6
Developmental genetics of mammals
Early differential tissue expression of a transposon-like family during
mouse embryogenesis
Philippe Brulet*, Service de Genetique Cellulaire, Inst. Pasteur, Paris, France
The early transposon (E.Tn) family of long repeated and dispersed sequences in the mouse genome
presents several structural characteristics
of retroviral proviruses. The proviral sequence has long terminal
repeats (LTR). The RNA transcript 5 ; and 3' ends are localized in the LTRS. A 12 bp direct repeat is found
at both ends of the RNA. By nucleotide sequencing, primer extension and Si mapping the E.Tn LTR has
the retroviral U3.R.U5 structure.
An inverted repeat ending in TG . . . CA borders the LTR. Two bp after
the 3' end of the 5' LTR is a lRNA binding site. A purine sequence is found 5' of the 3' LTR. A six bp
duplication of genomic DNA brackets the E.Tn sequence. Finally by heteroduplex electron microscopy
analysis, the RNA is colinear to the DNA.
By in situ hybridization on embryo sections using either the complete proviral sequence or only the LTR
sequence as probes, E.Tn RNA isfirstdetected in ICM cells. After implantation, the embryonic ectoderm is
strongly labelled up to day 7j . Between day 5 and 7j , some differentiated tissues like the extra embryonic
ectoderm express E.Tn. RNA, others do not. After day 8 when the three germ layers are set up no RNA
transcript is detected.
Chronobiochemical aspects of the hepatic DNA biosynthesis in
experimental animals under the action of some metals
Z. Garban, Iulia Eremia and Gabriela Daranyi, Laboratory of Embryology, Centre of Hygiene and Public
Health, Br. Mihai Viteazul24,1900 Timisoara, Romania
The hepatic DNA biosynthesis under the influence of some metal ions (Mg2*, Zn 2+ , Mil2"1") has been
studied in albino rats (90 ± lOg b.wt.) with special emphasis on its chronobiochemical aspects (the circadian
changes in the rate of biosynthesis). Some important
peculiarities have applicability in molecular biology
2
and in dismineralosis. Metal chlorides (25-10~ g % in physiological saline) were injected i.p. (1 ml/
100 g b.wt.). The substances were administered at 8-8.30 a.m. (3 groups - first series) and at 20-20.30 p.m.
(3 groups - second series) respectively. Each group (including also control, untreated animals) involved 8
specimens. DNA dosage was performed at 12 hours after the administration, both in experimental and
control groups. The results were statistically processed.
The experimental data showed that the above mentioned metal ions determine modifications in the
hepatic DNA concentration (/ng/mg wet tissue). Comparing the DNA concentration of the experimental
groups (from the first and second series) it may be concluded that it increases in the animals belonging to the
second series with +0-05 (ig/mg in the case of MgCl2; +0-08 /ug/mg in the case of ZnCl2 and with
+0-09 /Ltg/mg in the case of MnCl2 . The process is correlated with the normal rhythm of hepatic DNA
biosynthesis characterized by an augmentation in the morning and a remission in the evening (Mayersbach,
1977). If, e.g., the DNA concentration in the experimental groups and in the control group is compared in
the evening the following differences are found: -0«03 /xg/mg in the case of MgCl2; +0-04 /tg/mg in the case
of ZnCl2 and +006 /u,g/mg in the case of MnCl2 .
The differing effects may be explained by the well known fact that Mg24" binds preferentially to the
phosphodiesteric groups while Zn2* and Mn2+ bind to the bases of DNA. These interactions occur
during the cell cycle having an effect on the transmission of the information encoded in DNA.
Developmental genetics of mammals
Microinjection of oncoviral DNA into mouse eggs
K. Gazaryan* (Moscow)
No abstract for publication
Notched scapula (ns): A new mutation in the Syrian hamster
Mesocricetus auratus
S. A. A. Hassoon and Janet E. Hornby, Department of Pure & Applied Zoology, The University,
Whiteknights, Reading, Berkshire, RG62AJ
An autosomal recessive gene, notched scapula (ns), affecting the morphology and the histology of the
scapula is reported. The most characteristic features of the adult abnormal scapula are: the blade is
narrower due to a deep notch in the region of the antero-vertebral border; the remaining part of the blade is
slightly thicker than normal and shows some abnormalities; the part of the spine which runs from the middle
of the scapula to the vertebral border is missing in the abnormal scapula.
The development of the cartilage and bone in the normal and abnormal scapula is being studied with
alcian blue and alizarin red staining of the whole animals. The defect is recognizable in 14£ day embryos: the
cartilaginous region is much narrower than normal and the bony regions resemble the abnormal adult
scapulae. Abnormal scapulae were first detected in anopthalmic white (Wh) hamsters, and when present the
abnormalities are more severe than in golden animals. Histological examination shows abnormal
organization in the bone, and in the bone marrow cavities.
8
Developmental genetics of mammals
Retroviruses as probes for mammalian development
Rudolf Jaenisch*1, Michael Breindl \ Klaus Harbers \ Angelika Schnieke 1, Ju'rgen Lohler 1, and Klaus
Kratochwil 2. lHeinrich-Pette-Institutfur Experimentelle Virologie undlmmunologiean der Universitdt
Hamburg, Martinistrasse52, 2000 Hamburg 20, West Germany. 2Osterreichische Akademie der
Wissenschaften, Institutfiir Molekularbiologie, Billrothstrasse 11, A-5020 Salzburg, Austria
Experimental insertion of the Moloney leukemia virus into the germ line has resulted in an embryonic
recessive lethal mutation in Movl3 mice. Integration of the proviraJ genome occurred at the 5' end of the
arl(I) collagen gene blocking formation of stable mRNA. Sequence and SI mapping analyses were
performed to characterize the position of the proviral genome in relation to the transcriptional map of the
mutated gene. The results indicated that the virus has inserted into the first intron 19 bp 3' of the
intron-exon boundary.
To study the mechanism of how insertion of the provirus may interfere with gene transcription, the
chromatin structures of the mutated and the wild-type alleles were compared. Limited digestions with
DNasel revealed that a hypersensitive site located at 100-200 bp 5' of the cap site was strictly correlated with
collagen cd(l) transcription. Integration of the virus into the first intron prevents the developmental
appearance of this site, and this may interfere with proper activation of the gene during embryonic
development.
Embryos homozygous at the Movl3 locus are arrested in development between days 11 and 12 of
gestation. This is the time when abundant transcription of the arl(I) collagen gene starts, suggesting an
essential role of type I collagen for midgestation development. Histological examination of day 12 embryos
revealed a general cell necrosis without obvious malformation. Cell necrosis typically begins in erythropoietic cells of the liver. The embryos die, however, due to a sudden breakdown of the circulatory system.
In vitro organ explantations are being performed for functionally testing the role of collagen in
organogenesis.
Approaches to cloning the T-complex
H. Lehrach* and A. M. Frischaut
No abstract for publication
Developmental genetics of mammals
9
Evidence from genetic analysis of the mouse t-complex concerning its
role in development
M. F. Lyon*, MRC Radiobiology Unit, Harwell, Didcot, Oxon OXU ORD
Recent advances in knowledge of the t-complex have shown that the number of lethal complementation
groups is much larger than previously thought, and that the embryonic lethals concerned are distributed at
various loci at different points in the complex. Transmission ratio distortion involves multiple distorter loci
acting on a responder locus, and male sterility also involves various loci, which may be identical with the
distorter loci. The ratio distortion and male sterility loci are apparently unconnected with the lethal loci.
Crossover-suppression appears to depend partly on the presence of inversions, but probably not entirely so.
Although the number of known lethal loci in the complex is now large, it is probably not unusually so for
such a length of chromatin. Thus, this region of chromosome 17 may not have any special role in
development. The number of loci concerned in male fertility or spermatogenesis does appear rather
unusually high, however, and the t-complex may provide valuable material for studies of gene action in
spermatogenesis.
Chromosome rearrangements and spermatogenesis
C. A. Redi and S. Garagna, Dipartimento di Biologia Animate and Centro di Strudioper Vlstochimica del
C.N.R., Universita di Pavia, Piazza Botta, 10,27100 Pavia, Italy
It has been widely reported in mammals that the correct chromosome constitution is an essential
prerequisite for the regular occurrence of spermatogenesis. The high frequency of Robertsonian (Rb)
transformations makes the house mouse an excellent animal model for studying the influence of
chromosome structural rearrangements on the spermatogenetic process. Three karyotype constitutions have
been studied which are known to be reliable examples of normal fertility, subfertihty and sterility. Each
karyotype constitution was related both to the cytologic composition of the twelve stages of the seminiferous
epithelium and to the sperm DNA content. In Rb heterozygous mice it was observed: 1) a reduced ratio
between primary spermatocytes and spermatids, and a lower number of proliferating spermatogonia; 2) a
disrupted pattern of enzymatic activities in spermatogonia and spermatocytes; 3) no numerical change and
no enzyme alteration in Sertoli cells; 4) a high frequency of aneuploid spermatozoa and, consequently, an
increased number of unbalanced blastocysts. These results suggest that two main phenomena act m affecting
the fertility status in cases of chromosome variation:
a) the breakdown of spermatogenesis, which can take place at any level of the cytodifferentiative process of
spermatogonia (although massive cell losses occur at the spermatid stage);
b) the formation of genetically unbalanced spermatozoa, giving rise to zygotes which die early both before
and after implantation.
10
Developmental genetics of mammals
Chimeric mice containing homozygous T/t complex lethal genotypes for
l 8 5
0
7
3
Anna W. Seitz* and Dorothea Bennett, Laboratory of Developmental Genetics, Sloan-Kettering Institute for
Cancer Research, 1275 York Avenue, New York, N.Y. 10021, U.S.A.
The T/t complex in the mouse includes a series of recessive lethal developmental mutations, which block
early embryonic development at different stages. Attempts were made to obtain aggregation chimeras
consisting of homozygous lethal mutants and wild type. Since the t/t embryos could be obtained only from
mating +/t heterozygotes together, the experimental embryos used for chimerization included +/+, +/t and
t/t embryos paired with known +/+ embryos; the two groups were distinguishable by coat color phenotype.
Glyoxylase 1 isozymes were used to define chromosomes carrying t-mutations as distinct
from all wild type
chromosomes.
Four separate
sets of coat color chimeras were obtained: 85 for
the t wl8 genotype, 34 for
w5
w73
wl
t homozygotes, 4 contained
tw5 ,19 for t°, and 32 for t . When they were analyzed: 6 chimeras contained w73
t homozygotes, 3 contained t° homozygotes, but no chimeras containing t homozygotes were found.
However, only 10-20 % of t-homozygous genotypes were rescued by chimerism. None of the chimeras
containing t-homozygotes produced offspring from the t/t genotype; this is not surprising since all but two
were males, and thus the t/t contribution would be expected to be sterile.
Isozymes of phosphoglucose isomerase (GPI) were used as markers for the t and wild type genotype. The
tissues of chimeras were analyzed for contributions from each partner. AH +/t <-* wl8
+/+ w18
chimeras were found
to consist of approximately equal amounts of both
genotypes.
The
same
is
true
of
t
/t
<-» +/+ chimeras.
However, the average tissue contribution of tw5/t was less than half, and of t°/t° even less.
Nevertheless cells from all three t-homozygotes contributed to all tissues. These results suggest that the
defect of these stage specific mutations was overcome by the presence of normal healthy cells in the
chimeric embryos, and that once past the critical stage t/t cells were capable of normal embryonic
development, and functioned normally in the adult. The proportion of cells thus 'rescued' appeared to
depend on the time of the lethality of
the t-mutation. The earlier the defect, the smaller the proportion of its
contribution in the adult. In the tw73 genotype, the onset of the defect probably occurs too early to be
rescued by chimerism at the 8 cell stage.
Molecular probes for the structure and evolution of mouse t-haplotypes
L. M. Silver* (New York)
No abstract for publication
Developmental genetics of mammals
Influence of genes at the W locus on melanocyte distribution in
chimaeras
11
D. A. Stephenson 1>2, P. H. Glenister 2 and Janet E. Hornby l.1Department of Pure & Applied Zoology,
The University, Whiteknights, Reading, Berkshire, RG62AJ. 2MRC Radiobiology Unit, Harwell, Didcot,
•
Oxon,OXU0RD
Alleles at the dominant spotting (W) locus influence the distribution of melanocytes. Heterozygotes
display various degrees of spotting, with phenotypes
ranging from a head spot (viable dominant spotting,
W^ to a distinct band across the loins (sash, Wsh). Homozygotes have a black-eyed white phenotype due to
an absence of neural crest derived follicular melanocytes. The genetic absence of follicular melanocytes
provides an opportunity to investigate the influence of the W locus on the pattern of distribution of wildtype
melanocytes by constructing chimaeras.
Embryos homozygous or heterozygous for mutant alleles at the
dominant spotting locus (i.e. WshWsh, WshWv, or W sh ±) were fused with embryos homozygous for the wild
type allele. In each case the resultant chimaeras
displayed a phenotype resembling the sash heterozygote.
Despite this distribution of pigment, in the jy sh w sh «-• + + and W*hWy «-• + + animals a hair morphology
marker (fuzzy, fz) showed that the cells from the two embryos were distributed equally and randomly
through the skin, and a pigment marker (beige, bg) confirmed the absence of W* melanocytes (Stephenson,
1983). However, in the W ± <-> ± ± chimaeras pigment markers, (pink eyed dilution and ruby-2,p ru-2),
allowed the presence of Wsh ± melanocytes to be demonstrated as discrete patches of wild type
pigmentation. The implications of these observations will be considered.
STEPHENSON,
D. A. (1983). Pigment gene expression with particular reference to sash (Wsh) chimaeras.
Genet. Res. 41, 319.
Introduction of genes into mice and teratocarcinoma cells
C. L. Stewart* and E. F. Wagner, EMBL, Meyerhofstrasse 1, D-6900 Heidelberg, West Germany
We are using two approaches to introduce new genetic information into mice. Both approaches use
retroviral vectors carrying dominant selectable marker genes as well as structural genes such as /3-interferon.
We have infected various EC cell lines with these
vectors and have obtained a high frequency of resistant
clones under selectable conditions (efficiency 10"1—10~3). Currently we are infecting preimplantation mouse
embryos with these retroviruses to see if mouse embryos will also express these vectors. The second
approach has been to infect feeder dependent EC.EK cell lines. Clones have been isolated and some of
these have been re-introduced into mouse embryos. At present 26 mice have been born and four show overt
chimaerism. These mice are being analysed for the expression of the retroviral sequences in various
chimaeric tissues. The results from both approaches will be discussed and the advantages and disadvantages
of the approaches compared.
12
Developmental genetics of mammals
Intracisternal A-particle expression during mouse embryogenesis
D. Szollosi* (Jouy-en-Josas)
No abstract for publication
The development of the vagina in the Tfm mouse
K.-U. Thiedemann, R. Mauchand U. Drews, Anatomisches Institut, University of Tubingen, Osterbergstr. 3,
7400, Tubingen, West Germany
We have studied the development of the vaginal anlage in organ cultures of the genital tract with and
without testosterone and in vivo in normal and Tfm mouse embryos by three dimensional reconstruction of
serial sections.
In the organ culture study we observed that without testosterone Wolffian and Miillerian ducts fused and
formed the vaginal anlage. In the presence of testosterone fusion was prevented by male differentiation of
the Wolffian ducts. We conclude (1) the 'sinu-vaginal bulbs' are identical with the lower ends of the Wolffian
ducts, and (2) that testosterone inhibits formation of the vagina by preventing fusion of Wolffian and
Miillerian ducts.
By reconstructing the vagina from serial sections in normal and Tfm embryos we observed that in the
female and in the Tfm phenotype the vaginal anlage is formed by downgrowth of Wolffian and Miillerian
ducts behind the urogenital sinus, the Wolffian ducts being the leading structure. In the female the Wolffian
duct degenerates after downgrowth; in the Tfm, in addition, also the Miillerian derived structures
disappear, probably by action of AMH.
We conclude that the Tfm vagina is not a 'sinus vagina', but a variable leftover of the degeneration
process.
Developmental genetics of mammals
13
Complex electronmicroscopical investigations on the spermatozoa of
red deers
/. Veres 1, E. Nikode'musz 2, E. Ldncz-Mezlinyi and G. Torok. 1 Central Laboratory of the University of
Agricultural Sciences. 2Game Biology Station, H-2013 Godollo, Hungary
We have started a comparative study on the ultrastructure of the spermatozoa of man, domestic and wild
animals with particular reference to biomembranes.
The ultrastructure of the spermatozoa of the red deer, as well as its character and charge dispersion are
similar to those of the bull. The cells are smaller but all the morphological features observed on ultrathin
sections are the same as in the bull.
The net negative character of the entire surface is identical in both species when using AgJ label sol
technique combined with transmissional resp. scanning electron microscopy developed by one of us (Veres,
I.: In Hayat: Principles and Techniques of Electron Microscopy Vol. 9. p. 262-287, 1978). The charges are
concentrated toward the middle- and principal parts, particularly on the tail end-piece, enabling the
collisionfree movement of the gametes. There is a slight charge concentration also at the region of
perforatorium (similar to the bull spermatozoa) which have not been detected in the spermatozoa of the
other mammalian species examined.
Developmental staging of monoclonal antibody and cDNA markers of
mouse spermatogenesis with male sterile mutations
K. R. Willison*1, R. K. Dudley \ /. C. Summerhayes \ /. Potter 1 andM. F. Lyon 2. lChester Beatty
Laboratories, Institute of Cancer Research, FulhamRoad, London, SW36JB. 2MRC Radiobiology Unit,
Harwell, Didcot, Oxon
Thirteen monoclonal antibodies (T1-T13) which react with antigenic determinants present in adult mouse
semniferous tubules have been prepared (Willison, Summerhayes & Suzuki (1983), Cold Spring Harbor
Conferences on Cell Proliferation 10, 219-228). Some of the determinants recognized are localized
intracellularly and some extracellularly as judged by indirect immunofluorescence tests on unfixed frozen
sections and on live germ cells. We have been interested in developing rapid screening procedures for
producing monoclonal antibodies specific to cells at different stages of spermatogenesis and have tested
these thirteen antibodies for their ability to bind frozen sections of testes from a variety h45
of male sterile
mutants. The mutants screened were WAT, SXR/+, T(X;16)16H,v Tfrn/Y, T(7;19)145H, t /th45, t86/^18.
Phenotypically they range from
absence of germ cells (W/W , SXR/+) through meiotic arrest to
non-fertilizing spermatazoa (t86/^18). Various antibodies (T5, T8, T9, T12, T13) bind to all the mutants
suggesting that the determinants recognized are carried by Sertoli cells. Other antibodies however, only
bind to some of the mutants; Tl, for example, only binds strongly to the two t-haplotype compound steriles
showing that the determinant is associated with spermatids and/or spermatozoa. We have now also staged
testis specific cDNA clones in Northern transfer experiments with total RNA extracted from the testes of
SXR/+ and Tfm/Y mutants (Dudley et al. (1984), NAR 12,4281-4293). This has allowed the identification of
haploid stage expressed cDNA clones. The sequences and genetic mapping of some of these clones will be
described.