/ . Embryo!, exp. Morph. Vol. 28, 3, pp. 6J5-631, 1972
615
Printed in Great Britain
Evidence for a two-step mechanism operating
during in vitro mouse kidney tubulogenesis
By CAROL L. GOSSENS 1 AND BRIAN R. UNSWORTH 1
From the Department of Biology, Marquette University,
Milwaukee
SUMMARY
The process of kidney tubulogenesis was investigated in mouse metanephrogenic mesenchyme, differentiating in tissue culture under the influence of an inductive stimulus from
embryonic mouse brain. The metanephrogenic mesenchyme was separated from the brain by
a membrane filter. The time of exposure to the inductive stimulus was controlled by removing
the brain from the filter.
Restricting the period of transfilter association of metanephrogenic mesenchyme and brain
resulted in incomplete tubulogenesis. A 30 h interaction time with brain led to the formation
of small tubules. These small tubules were at an unstable stage of differentiation, and regressed
during the 6-day culture period. Stabilization and elongation was achieved by the addition of
mesenchymal tissues or chick embryo extract. Induction was clearly not a one-time triggering
event, and as the degree of differentiation increased so the specificity for mesenchymal requirement decreased.
Embryo extract did not mimick the inductive event that initiated tubulogenesis, but supported the later stages of tubulogenesis. This was interpreted to indicate a certain specificity
in the induction reaction.
The incidence of tubule elongation was related to both the initial mass of metanephrogenic
mesenchyme and to the total contact time with brain. The greater the initial mass of mesenchymal tissue, the less contact time with brain needed for complete tubulogenesis.
The morphology of the elongated tubules depended on the nature of the mesenchyme with
which the small tubules were associated. This suggested that the expression of tubule morphology may be under the control of the surrounding mesenchymal cells. Possible mechanisms
operating during tubule elongation were discussed.
It was concluded that an integrated two-step mechanism was operating during kidney
tubulogenesis. The first step, cell condensation, was induced by contact with brain. The second
step, tubule elongation, was dependent upon the association of the condensates with the
surrounding non-induced mesenchymal cells.
INTRODUCTION
Many organs form as the result of developmentally significant interaction
between tissues brought into association by morphogenetic movements that
occur during embryogenesis.
Kidney differentiation requires an association between ureteric epithelium
and metanephrogenic mesenchyme (Fraser, 1950; Gruenwald, 1952). The
1
Authors' address: Department of Biology, Marquette University, Milwaukee, Wisconsin
53233, U.S.A.
616
C. L. GOSSENS AND B. R. UNSWORTH
developmental necessity for this tissue interaction may be illustrated by considering the Sd-strain of mouse. In mutants of this Danforth's short-tailed strain,
failure of the ureteric bud to branch off the Wolffian duct and grow into the
metanephrogenic mesenchyme results in the formation of anephrogenic embryos
(Gluecksohn-Schoenheimer, 1945).
The metanephrogenic system of the mouse is an excellent model for the tissue
culture analysis of an inductive process. Metanephrogenic mesenchyme forms
tubules when combined in vitro with ureteric bud, spinal cord, or embryonic
brain (Grobstein, 1954; Saxen et al. 1968; Lombard & Grobstein, 1969). The
inductive influence of the spinal cord is transmitted across a Millipore membrane
which is believed to exclude cellular contact (Grobstein, 1955, 1956; Grobstein
& Dalton, 1957). Experiments involving radioactive labelling of the spinal cord
with tritiated ami no acids have implicated the transfer of large-molecular-weight
materials during inductive interaction (Koch & Grobstein, 1963). These types
of in vitro tissue separation and recombination experiments have provided
evidence to support the concept that cytodifferentiation is controlled by factors
extrinsic to the cell (Grobstein, 1964). What is not certain is whether these
extrinsic factors, or inducers, are non-specific and merely initiate, or trigger, a
permissive differentiation of an already determined organ primordium, or
whether they both trigger and direct the subsequent organogenesis.
Certain organ rudiments show a strong differentiative bias when transferred
to in vitro culture, and express this bias by differentiating upon exposure to cellfree tissue extracts or 'nutrient' factors. For example, somites will undergo
chondrogenesis if maintained continuously in the presence of medium supplemented with fetal calf serum and chick embryo extract (Lash, 1968; Ellison &
Lash, 1971); and pancreatic epithelium will differentiate if cultured in the
presence of a particle fraction from chick embryo extract (Rutter, Wessells &
Grobstein, 1964; Rutter et al. 1968). In these particular systems the 'natural'
inducers, notochord or spinal cord and pancreatic mesenchyme, respectively, are
considered not to supply an essential differentiative stimulus to the responding
tissue, which differentiates, in a non-directed or 'permissive' manner (Holtzer,
1968; Ellison & Lash, 1971). However, this is not universally the case, and many
organ systems are considered to respond to the influence of specific inducers,
which both initiate and direct the differentiative course (Wolff, 1968). Examples of
these latter systems include: the dependence of epidermal differentiation on the
nature of the underlying dermis (Saunders, 1958; McLoughlin, 1961; Billingham
& Silvers, 1968); differentiation of the liver (Le Douarin, 1967, 1968); and the
ability of certain epithelia to show altered morphology when associated with
different mesenchymes; gastric epithelium (David, 1967); mammary epithelium
(Kratochwil, 1969); thymus epithelium (Auerbach, 1960/?); tracheal epithelium
(Wessells, 1970); and ureteric epithelium (Bishop-Calame, 1966).
When considering inductive tissue interaction, it appears to be conceptually
beneficial to recognize that organogenetic interaction is not an instantaneous
Mouse kidney tubulogenesis
617
event, but a complex, extended process (Grobstein, 1967). The morphogenetic
sequence characterizing tubulogenesis in mouse metanephrogenic mesenchyme,
cultured in transfllter contact with spinal cord, extends over 6 days (Grobstein,
1956; Saxen et al. 1968), although chemodifferentiation continues until about
the tenth day in culture (Koskimies, 1967). A morphologically 'silent' period of
some 24 h precedes the aggregation of mesenchymal cells into condensates that
are apparent after 30 h culture and well established by 48 h. The condensates
form whorls of cells which gradually transform into tubule rudiments. S-shaped
tubules are abundant by day 4 and these structures elongate and differentiate
during the next 2 days of culture to form the thin, convoluted structures characteristic of 6-day tubules. The mechanism by which this series of events is controlled is obscure; however, unpublished observations indicate that limiting the
time of inductive contact may lead to the formation of 'smaller, less complex'
tubules (Auerbach, 1960a; Grobstein, 1967). We designed a series of experiments to confirm and extend this undocumented report. It appeared to be of
particular significance to determine whether these tubules were small because
they were arrested at an early developmental stage, and whether an inductive
stimulus was obligatory for their complete tubulogenesis.
Using a transfilter method, we established that limiting the period of association of mouse metanephrogenic mesenchyme and brain to 30 h led to the formation of small tubules. These small tubules were similar in size to structures
formed after 3 or 4 days of continuous contact with brain. The small tubules
completed their tubulogenesis upon association with certain mesenchymal
tissues or chick embryo extract. Evidence is presented to support the concept
that at least a two-step process is operating during kidney tubulogenesis. The
initial step, cell aggregation, requires association of metanephrogenic mesenchyme with inductively active tissue; the second step, tubule elongation and
morphogenesis, is dependent upon association of the cellular aggregates with
non-induced mesenchymal cells, or factor(s).
MATERIALS AND METHODS
Embryos were obtained from randomly mated CF strain Swiss white mice.
The day of discovery of a vaginal plug was considered day zero of gestation.
Kidney rudiment dissection, salivary mesenchyme dissection, tissue culture
methods and composition of the nutrient medium were standard procedures
adequately described elsewhere (Lombard & Grobstein, 1969; Unsworth &
Grobstein, 1970). The transfilter method was essentially that originally described by Grobstein (1956), except that the modified filter assembly of Auerbach
(19606) was utilized for greater convenience. Brain tissue was clotted on the
well side of the filter (Millipore 22 + 3 /an thick, 0-45 ptm pore diameter), with
a mixture of chilled chick plasma and embryo extract (2:1). Kidney mesenchyme from one rudiment (except where otherwise indicated in the text) was
placed without a clot, transfilter to the brain.
618
C. L. GOSSENS AND B. R. UNSWORTH
Mouse kidney tubulogenesis
619
Table 1. Tubulogenic response following limited periods
of inductive tissue association
Tubulogenic response after 6 days culture
Duration of
heterotypic
interaction
24 h
30 h
48 h
6 days
Total
no. of
cultures
21
78
23
67
K
,
,
Negative
Small
tubules*
Elongated
tubulesf
16(76%)
33(42%)
1 (4%)
5(7%)
2(10%)
33(42%)
2 (9%)
4(6%)
3(14%)
12(15%)
20 (87%)
58(87%)
* Small tubules - morphologically resembled 3- to 4-day controls.
f Elongated tubules - morphologically resembled 5- to 6-day controls.
Tissues were routinely maintained in culture medium (Gibco Co.) supplemented with 3 % chick embryo extract, although in some experiments explants
were supplied with medium supplemented with 20 % embryo extract.
The cultures were maintained in a high-humidity incubator gassed with 5 %
CO2 in air at 37 °C, and their progress was followed daily with a binocular
microscope. Photographic recordings were made with a Zeiss Ultraphot II
camera microscope.
At the conclusion of the culture period the cultures were washed three times
in Tyrode's salt solution, fixed with 2-5 % glutaraldehyde for 30 min and stored
in 70 % ethanol at 4 °C. All tissues were stained with Mayer's hematoxylin and
eosin, and whole mounts were routinely prepared.
FIGURES
1-4
Fig. 1. Whole mount showing typical S-shaped tubules {s) differentiated in metanephrogenic mesenchyme isolated from a single kidney rudiment and cultured in
transfilter contact with brain tissue for 4 days in vitro. H and E stain, x 200.
Fig. 2. Whole mount of metanephrogenic mesenchyme cultured transfilter to
brain for 5 days. The tubules (/) are elongating and are about 4 or 5 cells in
diameter. Glomeruli (g) can be recognized at this stage. H and E stain, x 384.
Fig. 3. Whole mount of metanephrogenic mesenchyme after 6 days transfilter contact with brain. The differentiated tubules are now long, thin and convoluted (t)
and glomeruli (g) are evident. H and E stain, x 260.
Fig. 4. Whole mount of metanephrogenic mesenchyme maintained in culture for 6
days, but from which the transfilter brain was removed after 30 h. The differentiated
structures (st) are small and not elongated and are referred to in the text as 'small
tubules'. The differentiated area shown is small and represents the entire area of
differentiation normally observed when the period of inductive interaction is
restricted to 30 h. H and E stain, x 200.
620
C. L. GOSSENS AND B. R. U N S W O R T H
F I G U R E S 5 AND 6
Fig. 5. Whole mount of metanephrogenic mesenchyme that received 30 h inductive
transfilter contact with brain tissue and was maintained in culture for a total of 6
days. The differentiated area is small and the mesenchyme frequently becomes
detached from the filter surface and surrounds the partially differentiated small
tubules. H and E stain, x 200.
Fig. 6. (A) Living culture showing the typical cellular condensates (arrows) formed
after 2 days transfilter contact with brain, x 70. (B) The same culture shown in (A)
after 8 days in culture. The brain tissue was removed after 30 h transfilter contact
and the small tubules, apparent by 4 days culture, were unstable and progressively
degenerated to form an unorganized mass of cells it), x 70.
Mouse kidney tubulogenesis
621
RESULTS
Temporal sequence of events during kidney tubule differentiation
The morphological stages characterizing tubulogenesis in metanephrogenic
mesenchyme interacting with brain were found to be identical to those described
in the introduction, for metanephrogenic mesenchyme in transfilter contact with
dorsal half of spinal cord (cf. Grobstein, 1956). Mesenchyme cultured without
brain, spread out on the filter and showed no morphological signs of differentiation. The later stages of tubule formation (days 4-6) are illustrated in Figs. 1-3.
This control series was established for the purpose of comparing the differentiation obtained when the time of association with brain was varied.
Limiting the duration of inductive transfilter contact
Metanephrogenic mesenchyme, from one rudiment, was cultured transfilter
to brain for either 24, 30 or 48 h. The kidney mesenchyme was reincubated, after
removal of the brain, for a total of 6 days. Tubules were compared morphologically with the control series and the effects of limited inductive interaction
are summarized in Table 1.
When the brain was removed after 24 h transfilter contact, the majority of
the kidney mesenchymes failed to differentiate, the cells remained randomly
oriented and spread over the filter surface. Extending the period of transfilter
interaction by 6 h increased the frequency of tubulogenesis. The structures that
differentiated after 30 h transfilter contact with brain were mainly small in size
and limited in number (Fig. 4). These incompletely differentiated tubules are
referred to as 'small tubules' in the text, and may be compared in size with the
stage of tubulogenesis observed after 3 or 4 days, in kidney mesenchyme cultured continuously in the presence of brain (Table 1, controls; and Fig. 1). After
30 h of transfilter interaction, over 50 % of the positive cultures differentiated
only relatively few small tubules, and these small tubules became surrounded
by undifferentiated mesenchymal tissue by the sixth day of culture (Fig. 5).
Mesenchymal differentiation usually appeared to be normal through day 2 of
culture, at which time condensates appeared (Fig. 6 A). However, as incubation
continued, following removal of the brain at 30 h, the small tubules that formed
'regressed' during the next 2 or 3 days in culture (Fig. 6B).
The most noticeable result of restricting the period of transfilter interaction
with brain to 30 h was a high incidence of small tubule formation (Table 1).
However, many mesenchymes failed to differentiate and the incidence of tubule
elongation was no greater than that observed after 24 h transfilter interaction.
The incidence of tubule elongation was increased to the level observed in
control cultures (Table 1, controls) by extending the period of transfilter interaction to 48 h. The majority of the cultures differentiated to the coiled, elongated
tubule stage, and the incidence of small tubule formation was not significantly
above control values. It was concluded that 48 h transfilter interaction with
622
C. L. GOSSENS AND B. R. UNSWORTH
Table 2. Addition of various types of mesenchyme
to induced kidney mesenchyme
Duration
heterotypic
interaction
with brain
Time
mesenchyme
added
(h)
24
30
24
30
30
of
Mesenchymal
type added
Kidney
Salivary
Tubulogenic response
after 6 days culture
(h)
Total
no. of
cultures
24
30
24
30
72
26
53
20
39
22
A
f
Negative
Small
tubules
Elongated
tubules
9(35%)
14(26%)
3(15%)
5(13%)
6(27%)
9(35%)
14(26%)
5(25%)
9(23%)
4(18%)
8(30%)
25(30%)
12(60%)
25(64%)
12(55%)*
* This is the only experimental value not significant at the 5 % level of probability using the
chi-square test of independence.
brain was the minimum period of time necessary to ensure complete tubulogenesis in a single metanephrogenic mesenchyme.
Induced kidney mesenchyme cultured with non-induced mesenchymes
It was established in the previous section that limited heterotypic interaction
resulted in the incomplete differentiation of kidney tubules. Small tubules predominantly differentiated when the period of transfilter interaction with brain
was restricted to 30 h. An attempt was therefore made to determine whether
mesenchymal tissue could substitute for the brain in promoting tubule elongation.
Kidney mesenchyme from one rudiment was cultured transfilter to brain for
either 24 or 30 h. The brain was removed and mesenchyme was added in direct
contact with the induced kidney mesenchyme. Either kidney mesenchyme from
two freshly dissected rudiments or condensed capsular mesenchyme from two
13-day salivary rudiments was added, according to Table 2.
Conditions for tubulogenesis were improved by the addition of either kidney
or salivary mesenchyme, to kidney mesenchyme that had received short periods
of exposure to brain.
Salivary mesenchyme proved to be more effective in promoting tubule
differentiation than was kidney mesenchyme, and delaying the addition of
salivary mesenchyme for 42 h after the removal of the transfilter brain did not
significantly alter its tubule elongating ability. The morphological appearance
of the tubules, formed under the influence of salivary mesenchyme, was consistently different from day-6 controls. Although a certain degree of elongation
occurred, the tubules were about five cells in diameter (Fig. 7), as compared
with the tubules of 3-cell diameter of day-6 controls (Fig. 3). Extending the
culture to 8 days produced neither thinner tubules nor greater elongation, the
tubules remained bulbous, unlike controls.
Mouse kidney tubulogenesis
623
The addition of kidney mesenchyme significantly enhanced tubule differentiation. However, the number of differentiated tubules per culture and the total
differentiated area per culture (Fig. 8) was generally considerably less than that
observed in mesenchyme cultured continuously with brain for 6 days (Fig 3).
Also, the elongated tubules that formed in this series of experiments usually
resembled the 5-day controls (Fig. 2), even when the cultured period was
extended to 8 days.
Addition of20% embryo extract to kidney mesenchyme
receiving limited periods of inductive contact
Since both kidney mesenchyme and salivary mesenchyme enhanced tubulogenesis, a possible growth-stimulating effect was postulated. Several years ago
a chick-embryo fraction extracted from predominantly mesenchymal tissue was
reported to promote differentiation and growth of embryonic pancreatic epithelia in vitro (Rutter et al. 1964). More recently it was reported that this extract
may act by promoting DNA synthesis and cell division (Rutter et al. 1968).
Chick embryo extract alone does not promote tubule differentiation in kidney
mesenchyme (Rutter et al. 1964); however, it was postulated that the extract
might supply 'growth' or 'environmental' factor(s) essential for tubulogenesis.
Cultures were set up in which kidney mesenchyme was incubated transfilter
to brain tissue, for either 24 or 30 h. These cultures were fed standard culture
medium, supplemented with 3 % embryo extract (E.E.), during the period of
transfilter association. After the brain was removed, this medium was replaced
by medium enriched with 20 % E.E. The results of addition of 20 % E.E. are
summarized in Table 3.
Addition of 20 % E.E. after 24 h transfilter culture with brain caused no significant improvement in tubulogenesis. Transfilter culturing for 30 h, followed by
the addition of 20 % E.E. at the time of brain removal, reduced the number of
mesenchymes differentiating to the small tubule stage and significantly increased the frequency of tubule elongation. The elongated tubules (Fig. 9)
morphologically resembled 5- and 6-day control tubules (Figs. 2, 3) but the
number of tubules per culture was less than in the controls. Removing the brain
at 30 h and delaying the addition of E.E. until 72 h decreased the incidence of
tubule elongation and supported the differentiation of relatively few elongated
tubules per positive culture (Fig. 10).
Limited periods of heterotypic interaction with three kidney
mesenchymes transfilter to brain
The present study demonstrated that conditions necessary for tubule elongation were created by the addition of embryonic mesenchymal tissues, or factor,
at the time that the transfilter inductive brain was removed from culture. This
suggests the possibility that the concentration of nutritional or 'environmental'
factors may be critical for the later stages of tubule differentiation, and such
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C. L. GOSSENS AND B. R. UNSWORTH
Mouse kidney tubulogenesis
625
Table 3. Addition of medium supplemented with 20% embryo
extract to induced kidney mesenchyme
Duration of
heterotypic
interaction
(h)
Time
20%E.E.*
added
(h)
Total
no. of
cultures
24
30
30
0
24
30
72
0
18
45
30
18
,
Tubulogenic response after 6 days culture
*
,
Small
Elongated
Negative
tubules
tubules
15(83%)
10(22%)
13(43%)
18(100%)
0(0%)
6(13%)
4(13%)
—
3(17%)
29(64%)
13(43%)
—
* Embryo extract (E.E.) prepared from 9-day chick embryos (1:1 in Tyrode's solution).
factors may be supplied if a larger mass of kidney mesenchymal tissue were
initially present. Therefore mesenchyme dissected from three kidney rudiments
was placed transfilter to brain, instead of the single rudiments used in the previously reported experiments. The heterotypic inducer was removed from the
system after either 24 or 30 h, and the stage of tubule differentiation recorded
at the end of the 6-day culture period (Table 4).
It is clear that only 30 h transfilter interaction with brain was sufficient to
ensure complete tubulogenesis in three kidney mesenchymes; whereas, a single
kidney mesenchyme required 48 h transfilter interaction to achieve a similar
FIGURES
7-11
Fig. 7. Whole mount showing typical bulbous-type (arrow) of tubules observed in
metanephrogenic mesenchyme after 6 days in culture. Brain removed and salivary
mesenchyme added at 30 h. H and E stain, x 200. Tubule morphology is different
from that observed in control cultures maintained in continuous transfilter contact
with brain for 6 days (Fig. 3).
Fig. 8. Whole mount, showing tubules formed after 6 days in culture. Brain removed
at 24 h and freshly dissected kidney mesenchyme added. Note that both the total
differentiated area and the number of tubules formed are not as great as controls.
H and E stain, x 200.
Fig. 9. Elongated tubules in metanephrogenic mesenchyme cultured for 6 days.
Transfilter brain removed after 30 h culture and 20 % chick embryo extract supplied at the time of brain removal. Whole mount, x 200.
Fig. 10. Small number of elongated tubules in metanephrogenic mesenchyme
maintained in culture for 8 days. Transfilter brain was removed after 30 h culture
and the addition of 20 % embryo extract was delayed until 72 hr of culture. Note:
the entire differentiated area is shown; tubule elongation is scarce. Whole mount,
x200.
Fig. 11. Elongated tubules observed when three kidney mesenchymes are cultured
transfilter to brain for 24 h. Six-day whole mount, x200. Note: the differentiated
area is extensive and the complexity of tubule structure appears to be comparable
with that observed in mesenchyme cultured continuously with brain for 6 days
(Fig. 3).
40
E M B 28
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C. L. GOSSENS AND B. R. UNSWORTH
Table 4. Inductive interaction with three kidney mesenchymes
transfilter to brain
Tubulogenic response after 6 days culture
Duration of
heterotypic
interaction
(h)
Total
no. of
cultures
Negative
Small
tubules
Elongated
tubules
24
30
25
22
5(20%)
2(9%)
3(12%)
1(5%)
17(68%)
19(86%)
incidence of tubule elongation. The resulting elongated 6-day tubules (Fig. 11)
were long, thin (three cells in diameter), noticeably convoluted, and abundant;
morphologically resembling 6-day control cultures (Fig. 3). Interestingly, the
temporal sequence of tubulogenesis in three kidney mesenchymes cultured
transfilter to brain was indistinguishable from the control series in which one
mesenchyme was used.
DISCUSSION
Our results confirm unpublished observations, indicating that if the period of
heterotypic interaction is restricted, tubulogenesis is incomplete (cited in
Auerbach, 1960#; Grobstein, 1967). However, contrary to these earlier reports
we found that 30 h inductive interaction was not adequate to ensure stability
of tubules in the mesenchyme. The predominantly small tubules that formed as
a result of 30 h transfilter contact with brain were unstable structures that
regressed during the 6-day culture period. Stability of tubules required 48 h of
transfilter interaction. This discrepancy may be explained by the greater mesenchymal tissue mass and the higher embryo extract concentration used by the
earlier workers (cf. Auerbach, 1960 a); we have now shown that both these
factors are of critical importance in tubulogenesis. The progressive stabilization
of tubules as differentiation proceeds is also reflected in the results of tubule
dissociation and reaggregation studies (Auerbach & Grobstein, 1958; Auerbach,
1960a).
It is clear from the present study that the condensates formed after 30 h
transfilter interaction generally differentiate only to the small tubule stage in the
absence of additional mesenchyme or factor(s). A small number of explants
undergo elongation after 30 h transfilter interaction, and this is probably due
to the fact that condensate formation is not a strictly synchronous event
(Koskimies, 1967). The cells that are first induced may receive a quantitatively
sufficient stimulus from the brain, in 30 h, to differentiate fully. This differentiative process may be contrasted with pancreatic epithelium, where the association of cells into a 'package', the proacinus, requires a certain minimum time
of inductive association. Once this package is formed and stabilized by association with inductive mesenchyme, secretion of zymogen granules occurs. Removal
Mouse kidney tubulogenesis
627
of the inducer prevents new proacinar formation, and the number of acini
differentiating is directly proportional to the duration of the inductive association (Grobstein, 1962), i.e. proacini, once formed, complete their differentiation.
The inductive mechanisms operating during kidney differentiation are but
poorly understood. In vitro observations of cultured metanephrogenic primordia suggest that only the tips of the ureteric bud are inductively active
(Wolff, 1968; Saxen & Kohonen, 1969). Time-lapse photography of entire
rudiments, together with tissue separation and recombination experiments
(Saxen & Wartiovaara, 1966), encouraged the concept that the initial cellular
condensation (or aggregation) reaction is induced by the tips of the ureteric bud.
Tubule differentiation was considered to follow autonomously. However, the
results presented in this paper suggest that the capacity for autonomous differentiation from condensate stage to elongated, coiled tubule stage is critically
dependent upon the mass of available non-induced mesenchymal tissue, or
factor(s).
The ability of various mesenchymal tissues or embryo extract to support
tubule elongation is of considerable interest. These observations indicate that
the fastidiousness of tissue requirement decreases with increasing degree of
differentiation. This phenomenon appears to be a basic property of organogenetic tissue interaction, and is reflected in the varying degrees of specificity
shown by different epithelia in their mesenchymal requirements for differentiation (Grobstein, 1967).
The fact that a cell-free extract can support later stages of tubulogenesis
suggests that mesenchymal factors may play an important role during tubule
elongation. However, embryo extract cannot mimick the inductive event responsible for condensate formation. This indicates that specificity for tubule
induction may reside in the chemistry of the inducer, a concept of importance
when considering mechanisms of kidney tubulogenesis.
A mesenchymal contribution appears to be obligatory for complete tubulogenesis, indicating that induction is not a one-time triggering event. Moreover,
condensates exposed to salivary mesenchyme differentiate morphologically
different tubules (larger in diameter, less convoluted), from condensates receiving
either embryo extract or kidney mesenchyme. The formation of larger-diameter
tubules in the presence of salivary mesenchyme may, in part, be due to the
reported growth-promoting ability of salivary mesenchyme (Attardi, Monticalini & Wenger, 1965). Salivary mesenchyme has also been shown to possess
tubule-inducing properties; however, the time course of tubulogenesis is
extended by several days when salivary mesenchyme is the inducing tissue
(Unsworth & Grobstein, 1970). In the present study no delay in the elongation response was observed when salivary mesenchyme was added to the small
tubules, thereby suggesting that the induction properties of salivary mesenchyme
may be separate from the elongation properties.
The 'mesenchymal effect', observed in the present study, is corroborated by
40-2
628
C. L. GOSSENS AND B. R. UNSWORTH
recent studies indicating that induced kidney mesenchymal cells, which remain
on the filter after the non-induced mesenchyme is removed by stripping, require
the addition of mesenchyme for their subsequent tubulogenesis (Saxen, 1970).
The added mesenchyme need not be species-specific, as heterologous chick
mesenchyme supports elongation, but the added cells are not incorporated into
the differentiating tubules (Saxen, 1970). Other studies also indicate that tubule
elongation is not achieved by incorporating surrounding mesenchymal cells into
the pretubular structures (Saxen & Sakselsa, 1971). Rapid mitotic growth of the
induced cells is a logical alternative mechanism to explain tubule elongation,
and this mitotic activity may be promoted by embryo extract (Rutter et ah 1968).
Further work is required to establish whether the molecular identity and mechanisms of action of the extract is identical in both tubule elongation and
pancreatic acinar formation.
Recently, relatively complex differentiation has been achieved by culturing
embryonic cells in improved media, such as Ham's F12 with fetal calf serum
(Ellison & Lash, 1971). This raises the question of whether improved methods
of cultivation would allow the complete expression of the tubulogenic properties of kidney mesenchyme, making the inductive event unnecessary. This
concept receives some support from the report that kidney mesenchyme, transplanted to the anterior chamber of the eye, can undergo tubulogenesis (Grobstein & Parker, 1958). To date, all efforts to achieve tubulogenesis in isolated
kidney mesenchyme cultured in improved media have proved unsuccessful
(Unsworth, unpublished observations), and increasing the tissue mass is alone
unable to promote tubulogenesis (Unsworth & Grobstein, 1970).
Culturing three kidney mesenchymes transfilter to brain for 30 h ensured an
incidence of tubule elongation equivalent to that observed in a single mesenchyme cultured continuously in the presence of brain. However, the addition of
two kidney mesenchymes at the time of brain removal, although creating a
similar tissue mass, resulted in only limited improvement in elongation. This
effect might be explained if there were an enhanced ability of mesenchyme
retained in culture for 30 h, to protect the cells from loss of tubule-elongating
factor(s) due to diffusion. At the present time we have no results that would
support this hypothesis. A possible alternative explanation would be to assume
that the brain not only induces mesenchymal cell aggregation, but also stimulates the synthesis of tubule elongation factor(s) in the non-induced mesenchymal cells. Further work is required to distinguish between these two possible
mechanisms of tubule elongation.
Support for a two-step process, operating during kidney tubulogenesis, is
provided by several observations. Isolated induced cells are morphologically
inactive in the presence of inductively active tissue, and require the addition of
mesenchyme to complete tubulogenesis (Saxen, 1970). The morphology of the
differentiated tubules can be altered by the type of mesenchyme with which the
induced cells are associated, and limiting the duration of inductive contact
Mouse kidney tubulogenesis
629
allows differentiation to only a small tubule stage, which is unstable. It therefore
would appear that the inductive interaction that initiates tubulogenesis is
followed by interaction between the induced mesenchymal cells and the surrounding non-induced mesenchymal cells. This latter reaction both stabilizes
and elongates the partially differentiated tubules. This may be considered as an
example of continued inductive tissue interaction, a phenomenon that has been
postulated to be of importance in the development and maintenance of many
organs (Auerbach, 1964; Tarin, 1972).
In summary, kidney induction is clearly not a one-step triggering process.
Based on the available experimental evidence, it appears appropriate to offer an
hypothesis for the mechanisms operating during kidney tubulogenesis. Transfilter contact with brain results in the formation of cellular aggregates or condensates. These aggregates require continued association with mesenchymal
tissue (or factor) to autonomously differentiate into elongated, coiled tubules,
characteristic of the fully differentiated kidney. Tubule elongation can be supported by a cell-free extract from chick embryo, suggesting that the factor is not
species-specific. It appears to be mechanistically beneficial to consider kidney
tubulogenesis as a two-step process. The first step, cell aggregation and pretubular formation; the second step, tubule elongation. Both steps may be either
directly, or indirectly, under the control of the inductively active tissue, allowing
the possibility that the inductive tissue interaction may be responsible for both
initiating and directing the course of tubulogenesis.
We wish to express our sincere thanks to Drs Robert Auerbach and Alapati Krishnakumaran for their critical comments during the preparation of this manuscript. The data in this
paper are taken from a thesis submitted in partial fulfillment of the requirements for the
Master of Science degree at Marquette University. This investigation was supported by grants
from the Marquette University Committee on Research, and the American Cancer Society,
Milwaukee Branch.
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