J. Embryo!, exp. Morph. Vol. 24, 2, pp. 367-380, 1970
2)61
Printed in Great Britain
The origin and movement
of nephrogenic cells in the chick embryo as
determined by radioautographic mapping
By GLENN C. ROSENQUIST 1
From the Department of Pediatrics, The Johns Hopkins University
SUMMARY
The origin of the presumptive nephrogenic cells in the epiblast of the chick embryo was
traced by radioautographic analysis of the movements of tritiated thymidine-labelled grafts
excised from medium-streak to 5-somite stage embryos and transplanted to epiblast, streak,
and the endoderm-mesoderm layer of similarly staged recipient embryos.
The nephrogenic cells originate near the area pellucida margin of the medium-streak-stage
embryo, migrate toward the streak, and are invaginated about one-third to one-half the
distance from the anterior to the posterior end of the streak, between the definitive-streak
and I - to 4-somite stages. Their route into mesoderm is along a relatively narrow pathway between the cells migrating to the paraxial or presomite mesoderm on one side, and those
destined for the proximal limbs of the lateral plate on the other.
The cells which will form the anterior part of the intermediate mesoderm are the most
medially placed cells in epiblast, reach the streak at an earlier stage of development, and are
the first nephrogenic cells to migrate into mesoderm. After about the 17- to 19-somite stage,
cells from this group which have formed the pronephric cord or duct begin to move
posteriorly in relation to the rest of the intermediate mesoderm, toward the future cloaca.
The last nephrogenic cells to leave epiblast and enter the streak and mesoderm are those
destined for the posterior end of the intermediate mesoderm. This group of cells surrounds
the posteriorly migrating pronephric (Wolffian) duct and differentiates into mesonephros.
INTRODUCTION
The radioautographic mapping of the primitive-streak to head-process stage
chick embryo (Rosenquist, 1966) demonstrated that the intermediate (presumptive nephrogenic) mesoderm originates in the epiblast layer lateral to the
primitive streak (Fig. 1, left side), migrates medially to the streak, and is invaginated at a position about one-third of the distance from the anterior to the
posterior end of the streak. Considerable mixing of the premesodermal cells takes
place at the streak, resulting in the crossover of some cells into the mesoderm
layer opposite the side of their origin. After entering the mesoderm, the cells
destined for intermediate mesoderm migrate anteriorly and laterally from the
streak into the isthmus which joins the paraxial accumulation of mesoderm to
1
Author's address: Cardiac Clinic, Children's Medical and Surgical Centre, The Johns
Hopkins Hospital, Baltimore, Maryland 21205, U.S.A.
368
G. C. ROSENQUIST
the somatic and splanchnic leaflets of the lateral plate (Fig. 1, right side). Since
the previous mapping study did not include embryos older than the 17-somite
stage, the differentiation of the nephros from the intermediate mesoderm
was not investigated.
4?
x
a
. - • • • •
Fig. 1. Diagram (after Rosenquist, 1966) which illustrates that at the medium-streak
stage, the strip of epiblast between cells which will form paraxial and those which
will form lateral plate mesoderm extends laterally from the streak, and is the origin
in epiblast of the nephrogenic cells (left). After these cells have been invaginated
at the streak, they migrate as intermediate mesoderm anteriorly and laterally from
the streak between the paraxial mesoderm (medially) and the lateral plate mesoderm (laterally). (Head-process stage, right.)
Using radioautographic analysis, the present investigation traces the movements of tritiated thymidine-labelled transplants from their original positions
in the nephrogenic regions of epiblast and streak of recipient embryos, into the
ducts, tubules, and undifferentiated intermediate mesoderm of the host
embryos.
Origin of nephrogenic cells
369
MATERIALS AND METHODS
Embryos were removed from white leghorn eggs incubated 10-26 h. The vitelline membranes to which the embryos remained attached were secured to a glass
ring and suspended over a pool of albumen (New, 1955). Embryos were staged
by the criteria below (Fig. 2; Rosenquist, 1970).
Medium-streak stage (MS). There is a slightly pear-shaped area pellucida, with
a moderately broad but well-formed primitive streak which may extend 5060 % of the length of the area pellucida (Hamburger & Hamilton, 1951, p. 54,
stage 3; plate 1, stage 3 + ).
Late medium-streak stage (LMS). There is a slightly pear-shaped area pellucida, with the anterior end wider than the posterior end. The streak is approximately 65 % of the length of the area pellucida, and the length of area pellucida
is greater than at the previous stage (Hamburger & Hamilton, 1951, p. 54,
stage 3).
Definitive-streak stage (DS). The length of the streak is 70-80 % of the length
of the area pellucida. The anterior end of the streak is compact and rounded,
and is more dense in appearance than at the late medium-streak stage. Hypoblast folds at anterior margins of area pellucida are well developed. The streak
splits easily at the midline into lateral halves (Willier & Rawles, 1931, DPS stage;
Rudnick, 1932, DPS stage; Spratt, 1942, DS stage; Hamburger & Hamilton,
1951, plate 2, stage 4).
Head-process stage (HP). A short straight head process extends anteriorly
from the anterior end of streak, which is 65-80 % of the length of the area
pellucida, depending upon the amount of increase in size of the latter. There is
no evidence of amniocardiac vesicles upon microscopic investigation. The streak
easily splits at the midline into lateral halves (Willier & Rawles, 1931, HP stage;
Rudnick, 1932, HP stage; Spratt, 1948, HP stage; Hamburger & Hamilton, 1951,
plate 2, stage 5).
Head-fold stage (HF). There is a long head process with anterior accumulation
of cells in a shallow depression (future site of the foregut) which has begun to
fold, forming an anterior intestinal portal (Rudnick, 1932, HF stage; Spratt,
1942, HF stage; Hamburger & Hamilton, 1951, plate 2, stage 6).
Somite stages. Stages after the head-fold stage are determined by the number
of pairs of somites present, e.g. stage 4S refers to an embryo with four pairs
of somites (Willier & Rawles, 1931, stage 4S; Hamburger & Hamilton, 1951,
plate 2, stage 8).
Early limb-bud stage (ELB). In the two embryos which had progressed to the
early limb-bud stage, the number of somites could not be determined accurately.
In both cases the head was slightly stunted in growth, and although the heart
was beating at the time the embryos were fixed, circulation appeared to have
stopped. (The early limb-bud stage is equivalent to Hamburger & Hamilton,
1951, plate 6, stages 17-19.)
370
G. C. ROSENQUIST
Donor embryos were labelled by placing 1 /<Ci [3H]thymidine in 0037 ml
physiologic saline solution on the hypoblast surface of the explanted embryo.
The donors were then reincubated until they reached the stages of the recipients
(1-5 h), and washed six times with 1-2 ml saline at room temperature. This
Fig. 2. Series of diagrams showing the approximate position of the anterior migrating
nephros (AMN) and the posterior migrating nephros (PMN) at various stages of
development. At the medium-streak (MS) stage, the AMN was located in the
epiblast (striped areas in left half of each figure). It migrated progressively toward
the streak, which it reached at about the late medium-streak (LMS) to definitivestreak (DS) stages. By the head-process (HP) stage, a part of the AMN had migrated
through the streak into the mesoderm layer (right half of each figure), reaching its
definitive position in the intermediate mesoderm at about the 8-somite stage. After the
17- to 19-somite (17-19S) stage, the pronephric (Wolffian) duct, as part of the AMN
(solid line extending posteriorly from the striped areas in 1 and J) began to grow
posteriorly in relation to the rest of the intermediate mesoderm. The PMN (shaded
areas) originated at the lateral margin of the area pellucida of the MS stage embryo,
and followed behind the AMN as it migrated through the epiblast, streak and mesoderm. By the early limb-bud (ELB) stage, the PMN had reached the posterior part
of the intermediate mesoderm, where it had been invaded by the posteriorly growing
pronephric duct. As in the text, an asterisk (*) after the embryo number indicates that
the position shown is the location of the graft after its migration in the host embryo.
Each graft is therefore shown twice—before and after incubation. HF = head-fold
stage.
Origin of nephrogenic cells
371
procedure labels 50-70% of the cells, which is sufficient to trace the movements
of the grafts with radioautography (Rosenquist, 1966). Grafts were excised from
the donor embryos and transplanted to identical sites in the epiblast, streak
or endoderm-mesoderm (hypoblast) layer of similarly staged recipient embryos,
and the graft position was recorded by photographs or sketches of the recipient
embryos (Rosenquist, 1966).
The recipient embryos were incubated in sealed moisturized cans containing
atmospheric air for 4-38 h following the grafting operation. Seventeen of the
embryos were then re-photographed or sketched, fixed in 10 % formol in physiologic saline, embedded in paraffin wax, sectioned serially at 10/A, radioautographed, stained and reconstructed after study (Rosenquist, 1966). The two
embryos which survived to limb-bud stages, and two which reached the 23 +
somite stage, were selected when they had developed good extra-embryonic circulation (16- to 19-somite stage); they were turned upside down on a physiologic agar
(Howard, 1953) and reincubated in sealed cans containing a moisturized mixture
of 95% oxygen and 5 % carbon dioxide. Additional chick albumen was added to
the exposed vitelline membrane from time to time. These four embryos had a total
incubation time of 41-63 h after the grafting procedure, and they were re-photographed, fixed, etc., in the same manner as were the first seventeen embryos.
Of the twenty-one embryos to be reported, none which received grafts at the
medium-streak stage developed all the way to the early limb-bud stage. Therefore,
to trace the movements of the nephrogenic cells from their origin in epiblast at the
medium-streak stage, to a position in intermediate mesoderm at somite stages, it
was necessary to combine the pathways followed by two or three labelled grafts.
For example, a graft transplanted to the epiblast about half-way between the
margin of the area pellucida and the streak (embryo 3, Fig. 2B) migrated to
the centre of the streak (embryo 3*, Fig. 2C); a graft placed at the centre of the
streak (embryo 11, Fig. 2 C) moved from that position into intermediate mesoderm
(embryo 11*, Fig. 2H).
Although part of each of the twenty-one transplants illustrated in Fig. 2 lay in
the nephrogenic region, the number of embryos investigated was relatively small,
and each transplant contained cells other than nephrogenic cells (as indicated
in Table 1). Therefore the positions of the transplants at each stage in Fig. 2
suggest the location of the nephrogenic region at that stage, but do not define it
precisely. The mapping of the nephrogenic region is based upon the following
assumptions: (1) that previous studies have established the general position of
the intermediate mesoderm in the epiblast, streak and mesoderm layers of the
chick blastoderm without mappingevery part of these layers at each stage (Rosenquist, 1966). Consequently, a small number of transplants, carefully placed, can
demonstrate the position of more specific portions of the intermediate mesoderm,
such as the kidney. (2) That graft positions in different embryos at the same stage
are homologous even if the embryos were incubated for different lengths of time,
and that the pathways followed by more than one accurately placed graft can be
24
KM R 2 4
372
G. C. ROSENQUIST
used to follow movements of a group of cells through several stages of development. (3) That maps of presumptive organ-forming regions of the embryo are
valid even if structures other than nephros in the recipient embryos contain
labelled cells.
Throughout the text and figures, an asterisk (*) after the embryo number
indicates that the position shown is that of the graft after its migration in the
host embryo.
RESULTS
The presumptive nephrogenic cells of the chick blastoderm are divided into
two categories. The first, the anterior migrating nephros (AMN), includes the
entire pronephros and the anterior part of the mesonephros as they were described by Abdel-Malek (1950). The AMN is so named because its definitive
position, after it migrates from the epiblast through the streak into mesoderm,
is anterior to that of the second category of presumptive nephrogenic cells,
the posterior migrating nephros (PMN). The latter category includes the major
portion of the mesonephros, and the metanephros (Abdel-Malek, 1950). In the
following description only the migration of the nephrogenic cells is described.
It must be remembered that cells from each graft contributed to other tissue as
well, as noted in Table 1.
Movement of the anterior migrating nephros (AMN). This is demonstrated by
transplants contributing to the intermediate mesoderm lateral to the 1st—16th
somites. AMN is represented in Fig. 2 as striped areas.
At the medium-streak stage, the AMN was found in the epiblast about halfway between the primitive streak and the lateral margin of the area pellucida,
as illustrated by the migrating grafts in embryos 1 and 2 (Table 1 and Fig. 2 A).
At the late medium-streak stage, the AMN had moved closer to the primitive
streak (embryos 1* and 3-10, Table 1 and Fig. 2B). At the definitive-streak
stage, it had reached the primitive streak at a position which was approximately
one-third to one-half the distance from the anterior to the posterior end of the
streak (embryos 3*, 11 and 12, Table 1 and Fig. 2C). At the head-process stage,
the AMN had begun to migrate into mesoderm adjacent to the streak (embryos
4* and 13, Table 1 and Fig. 2D). The route of the later migration from the
streak was anterior and lateral; the AMN was parallel to the mesoderm cells
destined for the lateral plate (which were located posterior and lateral to the
AMN, Fig. 1, right side) and also to those destined for paraxial mesoderm
(which were located anterior and medial to the AMN, Fig. 1, right side). At the
head-fold to 1-somite stage, the AMN had moved farther into the mesoderm
layer (embryos 5* and 6*, Table 1 and Fig. 2E), although some cells were still
in the primitive streak (embryos 14-16, Table 1 and Fig. 2E).
By the 4- to 6-somite stage, some of the AMN had reached its definitive position
lateral to the first 4-6 somites (embryos 2* and 7*, Table 1 and Fig. 2F). At the
8- to 9-somite stage, additional AMN had reached the definitive position in intermediate mesoderm (embryos 8* and 9*, Table 1 and Fig. 2G). At the 17- to 19-
373
Origin of nephrogenic cells
Table 1. Position of labelled cells in recipient embryos
carrying tritiated thymidine-labelled grafts*
Embryo
no.
Stage
grafted
Incubatedf
(h)
Stage
fixed!
1
2
3
4
5
6
7
8
MS
MS
LMS
LMS
LMS
LMS
LMS
LMS
4
20
8
4
18
16
20
19
LMS
6S
DS
HP
HF
HF
6S
8S
9
10
LMS
LMS
18
63
9S
ELB
11
12
DS
DS
32
26
17S
17S
13
HP
26
19S
14
HF
41
*
15
HF
43
16
IS
38
17
18
19
20
21
MS
MS
MS
DS
5S
18
18
21
8
50
4S
5S
4S
HP
ELB
Position of labelled cells
Ep near St
LP and I M anterolateral to St
Ep and LP M in and near St
LP M anterolateral to St
LP M anterolateral to St
LP M anterolateral to St
DA, LP and I M anterolateral to St
DA, LP, Px and I M anterolateral
to St
DA, LP and IM anterolateral to St
IM anterolateral to St; labelled PD
surrounded by non-labelled PMN
for 750 fi
DA, LP and I M anterolateral to St
DA, LP, Px and I M anterolateral
to St; YS En near St
DA, LP, Px and I M anterolateral
to St; YS En near St
DA, LP, Px and I M anterolateral
to St; labelled PD surrounded
by unlabelled PMN for 580 ft;
YS En near St
DA, LP, Px and I M anterolateral
to St; labelled PD surrounded
by unlabelled PMN for 340/*;
YS En near St
DA, LP, Px and I M anterolateral
to St; labelled PD surrounded
by unlabelled PMN for 540 ft;
YS En, Ep and M in and near St
Ep and M in and near St
Ep and M in and near St
Ep and M in and near St
Ep and M in and near St
DA, LP, Px and I M anterolateral
to St; unlabelled PD surrounded
by labelled PMN for 1970/A;
YS En and Ep near St
* Key to abbreviations: AMN, anterior migrating nephros; DA, dorsal aorta; DS,
definitive-streak stage; ELB, early limb-bud stage; En, endoderm; Ep, epiblast; HF, headfold stage; HP, head-process stage; I, Intermediate; LMS, late medium-streak stage; LP,
lateral plate; M, mesoderm; MS, medium-streak stage; PD, pronephric duct; PMN, posterior
migrating nephros; Px, paraxial; S, somite; St, streak; YS, yolk-sac.
t Hours of incubation after placement of the graft.
X Asterisk (*) refers to stage after 23-25 somites, but before the development of limb buds.
Exact number of somites could not be determined.
24-2
374
G. C. ROSENQUIST
somite stage, a ridge had formed along the dorsal surface of the intermediate
mesoderm, consisting of cells which had emerged from the 4th—16th nephromeres (Abdel-Malek, 1950), and which had fused to form the rudiments of the
Wolffian ducts (embryos 11*—13*, Table 1 and Fig. 2H). As yet none of the
labelled cells in this ridge appeared posterior to the labelled cells in the strip of
Al
Origin of nephrogenic cells
375
undifferentiated intermediate mesoderm which separated the somites and the
leaves of the lateral plate. By the 22- to 23-somite stage, parts of the AMN forming
the Wolffian duct had begun to extend posteriorly through non-labelled intermediate mesoderm, toward the hind gut (embryos 14*—16*, Table 1, Figs. 21
and 3 B). At the early limb-bud stage, the Wolffian duct had reached the future
position of the cloaca (embryo 10*, Table 1 and Fig. 2J). In embryos 10 and
14-16, occasional labelled cells clung to this posteriorly extended labelled cord
without being a part of its relatively compact mass; it is not clear whether these
cells were pulled passively toward the cloaca as the duct migrated posteriorly,
or whether they migrated from the cord into the non-labelled intermediate
mesoderm surrounding it.
Movement of the posterior migrating nephros (PMN). This is demonstrated by
transplants contributing to the intermediate mesoderm surrounding the
Wolffian duct, posterior to the 16th somite. PMN is represented in Fig. 2 as
shaded areas.
At the medium-streak stage, the PMN was located in the epiblast near the
lateral margins of the area pellucida, at about the level of the anterior end of the
streak (embryos 17-19, Table 1 and Fig. 2A). By the definitive-streak stage,
the PMN was still near the area pellucida margin, but it was more posteriorly
placed in relation to the streak (embryo 20, Table 1 and Fig. 2C); this apparent
posterior movement was probably related to the anterior growth of the streak in
relation to the anterolateral margin of the area pellucida (Rosenquist, 1966)
and was thus not an actual migration of cells. At the head-process stage, the
PMN had migrated toward the streak (embryo 20*, Table 1 and Fig. 2D), and
parts of it may have entered the centre of the streak at approximately the same
position at which the AMN entered (one-third to one-half the distance from the
anterior to the posterior end of the streak). The remainder of the PMN at this
stage extended laterally and anteriorly from the streak as a narrow strip of
labelled epiblast, demonstrating how a square-shaped graft transplanted to epiblast elongates during its migration toward the streak. By the 4- to 6-somite stage,
FIGURE 3
(A) Cross-section showing labelled cells (black dots over nuclei) from a graft placed
originally in epiblast lateral to the streak, which have moved into the primitive streak
at the 5-somite stage (embryo 18, Table 1 and Fig. 2F). Ep — epiblast; M— mesoderm;
En = endoderm layers, x 200.
(B) After the 19-somite stage, the pronephric (Wolffian) duct containing labelled
cells in embryo 16 began to grow posteriorly into the non-labelled intermediate
mesoderm. In this cross-section the Wolffian duct has no lumen, and is surrounded
by non-labelled cells from the posterior migrating nephros. WD = Wolffian
duct. x250.
(C) Cross-section of the intermediate mesoderm of embryo 21, showing the Wolffian
duct (unlabelled, and therefore composed of cells from the recipient embryo)
surrounded by the posterior migrating nephros (composed of labelled cells from the
transplant.) WD = Wolffian duct, x 250.
376
MS (4-6 h inc.)
G. C. ROSENQUIST
LMS (10 h inc.)
DS (12 nine.)
HP
HF
Origin of nephrogenic cells
311
the PMN had moved through the streak into the migration pathway which
leads to the intermediate mesoderm (embryos 17*—19* and 21, Table 1 and Fig.
2F). Fig. 3 A shows labelled cells in the streak of embryo 18. At the time of
fixing, part of the grafted tissue in this embryo had not yet entered the streak,
part was in the streak, and part had already left the streak for intermediate mesoderm. By the early limb-bud stage, the PMN had migrated from the streak into
a column of intermediate mesoderm posterior to the 23rd somite, where it had
begun to differentiate into nephric ducts and tubules (embryo 21*, Table 1,
Fig. 2J, 3C). It did not contribute to the Wolffian duct. Although no embryos
were available which showed the position of the PMN between the 4- to 6-somite
stage and the early limb-bud stage, it may be assumed that the PMN continued in
the route it was following at the 4- to 6-somite stage (embryos 17*—19*, Fig. 2F)
until it entered intermediate mesoderm and formed mesonephric tubules
(embryo 21*, Fig. 2J).
DISCUSSION
Locating the nephrogenic cells
The first successful attempts to locate the nephrogenic cells in the chick
blastoderm utilized a method of culturing blastoderm fragments on the chorioallantoic membrane of host embryos until they differentiated. Hoadley (1926)
divided embryos of 4-6 h incubation into five full-thickness (epiblast and hypoblast) horizontal strips (Fig. 4, first row), and transplanted each one to the
chorioallantoic membrane of a host embryo. After 7-9 days incubation in this
culture medium, convoluted tubules lined with columnar epithelium differentiated from the strip of embryo which included the anterior part of the streak.
Since Hoadley described the donor embryos as having a broad primitive streak,
they are probably comparable in staging to the medium-streak-stage recipient
embryos of the present investigation. The findings of the present study are consistent with those of Hoadley. The present study indicates that at the mediumstreak stage the nephrogenic cells are in the epiblast layer between the lateral
margin of the area pellucida and the streak; this site was present in the donor
strip of Hoadley which included the anterior part of the streak (compare Figs. 1
and 4).
Similar convoluted tubules differentiated in chorioallantoic membrane culFig. 4. Diagram showing the position of the presumptive nephrogenic tissue as
identified by previous investigators using grafts to chorioallantoic membrane
(striped areas). First row: Hoadley (1926); second row: Hunt (1931); third row:
Rudnick (1932); fourth row: Rawles (1936). The position of the nephrogenic areas
shown here is consistent with the findings of the present study; however, previous
investigators used very large grafts, and did not distinguish between the nephrogenic
cells of the epiblast and hypoblast. Their experiments were therefore unsuitable for
tracing morphogenetic movements of the nephrogenic cells. Stages: MS (medium
streak), LMS (late medium streak), DS (definitive streak), HP (head process), HF
(head fold).
378
G. C. ROSENQUIST
tures from fragments cut from definitive-streak-stage embryos (Hoadley, 1926;
Hunt, 1931; Rudnick, 1932), head-process-stage embryos (Hoadley, 1926;
Rudnick, 1932; Rawles, 1936), and head-fold-stage embryos (Hoadley, 1926;
Hunt, 1931). Rudnick and Rawles made longitudinal cuts in addition to the
horizontal incisions (Fig. 4, third and fourth rows), dividing each horizontal
strip into right, left, and medial fragments. Since nephrogenic material was
found in all three, it was concluded that at the definitive-streak to head-process
stages, nephrogenic cells are present near the anterior end of the streak, both in
the midline and on each side of the streak. This is also in agreement with the
present investigation (Figs. 1, 2C) because by the definitive-streak stage, some of
the nephrogenic cells have reached the streak, while others are in a position in
the epiblast lateral to the streak (Fig. 2). By the head-process stage, nephrogenic cells are distributed to the mesoderm layer in addition to the epiblast and
streak (Fig. 2D). This investigation indicates therefore that although the chorioallantoic membrane grafts were useful in the general localization of the nephrogenic areas of the chick blastoderm, they were not satisfactory for tracing
morphogenetic movements. First, they did not distinguish between nephrogenic cells in the epiblast and those in the hypoblast, and second, they were too
large to show the location of the nephrogenic cells as precisely as does the present
method of grafting.
Neither the present nor previous investigations provide definite information
about the extreme posterior end of the intermediate mesoderm. It is presumed
that some of each type of mesoderm (i.e. lateral plate, intermediate and paraxial)
continues to be invaginated as long as the streak is active. In embryo 21, the streak
was still visible at the time of fixing, although it was quite short; it is not clear
whether gastrulation was completed at this time. It is not clear whether the labelled
cells seen in the epiblast layer next to the streak in this embryo represent the end
of the intermediate mesoderm, i.e. part of the metanephros which has not yet
been invaginated, or whether they are ectodermal elements destined for epidermis
or neural tube.
Neither the present nor a previous radioautographic mapping study (Rosenquist, 1966) indicates the location of the migrating nephros prior to the mediumstreak stage. Its peripheral position in epiblast near the lateral margin of the area
pellucida, at the medium-streak stage (Fig. 2 A), suggests that at earlier stages it
may be even farther from the streak, perhaps beyond the area pellucida margin.
This is only speculation, however, since the critical early embryos which would
settle this point are not available for study at this time.
Differential movement of the Wolffian duct in the intermediate mesoderm
The Wolffian (pronephric) duct has long been considered to be formed by the
fused ends of the pronephric cords or tubules, and therefore to be derived from
cells originating in the 4th to 16th nephromeres. The posterior growth of this
pronephric duct in relation to the rest of the intermediate mesoderm may be
Origin of nephrogenic cells
379
traced in serial sections of embryos of various stages; it has been confirmed
experimentally by interrupting the posterior growth of this structure with
electrocautery or mechanical impediments (Boyden, 1924, 1927) and by microsurgery (Gruenwald, 1937; Waddington, 1938). In some of Boyden's embryos
the mesonephros became functional, but since the Wolffian duct did not connect
with the cloaca, there was accumulation of fluid proximal to the site of
cauterization, and the growth of the allantois and cloaca was stunted. Gruenwald and Waddington cut transversely through the intermediate mesoderm
posterior to the last somite of embryos which had not yet developed a mesonephros; the posterior growing tip of the pronephric duct was in each case unable
to cross the incision. Since neither Wolffian duct nor mesonephros was found
posterior to the incision in embryos incubated to later stages, it was concluded
that the mesonephros and metanephros needed contact with the Wolffian duct
to differentiate.
The present investigation indicates that the Wolffian duct begins its posterior
growth relative to the rest of the intermediate mesoderm some time between the
19- and 22-somite stages (Figs. 2H, 1). In Table 1 (embryos 10 and 14-16) the
position of this posteriorly growing duct is shown in relation to the position of
other labelled cells in the same embryo. There are numerous examples of labelled
cells in lateral plate, aorta, or endoderm, which are just as posteriorly placed
as the labelled cells in the Wolffian duct and which, like the intermediate mesoderm, originated in the labelled graft. The posterior position of these cells is
due probably to the fact that they migrated more slowly than did the pronephric
cells, and thus did not reach their definitive position (anterior to the Wolffian
duct) before the embryos were fixed. It is evident from the distribution of the cells
that they did not grow posteriorly in relation to the rest of the intermediate
mesoderm as did the pronephric duct.
RESUME
Determination par une cartographie radioautographique de Vorigine
et du mouvement des cellules nephrogenes de Poulet
L'origine des cellules nephrogenes presomptives dans 1'epiblaste de l'embryon de Poulet
est determinee par une analyse radioautographique des mouvements de greffons marques par
la thimidine tritiee, excises des embryons aux stades ligne primitive a 5 somites et transplanted dans Pepiblaste, la ligne primitive et la couche endoderme-mesoderme d'embryons
hotes des memes stades.
Les cellules nephrogenes ont une origine proche du bord de l'aire pellucide de l'embryon
au stade ligne primitive, elles migrent vers la ligne primitive, et sont invaginees environ du
tiers a la moitie de la distance entre les extremites anterieure et posterieure de la ligne, entre
les stades ligne primitive et 1 a 4 somites. Leur itineraire dans le mesoderme poursuit une voie
relativement etroite entre les cellules migrant vers le mesoderme paraxial ou presomitique d'une
part et les cellules destinees aux membres proximaux de la plaque laterale d'autre part.
Les cellules qui formeront la partie anterieure du mesoderme intermediate sont les
cellules les plus proches du plan median dans Pepiblaste, elles atteignent la ligne a un stade
de developpement plus precoce et sont les premieres cellules nephrogenes a migrer dans
le mesoderme. Apres le stade d'environ 17 a 19 somites, les cellules de ce groupe qui ont
380
G. C. ROSENQUIST
forme le cordon ou le canal pronephriques commencent a se mouvoir posterieurement par
rapport au reste du mesoderme intermediaire, vers le futur cloaque.
Les dernieres cellules nephrogenes a quitter l'epiblaste pour penetrer dans la ligne et le
mesoderme sont celles destinees a l'extremite posterieure du mesoderme intermediaire. Ce
groupe de cellules entoure le canal pronephretique (canal de Wolff) qui migre posterieurement,
et se differencie en mesonephros.
This investigation was supported by USPHS research grants HE 10.191 and KE HE 20074
from the National Heart Institute. The author wishes to thank James D. Ebert for his continued interest in this research, and Soame D. Christianson for help in the preparation of the
manuscript.
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{Manuscript received 16 January 1970)