/. Embryol. exp. Morph. 94, 173-188 (1986)
173
Printed in Great Britain © The Company of Biologists Limited 1986
Basal laminar thinning in branching morphogenesis of
the chick lung as demonstrated by lectin probes
BETTY C. GALLAGHER*
Department of Biological Sciences, Stanford University, Stanford, California, USA
SUMMARY
Three lectins, wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Ricinis
communis agglutinin I (RCA), were used to study the basement membrane of developing chick
lungs. Thinning of the basement membrane at the tips of newly formed bronchi was visualized
with all three lectins, but was particularly evident using SB A. Control sections established the
ability of the lectins to stain hyaluronic acid and chondroitin sulphate. Neuraminidase, bovine
testes hyaluronidase and Streptomyces hyaluronidase removed some of the staining, but none
were able to affect the staining of the basement membrane. Possible explanations for this are
discussed in the text. Incorporation of [3H]thymidine is enhanced at the tips relative to the
interbud area in stage-30 lungs, extending previous studies on stage-26 lungs. Evidence has been
presented here which demonstrates that mechanisms of morphogenesis used in avian embryos
are similar to those already elucidated in work on mammalian embryos.
INTRODUCTION
The development of many organs begins as an interaction between epithelium
and mesenchyme (Grobstein, 1954; Cunha, 1976; Wessells, 1977; Goldin, 1980;
Bernfield, Banerjee, Koda & Rapraeger, 1984). Greater complexity may characterize these events than that seen in earlier inductive interactions. One aspect of
this complexity may be the specificity of signals exchanged between the tissues;
therefore, the study of epithelial-mesenchymal interactions is likely to yield
insight into other developmental processes (Grobstein, 1954).
Studies of salivary gland morphogenesis have produced a detailed picture of
epithelial-mesenchymal interactions. The basement membrane, which lies at the
interface between the two tissue types, has two main components: the trilayered
basal lamina, directly adjacent to the basal surface of the epithelium, and the
fibrillar mesh work, containing type I collagen (Kallman & Grobstein, 1964;
Kallman, Evans & Wessells, 1967; Bloom & Fawcett, 1975). At the tips of growing
glands, glycosaminoglycan (GAG) turnover and cell proliferation are enhanced.
The clefts are sites of increased collagen deposition and of GAG accumulation due
* Present address: Department of Anatomy and Cell Biology, Box 439, University of Virginia
Medical Center, Charlottesville, Virginia 22908, USA.
Key words: chick lung, lectins, branching morphogenesis, basement membrane, wheat germ
agglutinin, soybean agglutinin.
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B. C. GALLAGHER
to stabilization by the collagen. GAG synthesis is reduced at the tips, leading to
the appearance in the light microscope of a thinner basement membrane at the tips
compared to that seen in the clefts (Bernfield & Banerjee, 1972; Bernfield,
Banerjee & Cohn, 1972; Bernfield, Cohn & Banerjee, 1973; Banerjee, Cohn &
Bernfield, 1977; David & Bernfield, 1979,1981; reviewed in Bernfield et al 1984).
Previously, developing organs that have been examined for heterogeneities in
the basement membrane have been derived from mammalian sources. It would be
instructive to learn whether avian tissues use similar strategies in development.
The chick lung has a monopodial pattern of branching, in which a single main stem
continues to extend at the apex in its original line of growth while giving off lateral
branches. This is different from the mammalian tissues in previous studies, which
have new branches arising from the tips of previous branches.
To study these events, lectins have been used as probes of cell-surface chemistry. Lectins are glycoproteins or sometimes proteins that have the property of
binding to specific sugars (Lis & Sharon, 1977). Wheat germ agglutinin (WGA)
binds to JV-acetylglucosamine (glcNAc) and sialic acid (SA). Because hyaluronic
acid (HA) is made up of repeating disaccharides which contain glcNAc, WGA is
likely to show affinity for this GAG. Likewise, chondroitin contains alternating
units of Af-acetylgalactosamine (galNAc), a sugar to which soy bean agglutinin
(SBA) binds. Ricinis communis agglutinin I (RCA) binds to galactose and therefore will bind to keratan sulphate, which contains this sugar. Commercially
available HA and CS were stained with WGA and SB A, respectively and confirm
the expectations in these two cases, as shown in the Results section.
The purpose of the present study was to visualize changes in the basement
membrane over a period of development when the branches are first emerging to a
time when the older buds are beginning to branch. Fluorescein-conjugated WGA
(Fl-WGA), SB A (Fl-SBA) and RCA (Fl-RCA) were used to stain the lungs.
Incorporation of [3H]thymidine was examined in stage-30 lungs to determine any
local differences in levels of cell proliferation that might correlate with lectinstaining patterns and, or, morphogenesis. Thinning of the basement membrane at
the tips of newly formed bronchi was visualized with all three lectins. Cell
proliferation was enhanced at the tips relative to the interbud area.
MATERIALS AND METHODS
Fl-WGA, Fl-SBA, and Fl-RCA were purchased from Vector Laboratories, Inc. Streptomyces
hyaluronidase (Hs) and glcNAc were obtained from Calbiochem-Behring. Bovine testes
hyaluronidase (Hbt), Type VI-S, Clostridium perfringens neuraminidase Type VI (<0 002
unitsmg"1 casein substrate, <0-003 unitsmg ./V-acetylneuraminic acid aldolase activity),
syntheticN, N',N" triacetylchitotriose, galactose, grade III galNAc, grade III mixed isomers of
whale and shark cartilage chondroitin sulphate and alcian blue 8-GX were purchased from
Sigma. [3H]thymidine was bought from New England Nuclear. Nuclear Track Emulsion (NTB2)
was from Eastman Kodak. Horse serum 4000, sterile and filtered, was from Irvine Scientific Co.
Nutrient mixture F-12 (HAM), specially formulated to contain twice the amount of amino acid
and pyruvate concentrations (special F-12) and penicillin-streptomycin solution were from
Basal laminar thinning demonstrated by lectin probes
175
Gibco Diagnostics Laboratories. Human umbilical cord hyaluronic acid was from Nutritional
Biochemical Corp. Nuclepore filters, polycarbonate disc membranes, 0-1 jum pore size, and
lOjum thickness, were from Nuclepore. Bacto agar was obtained from Difco.
Chick lungs from stages 26-30 were dissected in a 1:1 mixture of horse serum and Ham's F-12,
then fixed and dehydrated in cold ethanol (Sainte-Marie, 1972), transferred to cold methyl
benzoate and embedded in paraffin. WGA was applied to 7jum-thick mounted sections at a
concentration of SjUgml"1; SBA and RCA were applied at SO/zgmP1. All staining was carried
out in the dark at room temperature for ^h, followed by three washes of lOmin in phosphatebuffered saline (PBS). The sections were overlaid with a few drops of 80 % glycerol, 20 % PBS,
and cover slips placed on top. Hyaluronic acid (HA) and chondroitin sulphate (CS) were added
as dry powder to autoclaved Bacto agar (2 % in water) at a concentration of 1 mg ml"1. After the
agar mixture had cooled to room temperature, it was fixed and embedded according to the
schedule used for lung tissue. Agar without GAG was also fixed and embedded as a control.
To demonstrate the binding of a lectin to a particular sugar, the hapten sugars were added to
solutions of the appropriate lectin at the same lectin concentration as control solutions. Control
and hapten-containing solutions were allowed to stand for 1-2 h prior to staining. The concentrations of sugars used were: 0-2M-galNAc, 0-2M-gal, 0-2M-glcNAc and 0-002 M-triacetylchitotriose. Control solutions contained only lectin. Sections of lung tissue were then stained
with control solutions, and adjacent sections were stained with the hapten-saturated lectin.
Some sections were preincubated with either Hs at 100 TRU ml"1, pH 5-3 in PBS; Hbt at 3000
NFUml" 1 , pH 5-3 in PBS, or neuraminidase at 1 unit ml"1, pH 5-3 in 0-05 M-acetate buffer at
37°C for 18 h. Adjacent control sections were incubated in buffer without the enzyme. Enzyme
pretreatment was followed by three washes with stirring in PBS, before lectins were applied.
Sections were photographed with a Nikon Optiphot microscope equipped with epifluorescent
optics. Light with a major peak at 365 nm wavelength was used to illuminate the tissue. Kodak
Tri-X film pushed to ASA 1600 by developing in Diafine was used to record the staining
patterns.
For [3H]thymidine incorporation studies, lungs were dissected at stage 26, then placed on
Nuclepore filters supported on steel mesh grids in organ culture dishes. The organ cultures were
supplied with a medium containing 79% special F-12, 10% 9-day-old embryo extract, 10%
horse serum, and 1 % penicillin-streptomycin. The lungs were incubated for 2 days at 37 °C in a
5 % CO2, humidified atmosphere in order to allow them to flatten. 20 juCi ml" 1 of [3H]thymidine
were added to each culture dish for 2 h before removal of the tissue for fixation in Bouin's
solution, dehydration in ethanol and embedding in paraffin. 7jan sections were cut, then
hydrated before dipping in the NTB2 emulsion at 41 °C and allowed to air dry. The liquid
photographic emulsion had been diluted 1:1 with a solution of 2 % glycerol in water. The slides
were kept desiccated in light-tight boxes at 4°C for 10 days before developing in 1:1 Dektol,
2 min at 20°C. The sections were stained with 1 % alcian blue, pH 2-5, 0-1 mM-MgCl2 5 min and
counterstained with neutral fast red for lmin, dehydrated and mounted in Permount before
being photographed in a Nikon Optiphot microscope with Kodak Panatomic-X film developed
with HC110, dilution B. In all cases, several sections were examined for patterns of grain
distribution.
RESULTS
Lung development
The embryonic chick lung first emerges as two ventral outgrowths from the floor
of the oesophagus (Locy & Larsell, 1916). Each lung primordium consists of a
single blind tube lined with a pseudostratified epithelium of endodermal origin
surrounded by mesenchyme. This tube, called the mesobronchus, proceeds to
elongate, and at stage 24 (Hamburger & Hamilton, 1951), the first of the primary
buds appears from the dorsomedial side approximately midway down the length of
176
B. C. GALLAGHER
the tube. The next primary buds appear in anterior-posterior sequence along the
length of the mesobronchus, also from the dorsal side until the seventh bud
appears at stage 28. At this time, branching is initiated from the ventral surface. At
about the 10-bud stage (stage 30), the more anterior buds begin to branch,
although the first bud branches earlier.
The gross morphology of the chick lung can be seen at stages 24-30 in 2-stage
(1 day) intervals (Fig. 1). After stage 28, the posterior mesobronchus is displaced
laterally so that the first through sixth primary buds, or bronchi, remain on the
medial side of the lung, and the seventh through sixteenth bronchi are located
laterally.
Fig. 1. Development of the avian lung at five 1-day intervals. Line in corner of (D)
represents 1 mm. (A) Stage 24,0-1 buds. (B) Stage 26,3 buds. (C) Stage 28,5-7 buds.
(D) Stage 30,10-12 buds. (E) Stage 32,13 buds. There is much branching of the buds
at this stage. At stage 30 (D) and stage 32 (E), trypan blue has been injected into the
lumen to make the branching pattern visible. All photographs x20.
Basal laminar thinning demonstrated by lectin probes
Lectin staining
111
Fl-WGA
Fluorescence microscopy of stage-28 and -30 lungs stained with Fl-WGA reveals
a preferential binding of the lectin to the basement membrane of both the lung
epithelium per se and the epithelium covering the outer surface of the lung, the
mesothelium (Fig. 2). In all of the tissue examined, what appears to be a basement
membrane is staining, but the basal regions of the epithelial cells could also be the
site of staining. In this paper, the term 'basement membrane' refers to staining in a
linear pattern at the basal surface of epithelia. In any particular lung, a progression
of development can be seen in successive buds - the anterior buds are older than
the posterior buds.
In early stages of growth, the basement membrane at the tip of the bud will
exhibit less staining than the interbud area. Later, when the bud has begun to
branch, sparse amounts of lectin-binding material are present at the new tips,
whereas staining is more evident at the new interbud area (Fig. 3).
The basement membrane of the distal mesobronchus stains uniformly throughout the stages observed, although there is slightly less staining at the tip (Fig. 4).
This portion of the mesobronchus contains sites of future bud initiation on its
dorsal and ventral sides; however there appear to be no obvious discontinuities as
judged by the light microscope in either area of the basement membrane. The
apical surface of the mesothelium is another component of lung tissue which stains
with Fl-WGA. Occasionally, the luminal surface of the inner epithelium will also
stain, but not in a discernible pattern. Diffuse staining of the mesenchyme is also
seen, preferentially associated in the area near the bronchi rather than near the
mesothelium (Figs 2, 3).
Lectin staining Fl-SBA
SBA-binding sites are distributed in a pattern similar to that seen using WGA,
but with a more pronounced difference between the tips and interbud areas.
Initially, a bud shows little staining at the tip (Figs 5, 6); in a later bronchus the
stain is distributed uniformly around the surface. Finally a branching bronchus will
have sparse staining at the new tips, but the new interbud area, stalks and old
interbud area will bind the lectin (Fig. 7).
The mesothelium shows an affinity for Fl-SBA, as it does for Fl-WGA, and a
faint staining of the mesenchyme is seen, in a pattern similar to that achieved with
WGA. The only major difference in staining patterns using the two lectins is that
the luminal surface of the bronchial epithelium is not stained with Fl-SBA,
whereas it is stained by Fl-WGA. Finally, RCA also binds to sites that are
codistributed with those which bind SB A (Fig. 8). Because an initial survey of the
binding patterns displayed by RCA showed the pattern to be similar to SBA and
WGA, further studies were not conducted on this lectin.
Competition with hapten sugar
When the hapten sugar is combined with the lectin, 0-2M-galNAc and 0-2M-gal
completely abolish the staining otherwise produced by Fl-SBA (Figs 9, 10) and
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B. C. GALLAGHER
Basal laminar thinning demonstrated by lectin probes
179
Fl-RCA (data not shown). 0-2M-glcNAc abolishes all WGA staining except for
that in the basement membrane (Figs 11, 12) where the residual staining is
considerably diminished compared with that seen without the competing sugar.
The complete removal of staining can only be achieved using 0-002 M-triacetylchitotriose as the hapten sugar (Figs 13,14).
Lectin staining of GAGs embedded in agar
Preparations of 1 mgml" 1 hyaluronic acid or chondroitin sulphate in agar were
fixed and embedded in the same manner as the tissue. These were then sectioned
and stained with the lectins to determine whether these GAGs are stained with
WGA and SBA.
l m g m l " 1 HA stains with B^ugml"1 Fl-WGA, but no staining can be seen with
50 fig ml" 1 Fl-SBA (Figs 15,16). The converse is true for 1 mg ml" 1 CS: it does not
stain with WGA, but does stain with SBA (Figs 17, 18).
Enzymic digestions
After enzymic digestions of sections with 1 unit ml" 1 neuraminidase, the apical
surfaces of the bronchial epithelium are markedly reduced in their ability to
bind WGA. The mesenchyme is also only lightly stained in comparison with the
control. The basement membrane appears to be unaffected by the neuraminidase
digestion (Figs 19, 20). Streptomyces hyaluronidase (Hs) digestion of the sections
reduces the staining of the mesenchyme with Fl-WGA, but the basement membrane appears unchanged in its staining characteristics (Figs 21, 22). Bovine testes
Figs 2-4. Patterns of staining with WGA. Mag. xl34. Bar, 0-1 mm.
Fig. 2. The basement membranes of the buds are stained somewhat more intensely
at the interbud area than at the tips. Buds '2' and '3' are in an early stage of outgrowth,
and are thinner at their tips than bud ' i ' at 'ra'\ Bud T is beginning to branch. The new
interbud area, 'n/', on its left is stained more heavily than the new tips (marked with
arrows). In addition to the basement membranes, the mesothelium ('raes' on figure)
stains in its apical and basal portions. The tips of the fourth and fifth buds do not lie in
the plane of the section. The matrix stains diffusely.
Fig. 3. Bud 2 (the second bud to appear) at a later stage, when it is branching. Most
of the basement membrane stains intensely, but the new bud (arrow) has a very lightly
staining basement membrane. The staining in the matrix seems to be associated with
areas closer to the bronchi.
Fig. 4. The posterior extension of the mesobronchus is stained evenly over its
surface.
Figs 5-7. Patterns of staining with SBA. Mag. X134. Bar, 0-1 mm.
Fig. 5. In a 3-bud lung, SBA stains the basement membranes of the buds more
intensely at the interbud area than at the tips (arrows).
Fig. 6. In an older lung, buds that are just emerging follow the same pattern as buds
that have arisen earlier: lightly stained at the tips (arrow), heavier staining at the
interbud area.
Fig. 7. A branching tip (arrow) is lightly stained compared to the old and new
interbud area. The new interbud area is below and to the right of the arrow.
Fig. 8. RCA stains the basement membranes of bronchi. The tip of the second bud
from the left is branching (arrow), and stains less heavily than the interbud area, stalks
and primary bronchus (mesobronchus). Mag. x96. Bar, 0-1 mm.
180
B. C. GALLAGHER
hyaluronidase (Hbt) increases the overall Fl-SBA staining (data not shown).
When the agar-embedded GAGs are predigested with the enzymes, HA is no
longer stained with Fl-WGA after incubation in both types of hyaluronidase,
whereas the control is still stained (Figs 23, 24). Hbt reduces the staining of CS by
Fl-SBA (Figs 25, 26).
Autoradiography
3
H-Tdr is incorporated into a higher percentage of cells at the tips of buds than
the interbud areas (Fig. 27). This is characteristic of newly formed buds, but in
older buds, the difference in DNA synthesis is not as marked. The distal mesobronchus has a high rate of incorporation which extends back to the most posterior
bud. When an older bud branches, the correlation of new tip with higher incorporation than at the new interbud area is only vaguely apparent. At the
junction of the two anterior mesobronchi that forms the trachea, DNA synthesis
is reduced compared to the remainder of the epithelium, which shows heavy
incorporation of the label (Fig. 28).
DISCUSSION
The distribution of glcNAc and, or, sialic acid (SA) as visualized by Fl-WGA
staining is predominant in the basement membranes of both the lung epithelium
itself and the mesothelium. Somewhat lesser amounts of these compounds can be
seen at the tips of newly formed buds. After the buds enlarge, they reach a stage
in which the basement membrane is uniformly covered with glcNAc- and SAcontaining substances. Later, when the bronchi ramify, the new tips and interbud
area repeat the sequence followed by the primary branches.
The presence of galNAc and gal, also primarily in the basement membrane, is
seen by staining with Fl-SBA and Fl-RCA, respectively. The regional differences
in the basement membrane during morphogenesis appear using these probes also
and the same temporal changes in the pattern are seen as with Fl-WGA.
The competition experiment with the hapten sugars demonstrates the specificity
of the lectins for these sugars. The fact that Fl-WGA staining of the basal lamina is
not entirely prevented by the previous saturation of the lectin with 0-2M-glcNAc is
unexpected. There are several alternative explanations for this phenomenon. The
binding site for glcNAc might extend beyond the volume of the monomer, leaving
room for additional binding to glcNAc. Perhaps the binding sites for sialic acid and
glcNAc are spatially distinct, so that occupation of the site for glcNAc will not
affect binding to SA, which could also be present in the basement membrane.
There is evidence that the two binding sites in WGA are indeed noncooperative
and spatially distinct (Wright, 1984), but according to a recent study, SA and
glcNAc bind to both subsites (Kronis & Carver, 1985). If 0-2M-glcNAc does not
saturate both binding sites, the remaining site can remain free to bind either
hapten, but there is, at present, no evidence in favour of an exclusive binding site
for glcNAc or SA.
Basal laminar thinning demonstrated by lectin probes
Figs 9, 10. Binding affinity of SBA for N-acetyl galactosamine (galNAc). Mag. x88.
Bar in Fig. 10, 0-1 mm.
Fig. 9. (Control). Staining pattern with SOjugml"1 SBA after it has stood at room
temperature for the same amount of time as the experimental solution.
Fig. 10. An adjacent section stained with 50 fig ml"1 SBA which has been combined
with 0-2 M-galNAc and allowed to stand 1 h at room temperature before application to
sections. The staining is abolished.
Figs 11-14. Binding affinity of WGA for N-acetylglucosamine (glcNAc). Mag. x88.
Bar in Fig. 14, 0 1 mm.
Fig. 11. (Control). Staining pattern with SjUgmP1 WGA after it has stood at room
temperature for the same amount of time as the experimental solution.
Fig. 12. An adjacent section stained with SjUgml"1 WGA after it has stood l h at
room temperature with 0-2M-glcNAc before application to sections. All staining has
been abolished except for the basement membranes.
Fig. 13. (Control). Staining pattern with 3/igmr 1 after it has stood l h at room
temperature before staining.
Fig. 14. An adjacent section stained with 3 jug ml"1 after it has stood in solution with
0-002 M-triacetylchitotriose for 1 h before staining is completely abolished.
181
182
B. C. GALLAGHER
Alternatively, a polysaccharide may be present in the basement membrane in
molecules not yet biochemically identified. The putative molecule might bind
more tightly to WGA than glcNAc. For example, triacetylchitotriose, a trimer of
glcNAc (Barker, Foster, Stacey & Webber, 1958), eliminates the staining at 1/100
the concentration of the monomer.
As anticipated, both HA and CS at a concentration of 1 mgrnl" 1 in agar stained
with WGA and SB A respectively; thus it can be assumed that when these GAGs
Basal laminar thinning demonstrated by lectin probes
are present in the tissue, and are not prohibited sterically from binding to a lectin,
their presence can be detected by this method. One cannot attribute all staining to
these molecules, as other substances may contain the lectin-binding sugars.
Glycoproteins such as laminin (Timpl & Martin, 1982), fibronectin (Yamada,
1981) and type IV collagen (Kefalides, 1970) contain the hapten sugars studied in
this report. However, the density of these sugars in a microenvironment is low
when compared to GAG's such as HA and CS, which are composed of alternating
units of the sugars. Proteoglycans are molecules that consist of a core protein with
a large percent by weight (e.g. 80%) of GAG's and therefore are likely to bind
lectins.
Because the binding sites for the three lectins codistribute (except at the apical
surface of the epithelium), the lectins may be staining the same substance. To
explore this possibility, each agar-embedded GAG was stained with Fl-SBA and
Fl-WGA, but significant staining was seen only with the appropriate lectin.
Dermatan sulphate also contains galNAc and therefore could be stained by SBA
as well as CS.
Alcian blue staining at different Mg2+ concentrations and digestion with different hyaluronidases are two methods which are often used to separate the
presence of HA from CS (Quintarelli, Scott & Dellovo, 1964). In both cases,
quantification is achieved by substraction of the first from the second amount. In
addition, the alcian-blue-staining procedure is not specific for CS in the presence
of embryonic HA (Derby & Pintar, 1978). Because SBA and WGA bind CS and
HA, respectively, the exclusive distribution of each can be directly visualized.
The neuraminidase-treated sections showed that much of the apical surface of
the bronchial epithelium is coated with a sialic acid-containing substance. This has
also been demonstrated in the embryonic kidney with WGA (Ekblom, 1981) and
Figs 15-18. Affinity of WGA and SBA for hyaluronic acid (HA) and chondroitin
sulphate (CS). Mag.1 x88. Bar, 0-1 mm.
Fig. 15. lmgml" HA stained with SfigmV1 WGA. WGA stains HA at these
concentrations.
Fig. 16. 1 mgrnl"1 HA stained with SOjUgml"1 SBA. No staining is seen with these
concentrations of GAG
and lectin.
Fig. 17. 1 mgml"11 CS stained with 3 jUgmP1 of1 WGA. No staining is seen.
Fig. 18. lmgml" CS stained with 50/igml" SBA. SBA stains CS at these
concentrations. Figs 16 and 17 also demonstrate that the agar is not the cause of
staining seen in Figs 15 and 18.
Figs 19-22. The effects of neuraminidase and Streptomyces hyaluronidase on the
staining patterns of WGA. Mag. x88. Bar in Fig. 20, 0-1 mm.
Fig. 19. (Control). Section incubated in buffer only, in parallel with experimental
section, then stained with WGA.
Fig. 20. Adjacent section incubated with 1 units ml"1 neuraminidase 16 h at 37°C,
then stained with WGA. Mesenchyme and apical surfaces of epithelium (arrows) are
diminished in their staining.
Fig. 21. (Control). Section incubated in buffer only in parallel with experimental
sections, then stained with WGA.
Fig. 22. Adjacent section incubated with 100 units ml"1 hyaluronidase 16 h at 37°C,
then stained with WGA. Mesenchyme is reduced in its staining.
183
184
B. C. GALLAGHER
neural tube using ruthenium red (Hay, 1978). The decrease in mesenchymal
staining after neuraminidase treatment indicates that sialic acid is also present in
the mesenchymal spaces.
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Basal laminar thinning demonstrated by lectin probes
185
Streptomyces hyaluronidase (Hs) specifically digests hyaluronic acid (Ohya &
Kaneko, 1970; Derby & Pintar, 1978). The mesenchyme stained significantly less
after digestion with this enzyme, indicating that hyaluronic acid is present in the
mesenchyme in sufficient amounts to stain with the lectin.
The conclusions regarding neuraminidase- and Hs-sensitive material must be
tempered with caution, as the enzymes may be contaminated. Also, in the present
experiment, material may be stabilized by HA or SA, and thus could be removed
by Hs or neuraminidase without being in itself a substrate for the enzymes.
The concentrations of control HA and CS were 1 mg ml" 1 , which correlates with
a quantitative estimate of GAG levels in embryonic mouse interstitial matrix
(Derby, 1978). Both positive controls ceased to stain after treatment with the
appropriate enzyme. This property would extend to any HA and GAG in the
tissue, except where steric conditions prevent the access of enzymes to the GAG's.
Hbt digests GAG's into polymers of 4-14 sugar units (Weissman, Meyer, Sampson
& Linker, 1954). In the tissue, GAG may be anchored to a protein in the basement
membrane, and an oligosaccharide will remain bound to the protein after enzymic
treatment. This may be one reason why Hbt did not reduce the staining of Fl-SBA
in sections. On the other hand, the increase in staining seen after HBt may be due
to an increase in general affinity of the tissue for the lectin or the digested
polymers, perhaps mediated via endogenous lectins (Matsutani & Yamagata,
1982; Kobiler & Barondes, 1977; Pitts & Chang, 1981).
An oligosaccharide remaining bound to a protein core could also explain
the effect of Hs on staining with Fl-WGA. Although staining with Fl-WGA
was decreased by Hs in the mesenchyme, the basement membrane appeared
unaffected.
The autoradiography experiments indicate greater mitotic activity at the tips
than interbud areas. Several photomicrographs were scored for the number of
cells per length along the perimeter in the interbud area and the tips. No evidence
of different cell densities in either area was obtained; therefore the higher number
of thymidine (Tdr)-incorporating cells does not reflect a higher cell density. A
Figs 23-26. The effects of hyaluronidases on the staining of GAGs with lectins. Mag.
x88. Bar in Fig. 26, 0-1 mm. 1
Fig. 23. (Control). 1 mgml" HA incubated in buffer in parallel with experimental
sections before staining with WGA. Staining is apparent.
Fig. 24. lmgml"1 HA incubated with 3000 units ml"1 bovine testes hyaluronidase
(A) and 100 TRU units ml"1 Streptomyces hyaluronidase (B) for 16 h at 37 °C. WGA
does not stain the sections.
Fig. 25. lmgml"1 CS incubated with buffer in parallel with experimental sections
before staining with SB A. Staining can be seen.
Fig. 26. lmgml"1 CS incubated with bovine testes hyaluronidase for 16h at 37°C.
The staining with SB A is diminished.
Figs 27, 28. Patterns of thymidine incorporation in organ-cultured lungs.
Fig. 27. Incorporation of radioactive label is heavier at the tips (arrows) compared
to the interbud areas in recently emerged buds at the 13-bud stage, x 134. Bar, 0-1 mm.
Fig. 28. The junction of the two mesobronchi (arrow) is labelled lightly compared to
other areas of the lung. xlO4. Bar, 0-1 mm.
186
B. C. GALLAGHER
higher incorporation of 3H-Tdr in a single pulse at the tips could be due to a
smaller proportion of non-cycling cells relative to the interbud area, as was found
in the rodent lung (Lawson, 1983).
Greater incorporation of 3H-Tdr at the tips was also obtained in an experiment
on chick lung at an earlier stage (Goldin & Opperman, 1980). The present
investigation extended these results to cover the period of lectin staining studied.
At the tips, an inverse relationship is seen between the number of cells
incorporating 3H-Tdr and a well-defined basement membrane. The distal mesobronchus shows an elevated incorporation rate, yet the basement membrane
appears well stained with lectins (except for the tip), so that in areas of incipient
bud formation, the above relationship does not obtain. Within the limits of
resolution of this method, the distribution of DNA-synthesizing cells appears
uniform beyond the most newly formed bud, therefore the site of new bud
formation cannot be identified by 3H-Tdr labelling patterns.
CONCLUSIONS
Fluorescent WGA, SB A and RCAI were used to locate glcNAc, galNAc, and
gal moieties in the embryonic chick lung, in hopes of staining HA, CS and perhaps
KS. Positive control samples of HA and CS revealed that the GAG's were stained
by the lectins, and that the lectins showed a marked preference for specific GAG's.
All three lectins stained the basement membrane in a developmentally significant manner: sparsely at the tips and densely in the interbud areas. The pattern
was particularly evident when staining for galNAc.
The distribution of the hapten sugars correlates well with the model of
branching events as proposed for salivary glands. Cell proliferation is enhanced at
the tip relative to the interbud region, and therefore also agrees with the developmental programme seen in salivary glands.
Evidence has been presented here which demonstrates that similar events of
morphogenesis are used in organs which differ in their pattern of branching. These
similarities exist in epithelial-mesenchymal organs derived from avian as well as
from mammalian embryos.
I wish to thank Norman K. Wessells, Geoffrey Goldin, Paul Green and Steve Klein for
encouragement, support and thoughtful criticisms. I am grateful to Linda Jones for her expert
help in typing this manuscript. This work was supported by Grant no. HD-04708 to N.K.W. and
a predoctoral NSF minority fellowship.
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(Accepted 21 January 1986)