently used to find cis-regulatory DNA elements in gene loci. We therefore decided to apply this technique to the Sry locus.

56 3.2.2 Results

3.2.2.1 Thy-1 as a positive control

To establish the conditions for the DNAse I hypersensitive treatment, the Thy-1 gene was used as a positive control. Thymus DNA was extracted and digested with increasing concentrations of DNAse I (0-133.3 u/ml). It was then redigested with BamHI and hybridised with a 780 bp probe (thy-lAB) (kindly donated by F. Grosveld's lab), which includes the region from an Apal to a BamHI site (Fig. 3.1). A 1.1 kb band, which is produced by the BamHI restriction site and the DNAse I hypersensitive site, was detected. This revealed the DHS that maps inside exon three (Spanopoulou, et al., 1988).

3.2.2.2 Mapping of Sry hypersensitive sites

As Sry is expressed both in embryonic genital ridges and in round spermatids in the adult testis, we decided to look first for the DHSs of this locus using DNA extracted from adult testis, as more DNA can be obtained from this tissue that from embryonic genital ridges. Adult testes DNA was digested with DNAse in the same way as the thymus DNA. For the detection of Sry DHSs, both testes and thymus DNAs were redigested with Xbal and hybridized with probe 2XN2 (Fig.3.2 B). The Sry locus consists of a unique region surrounded by an inverted repeat and therefore this probe recognises two fragments (a 2.0 kb and a 3.1 kb) simultaneously (Fig. 3.2 A I and II). No DHS could be detected with this probe. Another probe, 5XN3, hybridizes to a 4.7 kb band (Fig. 3.2 A III and IV), which in reality represents two fragments, one on each side of Sry, within the inverted repeat. No DHSs could be detected with this probe either.

3.2.3 Discussion

It had been found that Sry has two different transcripts, one in the embryonic gonads, and another in the adult testes. It is still not known where the adult promoter and its regulatory regions are, but we now know that a transgene of 13.6 kb is able to drive the expression of Sry in adult testis (section 3.32). Therefore, it is assumed that all the regulatory regions which enable Sry to be expressed in the testes must lie within this 13.6 kb fragment. However, using DNA extracted from adult testis we could not detect any DNAse I hypersensitive site within the 7 kb surrounding the Sry ORF.

57 Figure 3.1: Mapping of DNAse I Hypersensitive Sites in the Thy-1 gene.

a) Thymus DNA was digested with increasing concentrations of DNAse I, redigested with BamHI and hybridized with probe thy-l-AB. This revealed a 1.1 kb frament. b) The genomic locus of Thy-1. Exons are depicted as boxes and introns and flanking regions as lines. Filled boxes represent the coding part of the gene. The DHS revealed by probe thy-l-AB is marked by an arrow

58 a)

DNAse U/ml 0 2 4 8 16 32 66.5 133.3 •I

DHS ##*■# (11 Kb)

b) - I DHS «J E III JJV_ n ü

1 Kb thy-1-AB Figure 3.2: Mapping of DNAse I Hypersensitive Sites in the Sry gene.

Thymus and testes DNA was digested with increasing concentrations of DNAse I, redigested with Xbal and hybridized with probes 2XN2 and 5XN3.

A) I and III, thymus DNA; II and IV, testes DNA. I and II, probe 2XN2; III and IV, probe 5XN3.

B) The genomic locus of Sry . The regions of homology with probes 2XN2 and 5XN3 are shown.

59 A I DNAse U/ml 0 2 4 16 32 66 5 133-3 0 2 16 32 66 5 133-3

31 kb

2 0 Kb

IV DNAse U/ml 0 32 66-5 133-3 0 2 16 32 66 5 133-3

., .w

4-7 kb

T - ; - / - " ; f ^ t

-. -,

îw '- .i S'

B

V //> inverted repeat ORF The finding of DNAse I hypersensitive sites has been correlated with regulatory regions. It is speculated that in these regions the DNA has to be accessible to transcription factors that bind to the DNA to activate transcription. These regions are therefore nucleosome-free. Nevertheless, the underlying mechanism by which specific transcription factors interact with the structural features of the chromatin is not yet fully understood. Several reasons for not finding DHSs in the Sry locus can be envisaged.

First, the contribution of DNA from round spermatids to the total testicular DNA used in the analysis could be too low to be detected. The analysis with purified round spermatids could be attempted.

Second, it might be that not all the regulatory regions are hypersensitive to DNAse I digestion. In some genes the transcription factors might not displace the nucleosomes for sufficient time to leave the DNA free to be attacked by the DNAse. A third explanation is that Sry lies in a constituvely "open" region of chromatin so no hypersensitive sites can be detected. However this is unlikely, as a gene lying in such a region would be expected to be highly and/or widely expressed (which is not the case for Sry). A fourth more likely explanation is that the Sry locus in adult testis DNA lies within a "closed" region of chromatin (as would be expected for theSry locus in thymus DNA). The fact that we could not detect any difference in DNAse I sensitivity between testis and thymus DNA indicates that this may be the case. It is known that genes in mature sperm do not show their usual tissue-specific DHSs (Groudine and Conkin, 1985). It seems that as DNA has to be packaged into the sperm head, the usual chromatin structure is lost, acquiring one resembling more the structure of supercoiled DNA. It is not known at which stage during spermatogenesis the normal DNA structure is lost. In the study mentioned above it was shown that the characteristic tissue-specific DHSs pattern is still found in spermatogonia, therefore DHSs are lost somewhere between this stage and mature sperms. Presumably it correlates with the replacement of histones with protamines in spermatids. Sry is expressed in round spermatids so the simplest interpretation of these results is that no DHS were found because the chromatin is already repackaged prior to Sry expression. This should be investigated further.

There is no obvious function to the adult testes transcript, and as it became clear that it used a different promoter compared to the genital ridge transcript, we decided to stop the analysis of the adult promoter and went on looking for regulatory regions of Sry in the embryo.

60 However, analysis of DHSs in genital ridges is hampered by the quantity of material required, which is about 100 |ig of DNA. Around 1 |ig of DNA is obtained from a pair of developing gonads of 11.5 dpc, so a modified version of the DHS assay was attempted. This procedure, which involved the digestion of the DNA with DNAse I, creation of blunt ends on the DNAse treated DNA, ligation of primers to this blunt ends, and amplification of the DNA, proved to be technically difficult. In addition, the inverted repeat surrounding the Sry locus added extra complications to the assay. After several attempts, the experiments were not successful. We decided to stop the in vitro assays and look for the regulatory regions in vivo, using transgenic approaches described in the following sections.

3.3 Transgenic Studies

3.3.1 Introduction

The in vivo approach undertaken to find regulatory elements for Sry expression was the analysis of transgenic mice carrying theE. coli 13-galactosidase (lacZ) gene as a reporter gene under the control of Sry flanking sequences. This approach has been widely used to map cis-acting elements controlling gene expression because it has several advantages over in vitro assays. First, for genes expressed in cells without tissue culture counterparts, (as is the case for Sry), this approach is the only current means of localizing regulatory elements. Second, the use of reporter genes driven by flanking sequences of the gene of interest is the only means of demonstrating that an element specifies expression exclusively in the appropriate cell type and with a correct pattern of expression during development. Third, in cases where the gene of interest is expressed in several different tissues, a transgenic approach is useful to analyze the expression of the gene in different places at the same time. And lastly, many cases have been reported where the cis-acting elements found in tissue culture systems were found to be different from the ones found acting in vivo (Guy, et al., 1996;Hsu, et al., 1995;Tripodi, et al., 1991;Zimmerman, et al., 1990).

The technique of finding regulatory regions with reporter genes consists, in brief, in using all the known flanking sequences of the gene of interest to drive the expression of a gene which is easily detectable, and which is not normally present in the mouse. One of the most commonly used reporter genes is the E. coli lacZ gene because the production of lacZ can be assayed very easily, in situ, as a blue precipitate when X-gal is used as its substrate (Sanes, et al., 1986). Once the correct pattern of expression of the gene of interest is correctly mimicked by lacZ, serial deletions of the sequence driving this expression can lead to the localization of cis-acting elements important for the tissue- specificity and timing of expression of the gene.

61 It had been shown that XX males were produced when a 14.5 kb genomic fragment of Sry was introduced as a transgene (Koopman, et ah, 1991). In this case, sex reversal was found in approximately 25% of the transgene carriers. Therefore, it was assumed that this 14 kb fragment (p741) must contain all the regulatory elements needed for the proper expression of Sry. Four constructs using different fragments of p741 fused to lacZ were used to obtain transgenic mice, pZlO, pZ ll, pLUGl, (Fig. 3.3) and HOXY-8 (Fig. 3.12). pZlO and pZll had been previously constructed in the lab by A. Economou.

3.3.2 Results

3.3.2.1 PZlO construct:

To produce transgenic animals, one cell embryos were collected from fertilized superovulated three week old females. The male pronuclei were injected with the different constructs, and the embryos were transferred to pseudo-pregnant females to function as foster mothers. To produce transgenic lines the embryos were allowed to develop to term and checked for transgene presence by genotyping at three weeks post partumipp). Positive mice from this genotyping are referred to as "founders", from which lines were derived if they were fertile. Offspring from founders or subsequent generations were analyzed at embryonic stages for lacZ expression. To produce transient transient transgenic embryos, the injected one-cell embryos were transferred to pseudo-pregnant females and analyzed at embryonic stages (usually between 11.5 and 14.5 days after injection).

Two lines carrying the pZlO construct were produced (Sophie Vriz, unpublished) and lacZ expression was analyzed. This construct contains the 5’ region of Sry up to the Sph site (8389) in the ORF region, where lacZ has been inserted in frame into the HMG box of Sry (Fig. 3.3). No 3’ sequences of Sry are present. Progeny from both of the lines carrying this construct, pZlO-C and pZlO-L, were analyzed. We expected to detect expression of lacZ with the endogenous pattern of expression of Sry, namely, within the genital ridge between 10.5 and 12.0 dpc peaking at 11.5 dpc. Neither of the two lines showed lacZ expression with this pattern. Line pZlO-C showed ectopic expression of lacZ in the vertebrae, somites, ribs and heart (Fig. 3.4). This demonstrated that the transgene carried a functional copy of lacZ. Line pZlO-L showed no lacZ expression in any tissue.

62 p741 clone which gives sex reversal j4 6 2 5 bp

AAAAA .

7480 8015 10219 12969

lac Z PZlO

lac Z P Z ll 8715 14625

Initiation and end of I AAAAA embryonic transcript P-geo PA pLUG 1 H ORF of Sry

■ HMG box

Fig 3. 3 Schem atic representation of constructs pZIO, pZ11 and pLUG 1 Inverted repeats 6 0

Figure 3.4: Ectopic expression of lacZ in line pZlO-C

12.5 dpc embryos stained with X-gal for lacZ detection left, transgenic; right non-transgenic

64 3.3.2.2 PZll construct:

This construct was made by adding to pZlO sequences from the 3' region of Sry which extend from the second EcoRV site (8715) to the end of the p741 clone (Fig. 3.3). Four transient transgenic embryos were obtained and assayed for lacZ activity at 11.5 dpc. None of them showed any lacZ activity.

One line (pZll-9) was derived with this construct and its progeny analysed at 11.5 dpc and as adults. No activity of lacZ was found by x-gal staining. Alternative assays to test for expression were carried out, namely RT-PCR and RNAse protection. RT-PCR was performed on RNA from embryos of 11.5 dpc lacZ transcripts were detected in all tissues sampled: gonads, limbs and head (Fig 3.5). PCR without reverse transcriptase was performed as a control for DNA contamination and no signal was detected (data not shown). RT-PCR was also performed on RNA from some adult tissues (kidneys, testes and ovaries). In adult tissues the expression seemed more specific, transcripts being detected at high levels in testes, at low levels in kidney and not at all in ovaries or kidneys of transgenic females (Fig. 3.6). The reason for the sex-specific expression in kidneys is not understood.

RNAse protection assays were performed to analyze the transcript (Fig. 3.7). Only XX embryos were used, so no signal from the endogenous Sry gene was expected. A band of 371 bp, which represents full protection of the Sry portion of the probe was found. This indicated that the transgene was expressed. However, if the transgene had been transcribed from the same initiation site as used by the endogenous Sry gene, we would have expected to find a band of 280 bp. As no such band was detected, we can conclude that the transcript did not start at the same site as the endogenous embryonic transcriptional start site, but further 5'. This could compromise use of the normal Sry translational initiation site; for example, an abnormal 5' site could be used. As there are translational stop codons in all three frames preceding the SRY-lacZ ORF, there would be no chance of producing a fusion protein containing lacZ. This could explain why no lacZ activity was detected in the animals carrying this transgene.

65 Figure 3.5: Expression of transgene pZll in embryonic tissues.

RT-PCR was performed on limbs (L), head (H) and gonads (G) of an 11.5 dpc pZ ll-9 transgenic embryo using primers pZ5’ and pZ3’. Hybridization was done using a specific oligo against Lac-Z. Expression of the transgene pZll was detected in all tissues. The negative control without reverse transcriptase showed no signal (not shown).

66 iM

461 bp

H G Figure 3,6: Expression of transgene pZll in adult tissues.

RT-PCR was performed on adult ovaries (O), testes (T) and male and female kidneys using the same primers and probe as in figure 5. Expression can be detected strongly in transgenic testes, at much lower levels in male kidneys, and not at all in ovaries and female kidneys. DNA from transgenic embryos was used as a positive control for the PCR reaction and hybridization (C). +:transgenic; -:non transgenic.

67 K idneys

+ + + " + -+ + + - + -C

461 bp

O O T T T T? ? cf cf Figure 3.7: Analysis of the transcript expressed by pZll transgene.

Upper panel: RNAse protection assays were performed in gonads (G), head (H) and limbs (L) of 11.5 dpc transgenic (+) or non-transgenic (-) female embryos. Probe 2 (see Chapter 2) was used to detect the transgene. A band of 371 bp was detected in all three tissues indicating that the transgene is expressed, although the transcript is not beginning at the normal Sry initiation site. Probe B (see Chapter 2), which protects a band of 200 bp in Sap 62 was used as a loading control.

Lower panel: Schematic representation of the genomic locus of the pZll transgene in the region where probe 2 hybridizes to it. The expected protected fragment sizes of the full protectected probe and the probe hybridizing to the mRNA of the transgene are shown.

68 404 bp

P rob e 2 371 bp

307 bp

242 bp

2 3 8 bp

217 bp

SAP 62 204 bp (200bp)

BspHl EcoRV 7924 8247

Full protected 371 Expected protected fragment (bp) mRNA 280

BspHI Transcript start site EcoRV Xbal 7924 8015 8247 8295 7 ^ 3.3.2.3 PLUGl:

The constructs described previously carried lacZ as a fusion protein, therefore we were concerned that a different transcriptional initiation site could render a premature termination of translation. To avoid this, a new construct (pLUGl) was made in which lacZ carried its own translation initiation signal, stop codon and polyadenylation sites. This was accomplished by cloning lacZ into the first EcoRV site (8244) of Sry. This fragment carries 8.2 kb of the 5’ flanking region of Sry (Fig. 3.3). The fi-geo (B- galactosidase fused to a neomycin resistance gene, neo) gene was used instead of the classical lacZ, because in this way we it would give us the option of isolating Sry expressing cells by their neomycin resistance properties. Four transient transgenic embryos were assayed at 11.5 dpc, but they failed to show any B-galactosidase activity. Ten founders were obtained bearing this construct. Four of them were XX females (VPLA, VPLB, VPLC and VPLD). VPLD did not transmit the transgene to progeny and VPLA died before producing offspring. The other six founders were XY males (VPL14, VPL17, VPL54, VPL43, VPL44 and VPL47), of which VPL47 did not transmit the transgene to its offspring. None of the seven transmitting lines produced the endogenous pattern of expression of Sry by X-gal staining.

Embryos from line VPL54 (Fig. 3.8) and VPL44 (Fig. 3.9) were assayed by RT-PCR for expression of the transgene. Each embryo was assayed individually. The transgene was found to be expressed in the genital ridges at 11.5 dpc. We performed RNAse protection assays on embryos of lines VPL54, VPL44 and VPL17 (Fig. 3.10). Four pairs of genital ridges or four limbs of transgenic animals were used for each lane. We were unable to detect pLUGl expression by this method, although endogenous Sry transcripts were detected, as can be seen by the faint band appearing at the 280 bp position. The bands appearing around 250 and 370 bp seem to be background bands as they appear in all tissues of both transgenic and non-transgenic animals. One line presented ectopic expression of lacZ (VPL43), revealing that the B-geo gene used in these lines is functional (Fig. 3.11). We speculated that the level of expression of the transgene was extremely low since expression could not be detected by RNAse protection assays, but could be detected by RT-PCR which is a more sensitive assay. Perhaps lacZ activity was not detected as expression was just too low. Figure 3.8: Expression of pLUGl transgene in VPL54 line.

RT-PCR was performed on 11.5 dpc genital ridges of a VPL54 male transgenic embryo using primers pZ5A and pZ3’ to detect the transgene; primers Y11A2 and Y11B2 to detect the endogenous Sry; and primers HPRTIA and HPRTIB to detect Hprt. A band of 461 was detected with primers pZ5A and pZ3’ showing expression of the transgene. Hprt was used as a positive control for RNA recovery.

70 Q.CO I pLUG1 461 bp

Hprt 352 bp

Sry 340 bp Figure 3.9: Expression of pLUGl transgene in VPL44 line.

RT-PCR was performed in heads (H), limbs (L) and gonads (G) of 11.5 dpc embryos using primers pll94.1 and pZ3' for the pLUGl. A 555 bp band shows expression of the transgene in limbs and gonads of male embryos and in gonads of female embryos. +:transgenic; -:non-transgenic

71 H L G H L G

L G H L G

555 bp 555 bp

? - + Figure 3.10 : Analysis of expression of pLUGl transgene in 11.5 dpc genital ridges. Upper panel: RNAse protection assays were performed on 11.5 genital ridges of male and female 11.5 dpc transgenic embryos of lines VPL54, VPL44 and VPL17. Non transgenic male and female 11.5 dpc gonads (-) were used as negative controls. Probe 2 was used to detect transcripts from both theSry gene and the pLUGl transgene. A band of 280 bases was detected in the male gonads indicating the expression of the endogenous Sry gene. Probe D (see Chapter 2) was used to detect the Sap 62 gene as a loading control, producing a band of 143 bases. No bands revealing the expression of pLUG were found. Lower panel: Schematic representation of the genomic locus of Sry and the pLUGl transgene in the region where probe 2 hybridizes to them. The full length protected probe gives a fragment of 371 bp. The endogenous Sry gene gives a protected band of 280 bases. The pLUGl transgene should give a protected band of 232 bases if the transcript is initiating in the same site as the endogenous Sry transcript. It could give a protected fragment of up to 323 bases if the transgene is expressed, but using an upstream initiation site.

72 VPL54 VPL44 VPL17

bp ? c7 (f' ? c7’??c7

404

307

Endogenous 280 Sry

242 238 217

204 190 180

160

147 SAP 62 (143 bp)

BspHI Transcript EcoRV Xbal 7924 8015------8244 8294 0 + 1 1----

Sry Endogenous mRNA |- 280 232 Expected pLUG mRNA Transgene; ------y protected 323 fragment Full protected probe [■ 371

BspHI EcoRV 7924 8244 6-oeo

\ Figure 3.11: Ectopic expression of lacZ in line VPL43

12.5 dpc transgenic embryo stained for lacZ activity.

73 3.3.2.4 HOXY-8

Another method used to identify tissue specific enhancer elements consists in using a “minimal promoter” fused to lacZ. A minimal promoter is by itself unable to drive the expression of lacZ, but will do so if an enhancer element is included nearby. Expression of lacZ will, in addition, be in accordance with any tissue specificity provided by the enhancer. This is particularly useful when the promoter of the gene of interest is a weak one. Therefore, we attempted to use this strategy using the Hox-B2 minimal promoter. This promoter has been well characterized by R, Krumlaufs laboratory, and when used on its own, only gives a minimal basal expression in the superior colliculus of the brain. The first 8.2 kb fragment of Sry (up to the 8244 EcoRV site) was fused to the Hox-B2 minimal promoter driving lacZ (pHOXY-8, Fig. 3.12). Eight transient transgenic embryos were assayed at 11.5 dpc but none of them showed expression of lacZ in the genital ridges. The basal expression of the head was detected in three of the embryos (although at low levels), showing that the gene was functional (data not shown).

74 i§ &

14.6 kb Sry clone 8244 14625

Lac Z HB2 pHOXY-8 8244

^ open reading frame of Sry

I5 I Hox B2 min prom

r*" Direction of transcript

Figure 3.12 Schematic representation of pHOXY-8 3.3.2.5 Adult Promoter

It has been found that Siy produces two different transcripts, one in the embryonic gonads and another in the adult testes, specifically within round spermatids (Hacker, et al., 1995). The initiation of transcription of the adult transcript has not been yet identified but it is known that it starts somewhere upstream of the embryonic transcript (Hacker, et al., 1995). It has been shown that the adult transcript of Sry is spliced to give a circular transcript (Capel, et al., 1993), which is unlikely to be translated as it is not found attached to polysomes.Sry is expressed in round spermatids in the testis. In an attempt to find regulatory regions of the embryonic transcript a search for homologous regions was made using the EMBL on-line FASTA program (Pearson and Lipman, 1988).

A region of high homology (83% in 165 bp) was found between bases 615 to 780 of Sry and bases 910 to 1070 of the gene for the Mus musculus testis-specific somatic Cytochrome C gene(Hake and Hecht, 1993)(Fig. 3.13). Cytochrome C is expressed in all cells, nevertheless, it has been found that this gene has a testis-specific mRNA. This specific mRNA is only produced in round spermatids of the spermiogenic phase. As both Sry and the Cytochrome C genes seem to be transcribed at the same spermiogenic stage, it was possible that this highly homologous region could be involved in the testis-specific activation. In order to determine if this region was necessary for the production of Sry in round spermatids, we produced transgenic mice lacking the first 880 bp of the 14.5 clone containing Sry. This new clone (741.mutB, Fig. 3.14) was engineered as to carry a conservative mutation in the ORF of Sry (an A was changed to a C to produce a PstI site at position 8423) to allow an easy way to distinguish the endogenous Sry from the transgene. Five transgenic lines were produced with this construct. Two of them showed XX female to male sex-reversal. Line 741.mutB-1467 has a complete penetrance (all XX transgenic mice develop as males) whereas line 741.mutB-1474 sex reverses XX individuals around 60% of the time. This showed that the transgene was still able to express the embryonic transcript in a functional manner. To see if the adult transcript was still produced, RT-PCR was performed on adult transgenic testes with two primers (Y41A and Y11B2) which only detect the circular transcripts produced in round spermatids as they are in opposite orientation with respect to each other. Digestion of the PCR product with PstI was performed in order to distinguish between the endogenous and transgenic transcripts. We were able to detect the circular transcript originating from the transgene (Fig. 3.15), so it was concluded that the first 880 bp, containing the

76 region of high homology with testis-specific Cytochrome C were not necessary for the transcription of Sry in the adult testes.

77 ID MMTSCYTC standard; DNA; ROD; 2020 BP. AC L00605; DT lO-MAY-1993 (Rel. 35, Created) DT 2 0-MAY-1993 (Rel. 35, Last updated. Version 3) DE Mus musculus testis-specific somatic cytochrome C gene, 5 flank KW cytochrome c; testis-specific mRNA. . . .

SCORES Initl: 354 Initn: 354 Opt: 417 83.0% identity in 165 bp overlap

800 790 780 770 760 750 741Sry CACAGGTCTTCTAGATTCTACCATGTTGACTTGAGATGCCATTATTTTTTTAAAGTTCAG III mill I I iiiiiii Mill Mmtscy AATAGATAGAGCAATGGCTTTGAGATAGGGTTGGGATGCC--GAGTCTTTTAAA-TTCAG 880 890 900 910 920 930

740 730 720 710 700 690 7 41Sry ACTCCATTTTAGAGAACCTGACCTAAGTTTGAACTCAGGTAAACTAACAGGCTGATACCT llllllllllllllllllllllllllllllllllllllllllllllllllllll Mill Mmtscy ACTCCATTTTAGAGAACCTGACCTAAGTTTGAACTCAGGTAAACTAACAGGCTGGTACCT 940 950 960 970 980 990

680 670 660 650 640 630 741Sry AGCATTAGTTTCCAAGTTGCCTCCCAACA-AAACCTGAGCCAAGCTCCAGTCTCTAGGCT MMMMMMMMMMI Mill I Mill MMM Ml I II II Mmtscy AGCATTAGTTTCCAAGTTGCCCCCCAAAAGAAACCCAAGCCAA-AACCAAACACTCCCCT 1000 1010 1020 1030 1040 1050

620 610 600 590 580 570 7 41Sry AATCCCCACCAAGACCAGAGTTTCCAGGTTACACCCTCAAGAAAACCAGCATCTCTAGGT M M M I I I Mmtscy --CCCCCACATACACACATAAAACACAAACAAGAGCAACTCACAGCCAACTCGTTTATAA 1060 1070 1080 1090 1100 1110

Figure 3.13: Region of homology between Mus musculus testis-specific somatic cytochrome C gene and Sry.

78 880 BamH 1 8244 EcoR V 8712 EcoR V I 3 ] p741. mut t PST I 4 ~ l T p741. mut B

I = Region of homology between cytochrome C and Sry Q = ORF of Sry

Figure 3.14 : Schematic representation of p741.mut B

Sry genomic locus showing region of homology with testis-specific cytochrome C The arrow indicates the position in which an A was changed to a C to create a Pst I site Figure 3.15: RT-PCR on adult testes of transgenic line 741.mutB-1467

RT-PCR was performed using RNA from adult testes of transgenic (+) and non- transgenic (-) mice, using primers Y41A and Y11B2. After the PCR reaction, the DNA was digested with Pst I in order to distinguish between the endogenous and the transgenic transcripts. 397 bp Endogenous 240 bp Transgene 3.3.3 Discussion.

In an attempt to find the regulatory elements of Sry we used transgenic mice carrying lacZ as a reporter gene under the control of some of its flanking sequences. A transgenic line carrying the pZll construct (8.2 kb of 5' sequences and 5 kb of 3' sequences) failed to show expression of lacZ in the genital ridges (as assayed by X-gal staining). This construct had lacZ inserted into the HMG box of SRY and was predicted to produce a fusion protein. It was found by RNAse protection assays that this transgene is expressed at low levels, apparently using a transcriptional start site 5' to the endogenous Sry embryonic transcriptional start site. The use of a different initiation site could account for the failure to detect lacZ protein (as assayed by x-gal staining) as it might give a truncated protein or no protein at all.

In order to avoid the possibility of “out of frame” translation, a construct with 8.2 kb of Sry 5’ flanking sequences attached to a lacZ gene carrying its own ATG and Kozac consensus sequence (pLUGl) was used. No sequences from the ORF of Sry were included in this construct. Seven transgenic lines were derived. pLUGl was unable to drive lacZ expression in the genital ridges as assayed by X-gal staining in any of these lines. No expression of this transgene was detected by RNAse protection assays either, but its expression was detected by RT-PCR, indicating that the expression of pLUGl is very low.

As the fusion of sequences to a minimal promoter has proven quite useful for the recognition of regulatory elements, this method was also tried. 8.2 kb 5’ sequences of Sry were fused to a HoxB2 minimal promoter/ lacZ construct (HOXY-8) and 8 transient transgenic embryos were obtained. None of the embryos showed any lacZ staining except for the constitutive staining given by the HoxB2 minimal promoter in the head.

In conclusion, it seems that lacZ is not a good reporter gene in the context of Sry sequences.

Why did we choose to use a reporter gene, rather than a functional test of Sry itself, i.e. sex reversal? The decision to not use sex-reversal was made considering several factors. It is known that the level of Sry expression is critical for its function. It has been shown for one of the Sry transgenic lines (32.10) that the Sry transgene in a CBA background is expressed at about 40% of the endogenous level (Hacker, et al., 1995). This is likely to account for the observed incomplete penetrance of sex-reversal. It is also known that when a Y chromosome from certain Mus musculus domesticus strains

81 (ydoni) jg introduced into the C57BL/6J strain, it causes XY sex reversal. Different threshold levels required to trigger male testis development have been implicated in this phenotype. Another observation is that XY females carrying Y deletions, but an intact Sry gene sometimes express low levels of Sry. Apart from levels of expression, genetic background seems to affect the sex-reversal phenotype as seen inSry transgenic animals with different genetic backgrounds (Nigel Vivian and Adam Hacker, unpublished results), and in the effect of the Ydo^n different strains. Therefore, lack of sex-reversal does not necessarily mean that Sry is not being expressed. Besides, in transgenic experiments it has been shown that the smaller the fragments, the more likely it is for them to be subject to position effects. This would be very difficult to control in deletion experiments and would necessitate deriving huge numbers of transgenics to be confident that a particular construct was not able to give sex-reversal. On the other hand, if elements required for "high" expression levels were removed, sex reversal would be lost, but tissue-specific elements may be missed.

The decision to use lacZ as a reporter gene was made on the following basis. With lacZ staining one can survey many tissues at once, and see more easily whether the transgenic mice have ectopic expression unique to an insertion site, or whether they have consistent ectopic expression due to removal of a negative regulator element. The lacZ enzyme is quite stable and is thought to persist for more than 24 hours after transcription has stopped. We suspect Sry is expressed in the precursors of Sertoli cells, but by the time Sertoli cells can be recognized, they no longer express Sry. The persistence of B-galactosidase would solve this problem. Also, it is very likely that Sertoli and follicle cells derive from the same cell lineage. If follicle cells show B-gal activity, then this would provide formal proof of this notion. The use of B-geo would allow further experiments. Genital ridges derived from mice carrying the T-antigen as a transforming gene and Sry-Bgeo would allow for the selection of cell lines capable of expressing Sry.

Considering all this, it was very unfortunate that the strategy failed. There are several reasons which may account for this.

First, we know that the endogenous level of Sry is low, so the low level of expression of the transgene might just be reflecting this.

Secondly, the genomic structure of Sry suggests that it might have complex regulation. For example, the unusually long 3’ UTR of Sry might indicate that this is a very unstable transcript, as destabilizing sequences are usually found in the 3' UTR. Indeed, the Sry 3' UTR includes a region with four ATTTA sequences often associated with message instability (Hacker, et al., 1995). There is now a great deal of evidence

82 that the 3’ UTR are extremely important in the regulation of genes (for review, Decker and Parker, 1995;Jackson, 1993). For example, 3' UTR can confer appropriate temporal regulation of translation, as in the case of the mRNA of the protamine-1 gene (Braun, et al., 1989); they can regulate polyadenylation, which is itself involved in translation, as in the case for sequences found in the 3 UTR of the mRNA of the C12 gene of Xenopus (Simon, et al., 1992); or they can contain binding sites for repressors, which need not necessarily be proteins but can be short RNAs, as in the case of the C. elegans lin-14 gene (Lee, et al., 1993;Wightman, et al., 1993). It could also be that regulatory elements exist within the ORF of Sry. These elements might bind activators of transcription, or function as stabilizers of the mRNA, increasing the level of transcription. Without these elements, the overall expression of Sry might be very inefficient. There have been reports in which regulatory elements have been found in the ORF of mRNAs (reviewed Sachs, 1993). Using all the known sequences of Sry in the reporter construct might override this problem. However, the long inverted repeat regions of Sry make the manipulation of such constructs difficult, as they tend to rearrange at high frequency. pZll did contain all such sequences, but it was a nightmare to work with and showed incorrect initiation of transcription.

A third explanation for the problem we experienced might be that the lacZ gene disturbs the spatial arrangement required for the interaction of different regulatory elements. This would certainly be the case if interaction between the two sides of the inverted repeat are needed. In a recent report (Guy, et al., 1996) it was shown that lacZ interferes with the position independent expression normally conferred by the locus control region (LCR) of the 8-globin gene, even when all the fi-globin gene is present in the construct driving lacZ . It was suggested that the presence of lacZ impedes the chromatin opening activity of the LCR structure because lacZ sequences (which are prokaryotic) may not allow passage of the necessary alteration in chromatin structure. The other explanation is that the lacZ gene is disturbing the spatial arrangement of sequences required for correct regulation.

Finally, it might be that lacZ sequences interfere directly with the activity of the Sry promoter itself. There have been several reports in which such a mechanism has been implicated in the failure of the detection of lacZ expression. It has been shown that the 3- hydroxy-3-methylglutarylCoA reductase promoter, which is normally an ubiquitous and constitutive promoter, loses this property when driving the lacZ gene. This does not happen when driving the chloramphenicol acetyltransferase (cat) gene (Paldi, et al., 1993). In this study, it was also shown that the lacZ construct interfered with the expression of the cat construct when both constructs were integrated at the same locus. They propose that lacZ might contain sequence motifs which act as weak

83 transcriptional silencers. This has also been proposed for the neo gene when used in mammalian cells (Artelt, et al., 1991). These reports show that prokaryotic sequences might not be absolutely compatible with eukaryotic gene expression, especially if used with weak promoters. As both lacZ and neo have been shown to decrease expression, using both in the same construct may indeed be suicidal!

Using a different reporter gene could be useful in future experiments. A commonly used reporter gene is that for human placental alkaline phosphatase. However, germ cells express high levels of alkaline phosphatase so in our case, there could be background problems that would make the recognition of the transgene expression more difficult. The use of cat as a reporter gene is also very common, but it has the disadvantage that “in situ” assays for the gene product are not very well established.

The most promising reporter gene now available seems to be the “green fluorescent protein” (GFP) (Prasher, et al., 1992). This protein, derived from the jellyfish Aequorea victoria has been successfully used recently in several organisms, including mouse (Amsterdam, et al., 1995;Chaffie, et al., 1994;Ikawa, et al., 1995;Yeh, et al., 1995). This reporter gene has the great advantage that it does not require exogenously added substrates so it can be detected in living cells. Besides, the GFP gene has recently being engineered in order to give different fluorescent colours with improved brightness (Heim and Tsien, 1996). This could allow for the simultaneous detection of several transgenes. Another way of obtaining a reporter gene is by tagging the endogenous gene with an epitope (see next chapter), and then following expression with an immunoreactive assay specific for the tag.

Ironically, the only experiment that yielded positive results was one where the 14 kb fragment was slightly reduced in size and no reporter was used. This experiment was carried out to look at the role of a sequence at the 5' end in the expression of the adult testis transcript. For this we could not risk disrupting the Sry sequence with a reporter, and used a construct engineered with a point mutation to distinguish between transgenic and endogenous expression. The expected band for the transgene was found indicating that the first 880 bp were not necessary for either adult testes or genital ridge expression.

84 Chapter 4: Srv mis-expression

4.1 Introduction

Transgenic studies have shown that Sry is the male trigger (Koopman, et al., 1991), but it is not yet known how it accomplishes this task. A striking characteristic of the expression of this gene is that it is only expressed for a brief period of time (10.5-12.0 dpc) in the genital ridge. In order to address the question of how Sry works, we were interested to know what would be the consequences of expressing Sry beyond its normal period of expression, as well as the consequences of mis-expressing it in other tissues. For these purposes, constructs were made using a constitutive promoter from the gene B- actin driving the expression of Sry.

4.2 Results

4.2.1 pFLAG

It has been difficult to raise good antibodies against SRY so a highly immunoreactive epitope (FLAG), which consists of six amino acids (Asp-Tyr-Lys-Asp-Asp-Asp) (Hopp, et al., 1988) was fused to the 5’ end of the SRY ORF to facilitate the detection of the protein with antiFLAG antibodies. A construct using the human B-actin promoter driving the FLAG-SRY protein was made (Amanda Swain, unpublished). This construct was named pFLAG (Fig. 4.1).

Eight transient transgenic embryos carrying pFLAG were obtained with this construct and analysed at 14.5 dpc. Five of the embryos were XY and three XX. Their phenotypic sex corresponded to their genotype irrespective of the transgene and no abnormal phenotypes were observed.

Three live born transgenics were obtained with this construct: FLAG-4 and FLAG-5 were normal fertile XX females, FLAG-2 was an XY male . The latter was sterile, completely lacking sperm.

85 o>g § i Oi co (i-actin promi SRY H f ] pFLAG

li-actin prom SRY pVACS

FLAG

HMG box

Figure 4.1 Schematic representation of FLAG and VACS constructs RNAse protection analysis was performed on RNA extracted from whole embryos (Fig. 4.2) and from genital ridges, limbs and tails of 11.5 dpc embryos of lines FLAG-4 and FLAG-5 (Fig. 4.3). A 400 bp protected band indicated that the transgene was expressed. Western analysis showed that a low level of protein was produced in 11.5 dpc gonads of embryos from both lines (Fig. 4.4). We have thus shown that the pFLAG construct, which contains the ORF of Sry under the control of B-actin promoter, is expressed in genital ridges, limbs and tails of 11.5 dpc embryos of two different lines, and that it is capable of producing low levels of protein.

No abnormal phenotype was apparent in animals from the two transgenic lines, or in any of the eight transient transgenic embryos. There was also no sign of sex-reversal to give XX males, as might be expected, or XY females. The latter is conceivable if the inclusion of the FLAG epitope into SRY interfered with endogenous SRY action (i.e.. acted as a dominant-negative). It seems most probable that the level of transgene expression was too low to have any phenotypic consequence. In the case of the FLAG-2 sterile transgenic male, it is conceivable that SRY protein from the transgene was interfering with spermatogenesis. While Sry is normally expressed in spermatids, it is the circular transcript which is produced and thus is probably not translated (Capel, et al., 1993). Indeed, the peculiar organization of the Sry locus which gives rise to the circular transcript could be maintained specifically to prevent SRY protein from being made during spermatogenesis if it is deleterious to this process. On the other band, sterility is frequently a consequence of transgene insertions. This can be due to interruption of the endogenous gene or, more likely, to chromosome breaks or translocations induced by physical damage during pronuclear injection.

87 Figure 4.2: Analysis of expression of the Flag transgene in whole embryos.

Upper panel:RNAse protection assays were performed for the detection of the Flag transgene. RNA from whole embryos was used. Probe Hha was used revealing a protected fragment of 400 bp demostrating that the transgene is expressed in these embryos. Probe A (see Capter 2) was used to detect Sap 62 as a loading control.

Lower panel: A schematic representation of the genomic organization of the Flag transgene in the region where probe Hha hybridizes to it. " b 04- D) O) e 03 03

622 527

^ ^ Flag transcript 404 (400 bp)

307

248 _____ 242 # # a - SAP 62 (227 bp) 217 f*f P-actin promoter Flag Sry ORF ______A ______A ______A ___

Hha . -/ ,xJ______c_____ -✓A 398 479 8294 8672

Flag Transcript I ------1 400 Probe Hha i ------1 481 Figure 4.3 ; Analysis of expression of Flag transgene. RNAse protection assays were performed to analyze the expression of the Flag transgene. Probe Hha was used and a protected fragment of 400 bases was detected confirming the expression of the Flag transgene in the two lines analyzed. Probe sapD, which protects a fragment of 227 bases was used to detect Sap 62 as a loading control. The lanes are; 1) 13.5 dpc limb of a male non-transgenic embryo; 2) 13.5 dpc limb of a male of a Flag5 embryo; 3) 13.5 dpc head of a male Flag5 embryo; 4) 13.5 dpc testes of a Flag5 embryo; 5) 13.5 dpc limb of a female Flag5 embryo; 6) 13.5 dpc head of a female Flag5 embryo; 7) 13.5 dpc ovaries of a Flag5 embryo; 8) 11.5 dpc gonads of a Flag4 male embryo; 9) 11.5 dpc gonads of non-transgenic embryo; 10) 11.5 dpc gonads of a Flag4 male embryo; 11) 11.5 dpc gonads of a male Flag4 embryo.

Lanes 4 to 11 are an overexposed photography.

A schematic representation of the genomic organization of the Flagl transgene in the area where probe Hha hybridizes is shown in Fig. 4.2. 1 2 3 4 5 7 8 9 10 11

bp

622 527

FLAG transcript 404 (400 bp) I * 307 #*

248 Sap transcript 242 (227 bo) t M* 217 Figure 4.4: Analysis of protein expression in Flag4 and Flag 5 lines.

Western analysis were performed to detect expression of protein from the Flag transgene. Flag antibody was used to detect the protein expressed in Flag4 and Flag 5 11.5 dpc embryos. Cos cells transiently transfected with the Flag construct were used as a positive control (C). A) A faint band migrating at the same position as the positive control was found in the Flag4 transgenic embryos and not in the non-transgenic (-) embryo. B) A faint band migrating at the same position as the positive control was found in FlagS transgenic embryo and not in the non-transgenic one.

90 Î Flag 4 Flag 4

Flag-Sry protein

B Flag 5

m

Flag-Sry protein

% 4.2.2 PVACS

As the FLAG epitope was inserted very close to the HMG box of SRY, (two amino acids away), we were also concerned that it might be interfering with SRY function. Therefore, a new construct was made without the FLAG epitope (pVACS). An Sry fragment from the Xbal site at position 8294 to the Spel site at position 9703 was cloned directly downstream of the 13-actin promoter (Fig. 4.1). Three transient transgenic embryos were obtained and analyzed at 13.5 dpc. Two of them were XY and one XX. Their phenotype corresponded to their genotype and no abnormal development was seen. Four live born transgenics were obtained carrying this construct (VACS-11, -27, -44 and -84). VACS-11, -27 and -44 were XY males and VACS-84 was female. VACS-84 was completely normal, fertile, and gave rise to normal offspring. VACS-11 and -44 were also completely normal and produced normal offspring. VACS-27 had growth problems, being very small with respect to its litter mates (Fig. 4.5). It died at three months without giving offspring. As there were three lines and three transgenic embryos with no abnormal phenotype this growth phenotype is most likely due to an insertion effect. We did not observe any sex-reversal in XX transgenic embryos derived from lines VACS-11 and -84. We used RT-PCR to look for the expression of the transgene in the genital ridges of 11.5 dpc embryos and found it to be expressed (Fig. 4.6). As we do not have antibodies against SRY protein, we can not be absolutely certain that any protein was being produced from this construct. Nevertheless, as the pFLAG construct has the same promoter as the pVACS construct, and is able to produce a low level of protein, we assumed that the pVACS construct would behave in the same way. Again, we do not know if the phenotype observed in VACS-27 is an insertional effect or attributable to SRY. This mouse did not have sperm either, but considering its impaired development, this could just be a side effect.

91 Figure 4.5 Transgenic male VACS-27, carrying construct pVACS.

VACS-27 had growth problems as can be seen when compared with one of its litter m ates.

92

Figure 4.6: Expression of VACS and Flag transgene.

RT-PCR was performed on head(H), limbs(L) and gonads (G) of 11.5 dpc embryos transgenic for VACS or Flag construct with primers BACTIN5' and SRYRV3' producing a fragment of 404 bp. Expression of the transgene can be detected in all tissues sampled. S84: line VACS-84; S ll: line VACS-11; F43: line FLAG-4

93 ÇS84 9S11 9F43

H L G HLGHLG

404 4.3 Discussion

One of the consequences expected for the mis-expression of Sry in XX genital ridges is that they should develop as testes. This was not the case. Several explanations might account for this result. First, even in this case where we used a known strong promoter to produce SRY, the levels of expression and protein production seem to be low. This supports the idea that Sry regulation is complex and that maybe the undisturbed locus is required for normal expression. The protein levels could simply be too low for it to function properly. Second, evidence suggests that Sry is expressed in the Sertoli cell lineage. We do not know that the transgene is being expressed in the correct cell type, or if it is being localized in the correct compartment of the cell (the nucleus, as expected for a transcription factor). Third, adding the FLAG epitope might have changed the structure of the protein and/or its localization signals. In the case of the pVACS construct, which does not carry any modification of the SRY protein, we expected to avoid this third problem; nevertheless it did not present any consistent phenotype. There was also the possibility that the FLAG epitope might be interfering with SRY function. It has previously been shown that SRY is able to activate transcription of a reporter gene in vitro (Dubin and Ostrer, 1994). We now know that the SRY protein with the FLAG epitope is still able to activate transcription of this reporter gene in vitro (Isabelle Bar, personal communication). Therefore, at least in vitro, as the FLAG epitope does not seem to interfere with the binding of SRY to DNA, this explanation is not very likely.

Another explanation for the lack of any observable phenotype is that embryos expressing sufficient SRY to function are dying early in gestation. Similar experiments to ectopically express theSry-related gene Sox-2 showed that this could severely disrupt embryogenesis (Larysa Pevny, personal communication). There are different ways to avoid this. One is to use a promoter that directs Sry expression to a particular tissue instead of using a general ubiquitous one. The use of the promoters for WT-1, Amh orDax-1 (see Chapter 5) would direct Sry expression mainly to the developing gonads, but would misexpress it at different stages. Another way to ectopically express a gene, but in a more controlled way, is to use a transgenic binary system. This method makes use of a transactivator gene under the control of a tissue specific enhancer and a transresponder gene which controls the expression of the gene of interest. Each of these transgenes is carried in two separate transgenic mice. Only offspring which carries both of these transgenes will express the gene of interest in the tissue specified by the transactivator. For this purpose, VP16, a protein from the herpex

94 simplex virus which activates transcription by interacting with a short sequence termed IE response element has been used to produce mice with temporal and spatial regulation of a transgene (Gardner, et al., 1996). Other methods make use of the bacteriophage PI Cre recombinase which promotes both intra- and intermolecular synapsis and recombination of DNA (Sauer and Henderson, 1988). Recombination occurs at a specific 34-base-pair site, called loxP, and does not require any other protein factors. Any sequence surrounded by two direct repeats of loxP sites, will de deleted by the recombination promoted by Cre. Therefore, if the gene of interest is inactive because of the insertion of sequences flanked by two lox sites, its activation will occur only where Cre is expressed.

95 Chapter 5; Dax-1 regulation

5.1 Introduction

In mammals, both XY males and XX females have only one transcriptionally active X chromosome thanks to the dosage compensation system which inactivates any extra X chromosomes present. However, it is possible to have a double dose of some X-linked gene products in cases where regions of the X, that do not include the X-inactive centre, are translocated to autosomes or when there is an intra-chromosomal duplication of such regions. In humans, duplications of the short arm of the X chromosome (Xp), have been correlated with XY male to female sex-reversal (Bardoni, et al., 1994;Ogata, et al., 1992). It has been suggested therefore that double dosage of a gene (or genes) contained within the duplicated region might be interfering with the male determining pathway. The duplicated locus has been called DSS for 'dosage sensitive sex-reversal'. Absence of this locus does not seem to interfere with testis formation, as XY patients with deletions including this region develop as males. Functional deficiency of a locus that maps in the same region as DSS correlates with the X-linked adrenal hypoplasia congenita (ARC) disorder, which is characterized by the absence of the permanent zone of the adrenal cortex and by hypogonadotropic hypogonadism. Searching for expressed sequences in this locus, several genes were found. Among them was the gene DAX-1 (for DSS-AHC critical region of the X chromosome). Inactivating point mutations within DAX-1 or deletions of the locus have been found in ARC patients showing that DAX-1 is the gene responsible for this syndrome. DAX-1 encodes a protein with three and a half repeats of novel DNA-binding domain followed by a domain which shows approximately 50% continuous similarity to the ligand-binding domain of the nuclear receptor superfamily (see Chapter 1, Fig. 1.9). As both the adrenals and the gonads have a common origin, DAX-1 has been proposed to be the DSS (Zanaria, et al., 1994).

Mouse Dax-1 has been recently isolated and its pattern of expression characterized (Swain, et al., 1996). Mouse Dax-1 contains one single long open reading frame with two possible start sites. RNAse protection assays showed that only the second ATG was used. This corresponded exactly to the start site of human DAX-1. Mouse Dax-1 encodes a protein of 472 aa, two aa more than human DAX-1. It has a single intron of approximately 2.5 kb which is located in the same relative position as in human DAX- 1. The overall conservation between human and mouse genes is low (75.5% nucleotide sequence identity and 66% aa identity. Chapter 1, Fig. 1.9).

96 Radioactive and whole-mount in situ hybridization showed that Dax-1 is expressed, as would be expected for a gene responsible for AHC, in the developing adrenals, specifically in the cortex. This is the affected area in AHC patients.

Expression of Dax-1 was also found in the developing hypothalamus and anterior pituitary. It is not known if HGG, which is commonly associated with AHC, is from hypothalamic or pituitary origin. As Dax-1 is expressed in both of these tissues it is possible that deficiency of DAX-1 is the responsible for this syndrome as well.

Dax-1 was also found expressed in the developing gonads with a sex-specific pattern. It is first expressed in the somatic component of the genital ridge at 10.5 dpc in both males and females, and its expression can be clearly seen at 11.5 dpc still in both males and females. After 12.5 dpc, the expression of Dax-1 becomes sexually dimorphic. In males, the levels of expression decrease dramatically, with the exception of a strong positive group of cells lying at the junction between the gonad and the mesonephros. In the female, Dax-1 continues to be expressed throughout the gonad after 12.5 dpc, but over the next two days it becomes restricted to the region proximal to the mesonephros as in the male but in a much wider region. The difference in levels between males and females was confirmed by RNAse protection assays. In the adult testis, expression of Dax-1 is restricted to Leydig cells and in the adult ovary, it appears to be in stromal cells, which may include theca cells. It was found not to be expressed in follicle cells or in the luteinising cells of Graffian follicles. This pattern of expression strongly suggests that Dax-1 might indeed be DSS. To test whether this is so, we sought to introduce Dax-1 as a transgene in mice, therefore mimicking the double dosage of DSS in humans, and looking to see if this caused XY male to female sex-reversal. As the transcriptional regulatory regions of Dax-1 were not known, we first had to define the regions needed to target Dax-1 expression to the gonads. In this way we would be sure that the transgene would be expressed in the correct fashion.

5.2 Results

To find the regulatory regions of Dax-1, we applied the same strategy used for the Sry promoter. Several constructs harbouring different lengths of flanking Dax-1 sequence were used to drive the expression of lacZ in transgenic mice (figure 5.1). All the constructs used in this and the next chapter were made by Amanda Swain, and Robin Lovell-Badge helped with some of the pronuclear injections of the next chapter.

97 A N5V Vi

N v : y _ i ___ I I I 4 È - /y Mouse Dax- Genomic Locus

Lac Z D-12/Z

Lac Z D-6/Z

L acZ D-4-5/Z

Lac Z D-3/Z

F(-18~^-4)

i F(+0.7->+13)

V ------—V----- 2 5 Kb 9 Kb

PA D-12/cDax Exons Rabbit p-globin intron and Poly A

Figure 5.1: Schematic representation of Dax-1 costructs used In transgenic mice 5.2.1 D-12/Z

This construct has the region from -12 to + 0.7 kb of Dax-1 and the lacZ gene such that the resulting protein is a fusion between DAX-1 and b-galactosidase. Five transient transgenic embryos were analysed at 11.5 dpc. Two of them showed lacZ expression in the genital ridges (D-12/Z-tl and D-12/Z-t2, Fig. 5.2). D-12/Z-tl showed weak expression of lacZ in the gonads and ectopic expression in the spinal cord, head and tips of the limbs (Fig 5.2-A). D-12/Z-t2 is shown in figure 5.2-B, where lacZ expression can be seen in a few cells of the genital ridge. Four founders were derived with this construct. D-12/Z-1322 and D-12/Z-1581 lines were derived from a male founder while lines D-12/Z-1317 and D-12/Z-1608 were derived from females. The expression of the transgene during development was analysed in these 4 lines.

No expression was seen at all in embryos or adults of line D-12/Z-1608. However, lines D-12/Z-1322, -1317 and -1581 showed a pattern of expression of lacZ in the gonads consistent with the endogenous pattern of expression of Dax-1. The three lines have the same general pattern of expression although they differ in the intensity of lacZ expression. The earliest time in development when expression can be detected is at 10.5 dpc in both males and females (Fig 5.3). The level of expression increases in both sexes at 11.5 dpc in lines D-12/Z-1317 (Fig. 5.3) and -1322 (Fig. 5.4). However, in line D- 12/Z-1581 (Fig. 5.5) the level of expression at 11.5 dpc is low, being observed only in few cell of the gonad. In all three lines, the expression of lacZ in males becomes restricted to the cells adjacent to the mesonephros (the presumptive rete-testis) from 12.5 dpc onwards (Figs. 5.3-5.5). The expression in the rete-testis remains strong at least until 17.5 dpc (the latest stage examined). In females, the levels of lacZ remain high at 12.5 dpc in the entire ovary. Its expression starts to be restricted to the cells adjacent to the mesonephros (the presumptive rete-ovarii) around 13.5 dpc. By 17.5 dpc it is predominantly expressed in the rete-ovarii. This can be observed more clearly in sections of the genital ridges of 11.5 to 14.5 dpc embryos (Fig. 5.6). In adult testes, lacZ expression is found in cells between the seminiferous tubules. These are probably the steroidogenic Leydig cells where Dax-1 was found to be expressed. In the adult ovary, expression of lacZ was also detected. We would expect the expression to be in the theca and interstitial gland cells, as these are the steroidogenic cells of the ovary. Nevertheless, we have not yet determined at cellular level which cells express lacZ. Sections of adult tissues will be done.

99 Figure 5.2: Expression of lacZ in B-12/Z-tl and B-12/Z-t2 embryos at 11.5 dpc.

A) B-12/Z-tl showing weak expression of lac Z in the genital ridges as revealed after dissectin (right panel) and ectopic expression in the spinal cord, head and tips of the limbs (left panel).

B) Genital ridge from B-12/Z-t2 showing weak expression of lacZ. No ectopic expression was found elsewhere.

100

Figure 5.3: Expression of lacZ in line B-12/Z-1317

Expression of lacZ can be detected from 10.5 dpc in the genital ridges of both XY and XX embryos. At this stage, ectopic expression at the tip of the tail can also be seen. At 12.5 dpc, lacZ expression becomes sexually dimorphic. In ovaries it is strong and expressed all over, while in testes it is weaker and only expressed in the region adjacent to the mesonephros. At 17.5 dpc, expression in the ovaries is strong mainly in the rete ovarii, but expression can also be detected in some cells of the ovary. In the testes, strong expression is restricted to the rete-testes. 0= ovary; T= testes

101 10.5 12.5 17.5 » Figure 5.4: Expression of lacZ in line D-12/Z-1322.

Expression of lacZ is strong in both sexes at 11.5 dpc. At 12.5 dpc expression in ovaries continues to be strong and throughout the ovary. In testes, the expression remains only in few cells adjacent to the mesonephros. This pattern remains the same at 13.5 dpc. At 14.5 dpc the expression in the ovary starts to become restricted to the rete-ovarii, and in males there is an increase in the expression in the rete-testis. At 17.5 dpc, expression in the ovary is mainly in the rete-ovarii, and in the testes in the rete-testis. In adult ovaries, expression of lacZ can be detected in some cells of the ovary. In adult testes expression of lacZ can be detected outside the seminiferous tubules. F= female; M= male 11.5 12.5 13.5 14.5 17.5 Adult

M % Figure 5.5; Expression of lacZ in line B-12/Z-1581.

At 11.5 dpc expression of lacZ in this line can only be detected in few cells in the genital ridges of both sexes. At 12.5 dpc, expression in ovaries increases, while in testes it remains only in few cells. At 13.5 dpc, expression in ovaries remains strong whereas in testes strong expression is detected only in few cells of the rete-testis. At 14.5 dpc expression of lacZ in the ovaries is restricted to cells adjacent to the mesonephros, and in the rete-ovarii. In testes, no expression could be detected at this stage. F= female; M= male 11.5 12.5 13.5 14.5

M Figure 5.6: Sections through the genital ridges of transgenic embryos from line D-12/Z-1322

A-D) 11.5 dpc; E) 12.5 dpc, XX; F) 12.5 dpc, XY; Expression can be detected at 11.5 dpc (A-D) throughout the genital ridge of both XY and XX embryos. At 12.5 dpc, expression can be seen throughout the ovary (E) while in the testes the expression is confined to the rete-testes (F). At 14.5 dpc the expression in both ovaries and testes can be seen in the rete.

104 B

D

* f i

E

ÜM ,

G It was observed that the levels of expression of the transgenes not only varied between lines, but between different individuals of the same line (Fig. 5.7). The reason for this is not known. The mice have a mixed genetic background (C57BL/10 x CBA) and we have evidence for background effects in expression of other transgenes, i.e. Sry (Nigel Vivian and Adam Hacker, unpublished).

In conclusion, we found that the construct D-12/Z, containing 12 kb of Dax-1 flanking sequences was able to drive expression of lacZ to the gonads with the same pattern of expression to the endogenous Dax-1 gene. Nevertheless, it does not drive expression of lacZ to the adrenals or to the hypothalamus. In order to define more precisely the region needed for the expression of Dax-1 in the gonads, shorter constructs were made and introduced into mice.

5.2.2 D-3/Z

The D-3/Z construct contains the region from -3 kb to +0.7 kb of Dax-1 fused to lacZ. Four transient transgenic embryos were analysed for lacZ activity at 11.5 dpc. None of them showed any staining in the gonads (data not shown). Three of the embryos revealed different levels of ectopic expression of lacZ in different places, e.g., head and spinal cord (not shown). We conclude that the first 3 kb of 5' flanking sequences are not sufficient to confer expression of lacZ to the gonads at 11.5 dpc.

5.2.3 D-4.5/Z

The D-4.5/Z construct contains the region from -4.5 to +0.7 kb of Dax-1 fused to lacZ. Ten transient transgenic embryos were analysed for lacZ activity at 11.5 dpc, three of which showed lacZ expression in the genital ridges which overlapped but was not identical to the endogenous Dax-1 pattern (D-4.5/Z-tl to t3. Fig. 5.8 A, lower panels). These embryos also showed lacZ expression in other regions of the embryo (D-4.5/Z-t4 and t5. Fig. 5.8 A, upper panels). Two further embryos showed no expression in the genital ridges, but had ectopic expression in different parts of the embryo (Fig. 5.8 B). The rest of the embryos did not show any lacZ activity in any area (not shown). The high levels of ectopic lacZ expression observed suggest that regulation of expression of this construct is extremely sensitive to its integration site or that a repressor element is absent.

105 Figure 5.7: Different levels of expression of lacZ in line B-12/Z-1322.

Varying levels of expression can be seen in sibling embryos of line B-12/Z-1322. The first two gonads are from two different males from the same litter. The last 3 gonads are ovaries from three different females of the same litter. Different levels of expression can be seen in each case.

106 Figure 5.8: Expression of lacZ in D-4.5/Z-tl to -t5 11.5 dpc embryos.

A) Embryos D-4.5/Z-tl, D-4.5/Z-t2 and D-4.5/Z-t3 expressed lacZ in the genital ridges, although they had ectopic expression of lac z in several other regions. Upper panels, whole embryos; lower panels, gonads of the upper embryo. B) Two additional embryos (D-4.5/Z-t4 and D-4.5/Z-t5) had ectopic expression of lacZ in some regions of their body, but did not have expression of lacZ in the genital ridges. 1 5.2.4 D-6/Z

The D-6/Z construct contains the region from -6 kb to +0.7of Dax-1 fused to lacZ. One transient embryo was obtained and it showed low expression of lacZ in ll.Sdpc gonads (not shown). Two lines were derived with this construct: D-6/Z-1634 and D-6/Z-1638. Line -1634 shows expression of lacZ in the genital ridges at 11.5 dpc in very few cells (Fig. 5.9). No expression was found in other parts of the body. We conclude that this 6 kb region is able to direct the expression of Dax-1 in the gonads, but that more 5' sequences might be required to achieve both higher and possibly more extensive expression in the genital ridge.

5.2.5 F(-18 ^-4) + D-12/Z

Although we were able to replicate the endogenous pattern of expression of Dax-1 in the gonads using a 12 kb genomic fragment upstream of the ORF, expression of Dax-1 in the adrenals and hypothalamus was not observed. We attempted to make construct using larger regions flanking Dax-1 but failed due to technical problems . It has been reported that coinjection of two fragments of DNA into eggs at the same time leads to their integration in the cell's DNA in a collinear fashion(Behringer, et al., 1989;Overbeek, et al., 1991). If the coinjected fragments share regions of homology, recombination between the fragments often occurs prior to integration. Therefore we coinjected D-12/Z with extra upstream or downstream Dax-1 regions and assayed for lacZ activity.

We coinjected D-12/Z with a fragment which overlaps the 12 kb 5' region between -5 to -12 kb and has a further 6kb upstream (-18 kb), F(-18-»-4) (Fig. 5.1). We obtained one transient transgenic embryo, F(-18-^-4) \D-12/Z-tl. It was analysed at 14.4 dpc and showed expression of lacZ in both the gonads and the adrenal glands (Fig. 5.10).

108 Figure 5.9: Expression of lacZ in 11.5 dpc genital ridges of D-6/Z-1634

LacZ expression can de detected in very few cells of the gonad.

109 Figure 5.10: Expression of lacZ in F(-18-> -4)\D-12/Z-tl 14.5 dpc embryo.

LacZ expression is seen in the gonads and adrenals. G: gonads; A: adrenals.

110 î

f i \ 4

A= Adrenal; 0=0vary 5.2.6 D-12/Z + F(+0.7^+13)

We coinjected the 12 kb D-12/Z construct with a fragment that extends from +0.7 kb to +13 kb oîDax-1 , F(+0.7->+13) (Fig. 5.1). We obtained 3 lines from this injection {D-12/Z\F(+0.7->+13) -1358, -1378 and -1372 . Line D-12/Z\F(+0.7->+13) -1358 showed no lacZ staining in any part of the body (not shown). Lines -1378 (Fig. 5.11) and -1372 (Fig. 5.12) both show the endogenous pattern of expression of Dax-1 in the gonads. In addition, line -1378 shows ectopic expression of lacZ in the kidneys, hands and nose (Fig. 5.13). We could not detect expression of lacZ in the adrenals or the hypothalamus.

5.3 Discussion

A summary of the results obtained with this series of transgenic animals is shown in Table 1. We have found that 12 kb of flanking sequences immediately upstream of Dax-1 are able to drive the expression of lacZ to the gonads with a similar expression pattern as the endogenous Dax-1 gene. Expression in the adrenals and hypothalamus is not observed with this same fragment. When assaying shorter regions to drive lacZ expression, we found that 3 kb of flanking sequences immediately upstream of Dax-1 are unable to direct expression of lacZ to any region. When a 4.5 kb fragment was used, three out of ten transient transgenic embryos expressed lacZ in the gonads. However, they also had high ectopic expression in many tissues. Therefore we are not sure if the expression in the gonads is specific to this construct or if it is just an effect of the integration site. When 6kb of flanking sequences immediately upstream of Dax-1 were used, specific expression of lacZ was observed in a few cells of the gonad. This contrasted with the expression obtained when using the 12 kb fragment (D-12/Z) which gives a widespread expression of lacZ in the gonad. Therefore we think that a gonad-specific element is present in the region between -4.5 to -6 kb but that enhancer elements might reside in the region between -6 to -12 kb.

I ll Figure 5.11: Expression of lacZ in line D-12/Z\F(+0.7-^ +13)-1378.

Expression of lacZ in urogenital ridges and gonadal region dissected from D-12/Z\F(+0.7-^ +13)-1378 embryos at 11,5 dpc, 12.5 dpc and 13.5 dpc.

112 11.5 12.5 13.5 15.5

f 4

I f Figure 5.12: Expression of lacZ in line D-12/Z\F(+0.7-^ +13)-1372.

Expression of lacZ in gonads dissected from 11.5 and 13.5 dpc transgenic embryos. M=male; F=female

113 II.! 12.5 13.5

% r %

M

m ...... LINES ...... GENITAL CONSTRUCT kb 5' kb 3' EXP OVARY/RTTESTES/RT ADRENALECTOPIC TRANSIENT RIDGE

0 F(-18^-4)\D-12/Z 18 0 1 1 + + + //+ + + + + + + 4 3 + + + + + + //+ + + —//+ + + + + D-12/Z 12 0 5 2 + + 2 1 + ?/+ ?/+ D -6/Z 6 0 1 1 + — 0 D- 4.5/Z 4.5 0 10 5 + + + 4-4- 0 D-3/Z 3 0 4 1 - + 3 2 + + + +++//+++ --// ++ + D-12/Z\F(+0,7^+13) 12 11 0

EXP: expression RT: rete

+++: strong expression ++: meduim expression +: low expression —: no expression

Table 1: Summary of lacZ expression obtained with the different Dax-1 constructs used We have started to sequence the distal region of construct B-6/Z and have found in this region a binding site for the WT-1 protein, three binding sites for SF-1 and a binding site for SRY (Amanda Swain, personal communication). It is interesting that a binding site for WT-1 is also found in Sry . As both Sry and Dax-1 are coexpressed during the initial stages of gonadal development, they could be regulated by the same transcription factors. WT-1 has been shown to be an important gene for the development of the gonads, so it is a good candidate for the regulation of both Dax-1 and Sry. Nevertheless, additional factors are involved in tissue specific expression of both genes because WT-1 is expressed in other tissues where neither Dax-1 norSry are expressed. SF-1 deficient mice continue to express Dax-1 in the gonads, so we think it is unlikely that SF-1 is required for the activation of Dax-1 in the gonads. However, it might be important in the activation of Dax-1 in other tissues such as the adrenals {Dax-1 expression in adrenals in SF-1 mice could not be checked because they lack adrenals). SRY is also not required for the initiation of Dax-1 expression as the latter starts in XX at the same time as in XY embryos. However, this site could be involved in the down- regulation of Dax-1 in the testes at 12.5 dpc. This is perhaps unlikely to be due to SRY itself, but could be due to the related gene, Sox-9, which is expressed at high levels at this stage in the testis, and will bind similar DNA sequences.

After 12.5dpc, Dax-1 expression in the gonads becomes restricted to the region adjacent to the mesonephros (this happens quicker in males than in females). This region seems to be the developing rete-testis or rete-ovarii. In the testes, the rete-testis connects the testicular cords with the epididymis (efferent ductules). In the ovary, the rete-ovarii is connected to the epoophoron, the homologue of the epididymis, which is vestigial. Both the rete-testis and the rete-ovarii give rise to steroidogenic cells. It has been shown that the efferent ductules and the epididymis have steroidogenic properties (Humar, et al., 1990;Simard, et al., 1991). In the ovaries, the rete-ovarii contributes to the interstitial gland (Byskov, 1975). This is interesting because it seems that Dax-1 expression correlates with steroidogenic tissues.

Endogenous Dax-1 is also expressed in the adrenal gland, but 12 kb of upstream sequence did not activate the expression of lacZ in this tissue. Instead, 18 kb were needed for this, therefore we think that an adrenal specific enhancer element must lie between -12 and -18 kb. More constructs containing this distal 6kb region are being analysed in search for adrenal-specific expression of lacZ.

115 Non of the injected constructs gave us expression of lacZ in the hypothalamus, therefore we concluded that any enhancer elements responsible for the expression of Dax-1 in this tissue must lie outside the analyzed region. Further analysis using even more Dax-1 flanking sequences, or intronic sequences are under way.

Most of the embryos obtained in these experiments have some degree of ectopic lacZ expression (Fig. 5.13). This can be explained by the position at which the transgene integrated in the genome. It is known that strong enhancers flanking the site of integration can affect the normal pattern of expression of transgenes (Wilson, et al., 1990). It has been speculated that using DNA elements which protect the transgene from these integration effects could be useful; nevertheless, the several elements conferring position independent expression which have been described so far can not be used as general protective elements because they also act as tissue specific enhancers (Chamberlain, et al., 1991;Greaves, et al., 1989;Grosveld, et al., 1987;Palmiter, et al., 1993). Attempts have been made to use scaffold attachment regions (SARs), which are thought to insulate gene expression domains via chromatin structure, but they do not seem to have the desired effect (De Sepulveda, et al., 1995).

116 Figure 5.13: Ectopic expression of lacZ in different transgenic lines.

Ectopic expression of lacZ could be detected in several of the lines produced: 1581= Line D-12/Z-1581, 12.5 dpc. 1317= Line D-12/Z-1317, 13.5 dpc. 1322= Line D-12/Z-1322, 11.5 dpc. 1378= Line D-12/Z\F(+0.7-> +13)-1378, limb 15.5 dpc, nose 12.5 dpc, and kidney 12.5 dpc. 1372= Line D-12/Z\F(+0.7-» +13)-1372, 12.5 dpc.

117 1581 1317 1322

1378 1378 1372

1378 C hapter 6; as a transgene

6.1 Introduction

Our aim was to test whether the male to female sex reversal found in humans carrying a duplication of the chromosomal region carrying DAX-1 could be replicated in mice. This would constitute proof that Dax-1 is the gene responsible for DSS.

6.1 Results

6.1.1 Mouse Dax-1 As the correct pattern of expression of Dax-1 in the gonads could be reproduced with 12 kb upstream of Dax-1, a construct was made with the 12 kb fragment attached to the Dax-1 cDNA with the last intron and polyadenylation sites of rabbit B-globin (D- 12/cDax, Fig. 5.1). Seven transient transgenic embryos obtained with this construct were analysed at 14.5 dpc. We looked for abnormalities in the gonads and checked if the genotype, as determined by PCR, corresponded to the phenotype observed. Three completely normal XX females (D-12/cDax-tl to -t3) and three completely normal XY males (D-12/cDax-t4 to t6) were observed. One XY embryo showed testes that looked slightly abnormal (D-12/cDax-t7), but however no obvious sex-reversal. We checked if the transgene was expressed in the gonads of the male embryos (D-12/cDaxt4 to t7) by RT-PCR. We detected of a band of 305 bp indicating that the transgene was expressed in the gonads of the four embryos and also in the limbs of D-12/cDax-t4 and D-12/cDax-t7 (Fig 6.1). Fourteen founders were also obtained with this construct (D-12/cDax-1812, -1979, -1983, -1994, -2006, -2159, -2162, -2165, -2170, -2173, -2175, -2182, -100, -102) All of them show normal external genitalia and their sexual phenotype correlates with their genotype. Some of the founders have produced litters, therefore they are fertile. Analysis by RT-PCR in samples from two of the lines have shown that the transgene is being expressed in the genital ridge of embryos at 11.5 dpc (Fig. 6.2). One line carrying D-12/cDax plus the fragment F(-18->-4) was obtained F(-18^-4)\D- 12/cDax-171. The founder of this line was a fertile XX female, although we were unable to analyze its progeny because the litter died just after birth and it was eaten by the mother.

118 Figure 6.1; Expression D-12/cDax transgene in 14.5 dpc embryos.

RT-PCR was performed on limbs and gonads dissected from embryos D-12/cDax-t4 to -t7. Expression of the transgene, represented by a 305 bp band, can be seen in the gonads of the four embryos. Embryos D-12/cDax-t5 and -t7 show expression of the transgene in the limb. The upper 800 bp band represents the signal derived from the genomic DNA of the transgene. +: with Reverse Transcriptase; without Reverse Transcriptase. FI: non-transgenic

119 L D-12/cDax-t4

G D-12/cDax-t4

L D-12/cDax-t5

G D-12/cDax-t5

L D-12/cDax-t7

G D-12/cDax-t7

L D-12/cDax-t6

G D-12/cDax-t6

LF1

G FI

I t il Figure 6.2: Expression of D-12/cDax transgene in genital ridges of 11.5 dpc embryos.

A) RT-PCR was performed on limbs and gonads dissected from 11.5 dpc embryos of line D-12/cDax-1812, using primers 6A and RGB. Expression of the transgene can be detected in genital ridges of both male and female embryos, as represented by a 310 bp band. The upper 890 bp band represents the genomic DNA transgene. HPRT was used as a positive control. +: with Reverse Transcriptase; without Reverse Transcriptase. T; Transgenic; NT: Non-transgenic.

B) RT-PCR was performed on limbs and genital ridges of 11.5 dpc embryos of line D-12/cDax-1979 using same primers as in A. Expression of the transgene can be detected in the genital ridges. L: limb; G: genital ridge

120 LIMBS GONADS

NT NT

0+ Of Of Of Of Of

I+I+I+I+I+I+

D-12/cDaxDNA (890 bp) HPRT (352 bp) D- 12/cDaxcDNA (310 bp)

B

o o

D- 12/cDax DNA D- 12/cDaxcDNA 6.1.2 Human DAX-1

Another attempt to duplicate the human sex-reversal condition in mice was made by injecting a human genomic fragment carrying the DAX-1 gene with 8 kb of flanking upstream sequence (HD-8). We obtained 4 transient transgenic embryos at 13.5 dpc (HD-8-tl to -t4). HD-8-tl was a normal XY male, -t2 was a normal XX female. Embryos HD-8-t3 and -t4 were delayed in their growth, resembling 11.5 dpc embryos. HD-8-t3 was XY HD-8-t4 was XX, as established by PCR genotyping . At this stage the gonads are undifferentiated, so we could not look for abnormal sexual phenotype in these two embryos. RT-PCR was performed in HD-8-tl, -t2 and -t3 embryos. It was found that the transgene was expressed in the XY male HD-8-tl embryo in the gonads, adrenals and limbs. The transgene was not expressed in the XX female HD-8-t2 embryo. HD-8-t3 expressed the transgene in the gonads (Fig. 6.3).

6.2 Discussion

Adding either mouse or human DAX-1 /Dax-1 as a transgene has not duplicated the human syndrome in which male to female sex-reversal occurs when a duplication of DAX-1 is present, even when we have found the transgene to be expressed. The lack of sex reversal in the transgenic mice could be accounted for in several ways.

It may be that the transgene is expressing too low levels of DAX-1 (i.e.. much less than the endogenous levels). Quantitative methods such as RNAse protection assays need to be used to resolve this question. A simple way of trying to increase the levels of the transgene will be by breeding the different lines to each other to make double or compound hemizygotes or to make animals homozygous.

121 Figure 6.3 RT-PCR of 11.5 dpc embryos carrying human DAX-1 as a transgene.

RT-PCR was performed on RNA samples from transient transgenic embryos carrying construct HD-8 using primers HDS'and HD3'. Expression of the transgene was detected in the gonads, adrenals and limbs of the XY T1 and in the gonads of the XY T3 embryo. No expression was detected in the XX T2 embryo.

122 A G L+ L- A G L+ L- A G L+ L- A G L+ L-

378 bp JL V ------NON-T T1 T2 T3 13 5 13 5 dpc g 13 5 dpc (2^ 11 5 dpc Another possibility is that the mouse Sry is more efficient than human SRY in activating male development. The finding that human and mouse S R Y /S ry are only conserved in their HMG boxes could mean that they function in slightly different ways. For example, mouse SRY has a glutamine-histidine domain which could be involved in transcriptional activation (Dubin and Ostrer, 1994). Human SRY does not have this domain, so human SRY could be less effective in activating transcription because of the lack of this domain. In this case, much more than a double dose of Dax-1 might be needed to overcome its action. An alternative to test this is to breed some of the Dax-1 lines with strains that are known to have a "weak" Sry (those carrying the Ydom, or Sry as a incompletely penetrant transgene, see section 3.33) expecting that this might facilitate the ability of Dax-1 to induce sex reversal. In case mouse SRY was extremely efficient, for example if mouse SRY, by its binding and bending of DNA blocked completely the binding of DAX-1 to its target, overdosing DAX-1 or weakening SRY might never lead to sex-reversal. However, this would not disprove a role for Dax-1 in the sex differentiation pathway.

Sequence conservation between human and mouse DAX-llDax-1 is only around 66% in amino acid identity and 75% in nucleotide identity, therefore this divergence could explain why the two proteins are functioning in different ways.

And finally, it could be possible that Dax-1 is not, after all, DSS. Within the 160 kb region where DSS was mapped, other expressed sequences have been cloned and genes from the MAGE family {MAGE-Xp, DAM 10 and DAM6 ) have been identify in this region (Dabovic, et al., 1995;Muscatelli, et al., 1995). The MAGE family was discovered through the finding of genes with homology to MAGE-1, a human gene that is expressed in testes and in tumours of various histological types (De Smet, et al., 1994;van der Bruggen, et al., 1991). MAGE-Xp is localized 50 kb distal to DAX-1. This gene was found to be expressed specifically in testis (as all the other members of the family) but in contrast with them, it was not expressed in any of the tumours tested. At least five copies of its fourth exon seem to be present in a 30 kb region, while the first, second and third exons seem to be present in single copies. The putative protein encoded by this gene resembles the mouse necdin proteins. No function has yet been assigned to these proteins, but necdin is a nuclear protein expressed at different developmental stages of the mouse central nervous system. It is possible that not DAX-1, but any of the genes found in this region are responsible for DSS. Pronuclear injection of YACS covering the whole region may answer the question on to whether there is any gene in the region capable of causing sex-reversal.

123 In any case, it will be interesting to see the effect of knocking out Dax-1 in developing ovaries and adrenals. At the very least, a mouse model for AHC might be expected. However, the important question is what would happen to ovarian development in the complete absence of DAX-1. Two models for the function of Dax-1 can be considered.

First, if Dax-1 was a necessary ovary determining gene, we would expect an XX individual lacking Dax-1 to develop no ovaries. However, whether this individual developed testes or just kept an indifferent gonad would depend on the role of Sry. If Sry is only needed to repress the ovarian pathway, we would expect testes to form in an XX null Dax-1 mouse. On the other hand, if Sry is required to actively start testes development, then we would expect no testes to form either.

Second, if Dax-1 was an "anti-testis" gene, then we would expect an XX Dax-1 knock­ out mouse to develop testes. Of course, this is considering just the most simple models, because it is possible that both Sry and Dax-1 might be activating their own sex determining pathway and repressing the other at the same time. The finding of genes downstream of Dax-1 and Sry will clarify their role in the process of sex determination.

124 C hapter 7; C oncluding Remarks

Almost four years have gone by since I joined this lab and started trying to understand S iy regulation, nevertheless we are still almost as ignorant in this respect as then. After all the different attempts to find regulatory elements of this gene, there is only one conclusion to make: Sry is a very complex gene! (could this account for MEN’s nature?) But still, we have learned some things.

An important lesson is to be very careful with the reporter genes to be used; but regarding Sry, it seems that the intact locus of Sry might be needed for its correct regulation, as disrupting its ORE with lacZ resulted in the use of a different initiation site for transcription. Therefore maybe the best way of monitoring Sry expression will be with the use of a small tag attached to it so as to disturb as little as possible of the Sry locus, instead of replacing it with a reporter gene.

During spermiogenesis the structure of DNA seems to be greatly modified (as seen by the disappearance of the DHSs). This may be modifying the transcriptional state of genes which depend on DNA structure for their regulation (as could be the case forSry ). Sry could be activated at this stage because of the structural modifications needed for the packaging of the DNA into sperm. If production of SRY protein at this point was deleterious, the production of the circular transcript would be a way of impeding its production. The expression of SRY in adult testes driven by a tissue specific promoter will tell us how harmful this is.

To understand a little more about the adult transcript, its promoter could be looked for. Cells transfected with the known 14.5 kb of Sry cloned near the SV40 enhancer express only the circular transcript, so these cells can be used to locate the promoter by performing serial deletions of Sry sequences.

The fact that Sry seems to be an intronless gene and encodes for a transcriptional regulator has made us think that the main mechanism for sex determination will be based on the transcriptional regulation of downstream genes. This mechanism is different in Drosophila where the main mechanism is the use of alternative splicing between male and female transcripts, or in C. elegans where the localization of the proteins seems to be important. Therefore we are concerned with the basic question of whether SRY functions as an activator or a repressor. This is crucial, as knowing this will guide us as to which type of downstream genes we should expect to find. The fact that human and mouse SR Y /S ry are so different makes us wonder if they still work in

125 a similar fashion. It will be interesting to add the glutamine-histidine rich repeat of mouse SRY to the human one find out if this is able to sex-reverse, (in mice of course!)

On the other hand, Dax-1, which has been proposed as an ovarian determining gene, is a relatively new gene, and seems to be a better behaved gene (again, possible reflecting w o m en ’s nature). In this case lacZ turned out to be a useful reporter gene. We were able to identify probable tissue-specific regulatory regions in tYieDax-l promoter. We have a nice promoter that will target gene expression to both male and female genital ridges at 11.5 dpc and then becomes female specific at 12.5 dpc. This may be very useful in assessing the role of other genes involved in sex differentiation. By putting Sry under theDax-1 promoter and obtaining XX sex-reversal, we will confirm that Dax-1 and Sry are in fact coexpressed in the same cell type. Putting the genes that are normally downregulated in females at 12.5 dpc (SF-1, Sox-9) under the control of Dax-1 promoter will give us an idea of their role in testis/ovary differentiation. Detailed analysis at cellular level of lacZ expression under the Dax-1 promoter can give us information about the process of gonad development, as it can be used as a marker of different cell types. However, we still need to define precisely the tissue-specific regulatory elements within these regions, and by doing so maybe some light will be shed on to the regulation of Sry if they share common regulators.

With respect to the role of Dax-1 in the sex determination, we still do not have an answer. Many questions remain before we can conclude anything, but it is interesting that in wood lemmings a mutant X chromosome seems to exist which overrides the masculinizing effect of the Y chromosome. In a way this is what seems to be happening in the Xp-duplicated patients. It would be interesting to look at the Dax-1 locus in wood lem m in g s.

If we consider the pattern of expression of Sry, Dax-1 and Sox-9 (three genes which have been found to give sex-reversal) we can speculate some kind of interactions happening between them. The three genes are known to be expressed in the genital ridges of both sexes at around 10.5. By 11.5 dpc Sox-9 has turned off in females, but continues on in males, while both Dax-1 and Sry continue to be expressed in both sexes. By 12.5 dpc, both Dax-1 and Sry have been turned off in males, while in females, Dax-1 continues to be expressed. At this same age, Sox-9 is off in females while it is on in males (Fig. 7.1). And we know that other important genes for gonad development (WT-1 and SF-1) have been found expressed at 11.5 dpc in the genital ridges. SF-1 then becoming downregulated in females. If we were to make a model for the sex determination pathway in mammals and compare it with the known models of Drosophila or C. elegans it may look a bit disheartening:

126 XY Sry Sox-9 Testes

WT-1;SF-1

XX Dax-1 —I Sox-9 Ovary

But considering that when I started this PhD the model looked rather like this:

XY Sry Testes

XY No Sry Ovary it can be appreciated that a lot of progress has been made in these years. Actually, with all these new genes appearing on the scene, this is an extremely exciting time to be studying this field. We can start studying their interactions and soon will be able to elucidate their roles.

However, the most important conclusion is that it does not matter how difficult the genes are, sex will always be the most exciting theme of study!

127 Figure 7.1: Expression pattern of Dax-1, Sry and Sox-9 .

Dax-1 expression, as revealed by lacZ staining. Sry expression as revealed by whole mount in situ hybridization (A. Swain, unpublished) Sox-9 expression as revealed by whole mount in situ hybridization (from Silva, et. al., 1996) It can be noticed that, at 11.5 dpc, when Sry and Dax-1 are on in both male and female genital ridges, Sox-9 expression has turned off in female genital ridges. At 12.5 dpc, Sox-9 continues to be male specific, while Dax-1 becomes female specific.

128 10.5 dpc 11.5 dpc 12.5 dpc

Dax-1

Sox-9 ■Ë . . %

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