/. Embryol. exp. Morph. Vol. 35, 3, pp. 607-616, 1976
Printed in Great Britain
607
The use of ' normal' and 6 transformed'
gynandromorphs in mapping the primordial germ
cells and the gonadal mesoderm
in Drosophila
By W. J. GEHRING, 1 E. WIESCHAUS, 2 AND M. HOLLIGER 1
From the Department of Cell Biology, Biozentrum der Universitat,
Basel and the Zoologisches Tnstitut der Universitat, Zurich
SUMMARY
The primordial germ cells and the gonadal mesoderm were mapped in the Drosophila
embryo by analyzing the patterns of mosaicism in 'normal' and 'transformed' gynandromorphs. Relative to the adult cuticular markers the germ cells map as the posterior most
structure, which coincides with their known location in the blastoderm embryo. These data
support the hypothesis that the gynandromorph map reflects the real position of the primordia in the embryo. Since after the blastoderm stage the primordial germ cells migrate
anteriorly these data also indicate that the map in fact corresponds to the blastoderm stage
and not to a later stage of development. The genital disc maps as a single median primordium
anterior and ventral to the germ cells, the gonadal mesoderm is located anterior to the genital
disc and also forms a single median primordium on the ventral side of the embryo. The
primordia for the genital disc and the gonadal mesoderm are unusually large in size, which
presumably reflects some indeterminacy of the cell lineage leading to an 'expansion' of the
map.
INTRODUCTION
The location of the primordial cells for the various larval and adult structures
in the early Drosophila embryo can be deduced from an analysis of gynandromorphs and other genetic mosaics (Sturtevant, 1929). The gynandromorphs
generally used in these studies arise from female zygotes in which one of the two
X chromosomes is lost during the first nuclear division, giving rise to flies which
are mosaics of approximately 50 % female (XX) and 50 % male (XO) cells
(Hinton, 1955; Lifschytz & Falk, 1969). Using appropriate recessive markers on
the X chromosome which is retained in the XO cells, allows identification of
these cells in any kind of tissue (Janning, 1972; Hall, Gelbart & Kankel, 1975).
In the adult gynandromorphs the male and female cells are not freely intermingled, but instead the cells of one genotype are clustered together and cover
1
Authors'1 address: Dept. of Cell Biology, Biozentrum der Universitat, Klingelbergstr. 70,
CH-4056 Basel/Switzerland.
2
Author's address: Zoologisches Institut der Universitat, Kiinstlergasse 16, CH-8006
Zurich/Switzerland.
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W. J. GEHRING, E. WIESCHAUS AND M. HOLLIGER
large continuous areas of the epidermis. The orientation of the genotypic
boundary between XX and XO cells is largely random. The probability of this
boundary passing between two cells at the blastoderm stage, when the embryo
consists essentially of a monolayer of cells, is proportional to the (arc) distance
between them (Sturtevant, 1929; Garcia-Bellido & Merriam, 1969). Since the
location of a cell within the blastoderm largely decides upon its fate, the frequency of the genotypic boundary passing between two adult cells, can be taken
as a measure for the (arc) distance between their primordial cells in the blastoderm (Hotta & Benzer, 1972,1973). By calculating such distances and triangulation, several fate maps of this kind have been constructed (Garcia-Bellido &
Merriam, 1969; Hotta & Benzer, 1972, 1973; Ripoll, 1972; Janning, 19746;
Gelbart, 1974; Baker, 1975). Such gyandromorph maps are generally in agreement with the available embryological data (Poulson, 1965), but in most cases
direct evidence on the location of the primordial cells is scarce.
One cell type which provides an excellent test system for the mapping techniques is the pole cell. The pole cells can easily be identified as a group of round
cells located at the posterior pole of the egg, outside the monolayer of blastoderm
cells, and therefore, they should map as the most posterior structure. At the time
of their formation the pole cells become determined to form the primordial
germ cells (Illmensee & Mahowald, 1974) and possibly the cuprophilic cells of
the midgut (Poulson & Waterhouse, 1960). In the course of development they
migrate into the interior of the embryo and associate with mesodermal cells to
form the gonad.
Gynandromorph mapping of the gonad gave conflicting results: Falk, Orevi
& Menzel (1973) obtained a median and dorsal map position for the larval
gonad. Using different reference structures, Hotta & Benzer (1973) mapped it
close to the posterior end. A posterior location was also found for the adult
gonad (Janning, 19746). In any case, the map position of the gonad as determined in these studies most probably reflects the location of the primordial cells
for the gonadal mesoderm rather than the pole cells. Therefore, we decided to
map the two components of the gonad, germ cells and mesoderm, separately.
In the course of the present study we found that the gonads in gynandromorphs frequently lack mature germ cells. This suggested to us that in these
cases the primordial germ cells were of the opposite sex to the gonad mesoderm,
and therefore, they could not differentiate into mature germ cells. Meanwhile,
this hypothesis has been tested directly by pole cell transplantation. It has been
found that heterosexual transplantations of pole cells are unsuccessful, whereas
homosexual transfers frequently lead to the formation of fertile gametes derived
from the donor pole cells (Illmensee, personal communication; Van Deusen &
Gehring, in preparation). Therefore, the presence or absence of germ cells in a
gonad of a gynandromorph indicates whether or not the genotype of the germ
cells differs from that of the gonadal mesoderm. This gives us a method to map
the germ cells.
Gynandromorphs in Drosophila
609
The mapping data obtained in this way were confirmed by analyzing 'transformed gynandromorphs' (Novitski, 1951; Seidel, 1963) which are homozygous
for the recessive mutant transformer, tra (Sturtevant, 1945; Lindsley & Grell,
1968), Since tra transforms XX females into sterile males, such gynandromorphs look like male mosaics. Because they are all male, the morphology of
the sexual apparatus is normal and easier to interpret. Using the white marker
(see Lindsley & Grell, 1968) the genotype of the mesoderm can be recognized by
the coloration of the testicular sheath. Since XX trajtra pole cells cannot form
sperm (Brown & King, 1961; Seidel, 1963), whereas XO tra/tra pole cells are
able to differentiate into mature sperm (Seidel, 1963) the presence or absence of
mature sperm is an indication of the genotype of the germ cells. Using both
'normal' and 'transformed' gynandromorphs very similar fate maps were
obtained.
METHODS
The gynandromorphs used in this paper were obtained from zygotes of the
following genotypes: In(l)wYCly w/ 36a ('normal' gynandromorph) and 7«(7)wvC/
y w/ 3 6 ; trajtra ('transformed' gynandromorphs) by loss of the unstable ring-X
chromosome, In(l)wyG, during the first nuclear divisions (Hall, et al. 1975).
Thus, the XO-male areas were marked with y, for yellow bristles and chitin
colour,/ 36a , for forked bristles, and w {white) which affects the eye colour and
also the colour of the testis sheath, the vasa deferentia and the Malphighian
tubules (Lindsley & Grell, 1968).
About 30 % of the flies carrying the ring-X chromosome were gynandromorphs. The stocks were maintained at 25 °C on standard food and the adult
flies aged for 3 days before examination, to ensure complete pigmentation of the
testis sheaths. The flies were examined for external mosaicism under a dissecting microscope and the distribution of male and female tissue recorded on
drawings. Subsequently, the gynandromorphs were dissected and drawings were
made of the interior genitalia and the gonads. The presence or absence of germ
cells in the gonads was checked under a compound microscope if necessary.
From both kinds of gynandromorphs, 'normal' and 'transformed', 209
specimens each were examined. The gynandromorphs were selected on the
basis of external mosaicism. A sample of females showing no XO tissue in their
cuticle was also dissected, but no mosaicism affecting the internal genitalia or
the gonads was detected. As a further control 100 XO males were dissected, and
no internal mosaicism was found. Of the 209 'normal' gynandromorphs 109
were selected on the basis of abdominal mosaicism. This leads to a slight
increase in all the sturt distances of the fate map. However, it does not significantly influence the results, since we are interested in relative rather than
absolute distances of abdominal structures. If the chromosome elimination
occurs during the first nuclear division, which is randomly oriented, and if the
two daughter nuclei divide at the same rate, one would expect a given structure
610
W. J. GEHRING, E. WIESCHAUS AND M. HOLLIGER
Table 1. Sturt distances between the germ cells, gonadal mesoderm and adult
cuticular structures of the abdomen for 'normal' and 'transformed' gynandromorphs
T2 S2 T3 S3 T4 S4 T5 S5 T6 S6 T7 S7 GD GN GE
' Normal'
GD*
GNf
GEJ
'Transformed'
GD§
GN||
GEJ
43 38 43 35 39 32 38 32 35 28
40 31 41 31 38 27 38 27 35 23
42 41 43 38 42 36 41 35 38 32
35 26 — 15 25
35 22 15 — 18
34 31 25 18 —
33 31 34 32 33 30 32 28 32 — — —
38 35 39 37 40 35 41 34 40 — — —
40 39 38 38 36 36 33 33 31 — — —
— 21 24
21 — 33
24 33 —
Abbreviations: T2-T7 = 2nd-7th tergite; S2-S7 = 2nd-7th sternite; GD = genitalia
derived from genital disc; GN = gonadal mesoderm; GE = germ cells.
Criteria for mapping:
* External and internal genitalia were scored for their sex and/or ywf.
t The sex of the gonads was scored. Mosaic testes with w+ and w cells in their sheaths can
be recognized and were recorded as mosaics.
$ Presence or absence of mature germ cells was recorded.
§ Only external genitalia can be scored.
|| The testicular sheaths were examined for chromosomal mosaicism which can be distinguished from vt>/Hte-variegation.
X Presence or absence of sperm was recorded.
Based upon 209 normal and the same number of transformed gynandromorphs, i.e. 418
sides each. Structures which are mosaics of JOfand XO cells were counted as half female and
half male.
to be male in 50 % of the gynandromorphs. For 'normal' gynandromorphs we
found an average of 49 %, for 'transformed' a somewhat lower value of 39 %
was obtained.
RESULTS
In gynandromorphs, the probability that the randomly oriented genotypic
boundary passes between two adult structures, can be taken as a measure for
their distance in the embryonic fate map (Sturtevant, 1929; Garcia-Bellido &
Merriam, 1969; Hotta & Benzer, 1972). A probability of 1 % is designated as 1
sturt unit (Hotta & Benzer, 1972). The germ cells and gonadal mesoderm were
mapped relative to the abdominal tergites and sternites, each of which is formed
by a group of histoblasts, and relative to the genitalia which are derived from
the genital disc. All these structures are multicellular, but they originate from
separate primordia. Table 1 gives the calculated distances in sturts for a given
pair of structures in both 'normal' and 'transformed' gynandromorphs. These
data can be summarized as follows:
(1) The distance between the germ cells (GE) and the sternites (S) or the
Gynandromorphs in Drosophila
S2
I
611
S3
10 STURTS
Fig. 1. Fate map of the germ cells, gonadal mesoderm, and cuticular structures of the
abdomen derived from the analysis of (A) 'normal', and (B) 'transformed'
gynandromorphs (lateral view).
This map was constructed by first triangulating the cuticular structures relative
to their nearest neighbour. Subsequently, the germ cells (GE) and the gonadal mesoderm (GN) were mapped relative to the three nearest cuticular markers. Depending
upon which pair of cuticular markers are used for triangulation three different map
positions are obtained (shaded area). For abbreviations see Table 1.
tergites (T) decreased with increasing segment number. The closest cuticular
markers are the genitalia (GD) which map posterior to the last tergite and
sternite. Therefore, the germ cells map as the posterior most of these structures.
(2) In the 'normal' gynandromorphs GE consistently maps closer to S than
to T for a given segment, but the difference is small. In 'transformed' gynandromorphs the GE-S distances are approximately equal to the GE-T values
for a given segment, which indicates that GE is located in the median plane or
slightly ventral to it.
(3) The gonadal mesoderm (GN) also maps farthest from the first and closest
to the last abdominal segment, but in this case the distances between S and GN
are always considerably shorter than the distances relative to T. Therefore, the
mesoderm maps close to the posterior end, on the ventral side.
Fate maps derived from these data by triangulation are shown in Fig. 1. The
triangulation method gives slightly ambiguous maps since the map distances are
not strictly additive, but even when different reference points are used, the
612
W. J. GEHRING, E. WIESCHAUS AND M. HOLLIGER
Table 2. Left-right distances, frequency of perfect left-right division,
and frequency of mosaicism within a given structure
Structures
r
A
1
Normal Transformed
Frequency oft
mosaicism (;%)
Frequency of perfect
L-R division (%)
L-R distances*
\
Normal
Transformed
33
37
32
26
26
—
44
44
44
39
—
—
3
0
T2
T3
T4
T5
T6
T7
45
48
46
44
43
36
38
40
39
39
37
—
39
43
43
40
37
S2
50
44
S3
S4
S5
S6
S7
GD
GN
GE
49
48
48
44
43
13
13
13
46
45
42
—
—
10
12
47
47
47
49
46
46
2
5
13
34
Normal % Transformed
4
5
3
2
5
1
1
1
1
0
0
0
21
191
7
10
10
11
10
—
2
3
3
4
—
—
11§
37
12
* In sturts.
t In order to calculate the frequency of mosaicism, GD, GN and GE were treated as
single primordia.
% Based upon 100 'unselected' gynandromorphs.
§ Only the external genitalia can be scored.
|| The ovarian sheath cannot be scored.
relative positions of GE and GN are the same: GE is the posterior most
marker, GN is more ventral and GD is intermediate, between GE and GN
(shaded areas in Fig. 1).
Information about the relative distance from the ventral or dorsal midline
can be obtained by calculating left-right distances. For a pair of bilaterally
symmetrical structures, the left-right distance represents twice the (arc) distance
to the midline (Hotta & Benzer, 1972). In accordance with previous studies
(Hotta & Benzer, 1973) we found that the left-right distances varied between
36-45 sturts for the tergites and between 42-50 sturts for the sternites, the last
sternite and tergite giving the smallest value (Table 2). In. contrast to these large
left-right distances for S and T, the values for GD, GN and GE are considerably
smaller ranging from 10-13 sturts, indicating that they map very close to the
midline (5-7 sturts). This suggests that there is a single primordium near the
ventral midline rather than a symmetrical pair of separate primordia. This
assumption was tested further by examining the genotypic boundary separating
the structures from left to right, and asking how frequently this boundary
passes precisely between the left and right structure without cutting into either
of the two structures. As indicated in Table 2 the frequency of perfect left-right
Gyncmdromorphs in Drosophila
613
division is high for S and T but extremely low for GD, GN and GE. Since the
frequency of perfect division is related to the number of cells separating two
given primordia (Wieschaus & Gehring, 19766) these data strongly support the
assumption that there is a single median primordium for each of these three
structures.
For a given structure, the frequency of mosaicism is a measure for the
relative size of its primordium or the number of primordial cells. The larger a
primordium, the higher the probability that the genotypic boundary crosses it.
Assuming a circular primordium, the radius (r) of the primordium equals the
frequency of mosaicism (/) divided by n (Hotta & Benzer, 1973). For the
tergites and sternites the frequency of mosaicism is smaller than 11 % (Table 2),
indicating relatively small primordia of less than 7 sturts in diameter. In contrast,
GD, GN and GE show much higher frequencies of mosaicism. Especially the
frequencies for the gonadal mesoderm (37 %) and the genitalia (21 %) are high,
which suggests unusually large primordia. Alternatively, it could be assumed
that the lineage of these cells is not yet determined at the blastoderm stage,
leading to an 'expansion' of the map (see discussion), if some of the cells within
the primordium give rise to other structures as well.
DISCUSSION
Different criteria for mapping which have been used in this study need some
evaluation. Autonomous recessive marker genes were available for the cuticular
structures (y w/ 36a ) which allows an unambiguous identification. In the
'normal' gynandromorphs the sexual phenotype was used as a marker for the
inner genitalia and as an additional marker for the outer ones. Since the genitalia differentiate autonomously (Kroeger, 1959; Nothiger, 1964) this is justified.
The sex of the gonad was used as a marker for the gonadal mesoderm. In the
testicular sheath the w+ marker can be used to identify XX female cells, which
are capable of non-autonomous differentiation into phenotypically male cells
(Dobzhansky, 1932; Janning, 1974a). The testicular sheath cells also cover the
vasa deferentia (Stern & Hadorn, 1939). No such marker was available for the
ovarian sheath. The germ cells were mapped on the basis of the presence or
absence of mature germ cells. It is more precise to say that we mapped the site
for the ability to differentiate germ cells. However, we have shown by pole cell
transplantation, that the pole cells differentiate autonomously according to
their chromosomal constitution and are incapable of differentiating into germ
cells of the opposite sex (Van Deusen & Gehring, in preparation). Therefore, the
site for the ability to form mature germ cells coincides with the location of the
pole cells, provided that the germ cells are surrounded by a normal mesoderm.
In all cases where the sexual phenotype was used as a marker, small mosaic
areas are likely to be missed since a critical cell number may be needed for a
given structure to differentiate and to be identified as being male or female. In
39
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35
614
W. J. GEHRING, E. WIESCHAUS AND M. HOLLIGER
our sample of gynandromorphs missing structures were relatively rare: only one
animal had no gonads, and a single gonad was present in only 3 % of the cases.
The use of ' transformed' gynandromorphs has several advantages. Since they
are all male in their somatic tissues, their sexual apparatus is normal and easier
to interpret than normal gynandromorphs which are a complex mixture of male
and female parts. All gonads are testes and the genotype of the mesoderm
sheath cells can be recognized by their colour, XX showing yellow colour and
XO cells being white. The chromosomal mosaicism can clearly be distinguished
from white-variegation due to the wvC marker. The white-variegation results in
a 'salt and pepper' type intermingling of phenotypically w and w+ cells, whereas
the chromosomal mosaicism results in the formation of large patches of tissue
of one genotype. XX tra/tra pole cells are incapable of forming mature sperm
(Brown & King, 1961; Seidel, 1963). XO traftra germ cells give rise to mature
sperm which, however, is immotile due to the absence of a 7 chromosome
(Seidel, 1963). Therefore, the presence or absence of sperm indicates the genotype of the pole cells. The use of transformed gynandromorphs also has some
disadvantages, since the internal genitalia, which are all male and unmarked,
cannot be mapped. Also the male lacks the 7th tergite and both the 6th and 7th
sternites which makes mapping of the posterior structures more difficult. Nevertheless, the two methods together give consistent mapping data.
The pole cells are of particular interest since it has been shown by cytoplasmic
transplantation experiments that there are 'determinative factors' in the egg
cytoplasm near the posterior pole which induce the formation of pole cells
(Illmensee & Mahowald, 1974), providing the best experimental evidence for
•cytoplasmic localization. The present study clearly shows that the germ cells
map as the posterior most structure, which coincides with the position of the
pole cells at the blastoderm stage. Therefore, there is no need to invoke a mechanism of determination operating prior to the blastoderm stage as suggested by
Falk et al. (1973). Since immediately after blastoderm formation, the pole cells
migrate anteriorly and penetrate into the embryo, this map location provides
some direct evidence that the map does in fact relate to the blastoderm stage and
not to a later stage of development. Furthermore, it indicates that the mapping
•data correlate well with the actual position of the primordial cells.
The gonadal mesoderm, the genital disc, and the pole cells each map as separate primordia along the ventral midline in this anterior-posterior order, behind
the last abdominal sternites and tergites. A surprisingly high frequency of
mosaicism was found for the gonadal mesoderm (37 %) as compared to less than
4 % for the sternites and less than 11 % for the tergites. The value for the genital
disc (21 %) also appears very high, since it is known from histological studies
that the disc originates from a very small number of cells (Lauge, 1967). The
large frequencies of mosaicism for these structures can be explained by assuming
that the cell lineage of the blastoderm cells in those regions is not strictly fixed
and they can give rise to structures other than the genitalia. As pointed out
Gynandromorphs in Drosophila
615
clearly by Hotta & Benzer (1973), an indeterminacy of the cell lineage causes an
expansion of the map. Such a map expansion has been found in ' fine-structure'
maps of the leg discs (Wieschaus & Gehring, 1976a). The data presented above
suggest that map expansion also occurs over short distances between separate
primordia.
The gynandromorph mapping technique is based upon the assumption that
the location of a cell within the blastoderm largely decides upon its fate, but is
not known how precisely the location correlates with the fate. The fact that a
self-consistent map can be constructed confirms in a general way the validity of
this assumption. There is some direct evidence on this point, since we have
shown by dissociation and reaggregation experiments that cells from the
anterior region of the blastoderm can produce anterior adult structures but not
posterior ones, and vice versa (Chan & Gehring, 1971). This was confirmed by
transplantation of single blastoderm cells (Illmensee, unpublished). However,
recent experiments indicate that at least some blastoderm cells can still give rise
to cells from two kinds of imaginal discs and are not yet disc-specifically
determined (Wieschaus & Gehring, 19766). Such an indeterminacy of the cell
lineage is expected to lead to a map expansion. The map expansion found for
the genital disc and the gonadal mesoderm suggests that some indeterminacy of
the cell lineage also exists for these structures.
ZUSAMMENFASSUNG
Die Mosaikmuster von 'normalen' und ' transformierten' Gynandern wurden analysiert
und die Lage der Urkeimzellen und des Gonaden-Mesoderms im Anlageplan bestimmt. Im
Bezug auf die adulten Kutikularstrukturen kartieren die Urkeimzellen als kaudalste Struktur
im Anlageplan, was ihrer Position am Hinterpol des Blastoderm-Embryos entspricht.
Dieser Befund bestatigt die Hypothese, dass die Gynanderkarte die wirkliche Lage der
Primoridalzellen im Embryo angibt, und dass diese Karte dem Blastodermstadium entspricht.
Die Primordialzellen fur die Genitalscheibe und das Gonadenmesoderm liegen ventral in der
Medianebene vor den Urkeimzellen. Die Anlagen fur die Genitalscheibe und das Gonadenmesoderm umfassen relativ zu grosse Areale, was daraufhin deutet, dass diese Primordialzellen auf dem Blastodermstadium noch nicht determiniert sind, oder dass noch Primordialzellen fur andere Strukturen in diesen Arealen lokalisiert sind.
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