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Small-granule APUD cells in relation to airway branching and growthA quantitative cartographic study in syrian golden hamsters.

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THE ANATOMICAL RECORD 213:410-420 (1985)
Small-Granule APUD Cells in Relation to Airway
Branching and Growth: A Quantitative,
Cartographic Study in Syrian Golden Hamsters
STEPHEN N. SARIKAS, RICHARD F. HOYT, JR.,AND SERGE1 P. SOROKIN
Department of Anatomy, Boston University School of Medicine, Boston MA 02118
ABSTRACT
Small-granule APUD cell clusters and their clear-cell precursors
were mapped in serial 2-pm glycol methacrylate-embedded, periodic acid-Schiff(PAS)lead hematoxylin-stained sections of 13-, 14-, and 15-day fetal hamster lungs. Every
sixth section was drawn from a camera lucida projection on tracing paper. Each
tracing included the profiles of nonalveolated air passages and the locations of smallgranule cell clusters and solitary clear cells. Airways containing ciliated cells and
those surrounded by condensed mesoderm were also labeled. Single clear cells were
rare in fetal hamster lung. Of 2,368 endocrine cell loci identified in the three fetal
age groups examined, only 14 were single clear cells. A preliminary survey of the
entire left and right lungs showed that the pattern of airway and small-granule cell
development in the infracardiac lobe was similar to that occurring in the remainder
of the lung; this lobe was accordingly considered a model for the whole lung, and the
ontogeny of its small-granule cell population was quantitated and compared with
results of similar quantitative mapping of this lobe in a n adult animal (Hoyt et al.,
1982a,b). Along the lobar bronchus of the 13-day infracardiac lobe and proximal
portions of its main branches, small-granule cell clusters occurred most often near
airway intersections. As the number and density increased in subsequent fetal
stages, small-granule cell clusters became conspicuous along internodal bronchial
segments. In distributing bronchioles, the population density of small-granule cell
clusters decreased between 13 and 14 days but more than doubled by day 15. Unlike
human lungs, where centrifugally developing small-granule cell clusters are firmly
established in terminal bronchioles well before birth, most peripheral bronchioles in
fetal hamster were devoid of small-granule cell clusters, even a t 15 days, one day
before birth. Comparison of numerical population densities in this lobe of fetal and
adult lungs indicates that small-granule cell clusters continue to form past day 15
and suggests that they are considerably more numerous in adult than fetal lung.
To date, very little quantitative information has been
published on the number, density, and distribution of
pulmonary neuroepithelial bodies and small-granule cell
clusters in any species during fetal development, although the pattern of their differentiation is now known
in several. Where these data can be compared with
counts of similar cells in adult lungs, they not only
extend our understanding of this putative pulmonary
endocrine or paracrine system but may help to indicate
a t what periods of life it is likely to be active.
Currently, the only fully quantitative estimate of
small-granule cell populations in adult lungs is that of
Hoyt et al. (1982a,b),derived from a serial survey of the
infracardiac lobe from one adult hamster lung a t 3-pm
intervals in glycol methacrylate sections stained by PASlead hematoxylin. Dimensions of the bronchial tree, locations of endocrine cell loci, and population densities of
clustered and solitary endocrine cells were closely examined along all levels of the conducting airways.
The present study has been undertaken to obtain similar quantitative data describing the population density
0 1985 ALAN R. LISS, INC.
and distribution of small-granule cell clusters in the
developing fetal hamster infracardiac lobe, built upon a
general understanding of small-granule cell ontogeny in
this species (Sarikas et al., 1985)and demonstrating that
in this respect the lobe serves as a n adequate model for
remaining parts of the lungs. The data provide the basis
for comparisons made between the developing population of small-granule cell clusters in fetal lungs and that
in the stable, mature organ.
MATERIALS AND METHODS
Preparation of Tissue Specimens
The quantitative results to be described were based
upon study of one fetal hamster for each developmental
Received February 25, 1985; accepted May 29, 1985.
Dr. Sarikas’s present address is: Department of Biochemistry, University of Massachusetts Medical Center, 55 Lake Avenue North,
Worcester, MA 01605.
Address reprint requests t o Dr. Hoyt.
411
HAMSTER SMALL-GRANULE CELLS AND AIRWAY GROWTH
Fig. 1. Representative line drawing of a transverse 2-pm section of plastic-embeddedlung, 14day fetal hamster. Profiles of nonalveolated airways are marked to show small-granule cell
clusters (solid black spots) and investment by condensing mesoderddeveloping smooth muscle
(dashes). Ciliated cells were absent. L = left lung; RU, RM, RL = right upper, middle, lower
lobes.
stage a t 13, 14, and 15 days of gestation. Each specimen
was selected as fully representative of its age from a
much larger sampling of fetuses used for the previous
qualitative study (Sarikas et al., 1985). The thoraxes
were dissected out, fixed by immersion for 24-48 hr, and
processed for light microscopy as described in that report. The entire lung of each fetus was serially sectioned
in situ a t 2 pm on a Jl3-4 microtome. Accurate cutting
records were made of lost sections and knife changes.
Two sections were collected on each precleaned glass
slide and every third slide was stained with PAS-lead
hematoxylin (Sorokin and Hoyt, 1978).
--To_ -1_ -
Serial Tracings of Fetal Hamster Lungs
Lung profiles of every sixth section (ca. 12-pm inter~ white
vals) were drawn a t a magnification of 1 2 5 on
tracing paper using a Zeiss binocular microscope and
drawing tube. Each tracing included the outlines of all
conducting airways and the location of small-granule
cell clusters and their “clear cell” precursors (Fig. 1)as
confirmed by microscopic examination a t 1,000 x . Airways surrounded by condensed mesoderm as well as
those containing ciliated cells were also marked appropriately in the drawings. A total of 619 tracings were
made: 152 from the 13-day, 205 from the 14-day, and 262
from the 15-day fetal lung.
Bronchi and bronchioles were subdivided into unit
airways (Fig. 2), which were defined as the distance
between successive branch points (Hoyt et al., 1982b).
Then, for each age group, the length of unit airways
containing small-granule cell clusters in all pulmonary
lobes was calculated and recorded in millimeters as follows: Using the serial tracings, the lung profiles were
superimposed by best overall fit of the major airways in
all the lobes. The horizontal displacement (Fig. 3) between the centers of airway profiles representing the
beginning and end of a given unit airway was determined by measuring the distance between each center
fig. 2. In this diagram, each daughter airway joins its parent along
a ring of junction (dashed circle) including the carinal point (black
spot). Successive branch points define a unit airway (A).
point and dividing that value by the magnification. The
corresponding vertical displacement (Fig. 3) was approximated by multiplying the section thickness (2 pm) by
the number of sections cut between the appropriate airway profiles determined from the cutting records.
The lateral and vertical displacements formed two
sides of a right triangle. The hypotenuse of this triangle
approximated the axial length of the unit airway and
was determined by application of the Pythagorean theorem (a2 b2 = c2). The total length of any given airway
(e.g., the lobar bronchus) could be calculated by adding
together all the units comprising that airway (Fig. 3).
+
412
S.N. SARIKAS, R.F. HOYT, JR.,AND S.P.SOROKIN
A
\a"
I
413
414
S.N. SARIKAS, R.F. HOYT, JR.,AND S.P. SOROKIN
basis for the studies of Hoyt et al. (1982a,b). Measurements of airway length, number of small-granule cell
clusters, and their population densities were compared
with corresponding data tabulated from the adult lobe
in these same two studies.
RESULTS
w
0
'
1
i;
1
c/
In hamsters, the left lung consists of a single large
lobe; the right comprises the right upper, right middle,
right lower, and infracardiac lobes. Each lobe is aerated
by a lobar bronchus that forms a central longitudinal
axis and gives rise to lateral airways called divisional
distributing bronchioles by Hoyt et al. (1982b). These
latter branch directly or indirectly to terminal bronchioles leading into alveolar ducts.
Formation of the Hamster Airway
-jb
a
Fig. 3. Approximation of airway length. Serial tracings (viewed from
above at top, edge-on at bottom) were superimposed for best fit for all
main airways in both lungs. Lateral displacement (a), measured between center points (black spots) of profiles marking the ends of a unit
airway, was corrected for magnification. Vertical displacement (b) was
determined by multiplying section thickness (2 pm) by the number of
sections cut between each profile. Airway length (c) was calculated by
the Pythagorean theorem (a2 + b2 = c2).
Airway Maps
By carefully superimposing the serial tracings and
allowing for the distance between them, we could reconstruct the developing lobar airway and represent it in
two dimensions. The position of small-granule cell clusters, the airways encircled by condensed mesoderm, and
in some lobes those containing ciliated cells were plotted
on the resultant airways maps (Figs. 4 and 51, one of
which was made for every lung lobe a t each developmental stage. It was then possible to count the number
of small-granule cell clusters and their clear-cell precursors and assign them to their appropriate unit airways.
Detailed Analysis of the tnfracardiac Lobe
Preliminary examination of airway maps and cell
cluster counts revealed a similar developmental pattern
in the left lung and all four lobes of the right lung.
Therefore, the infracardiac lobe, being the smallest and
also the subject of exhaustive study in the adult, was
chosen for more detailed quantitative analysis as follows: 1)The length of each unit airway in the 13-, 14-,
and 15-day fetal infracardiac lobes was measured as
described above, and the airway maps were redrawn to
scale. 2) The population density of small-granule cell
clusters was determined in the lobar bronchus, divisional distributing bronchioles, and more peripheral airways by dividing the number of clusters a t each level by
the total length of the appropriate unit airways. 3 ) Microscopic slides, serial tracings, and airway maps were
reexamined to determine how many small-granule cell
clusters were associated with the rings of intersection of
parent and daughter airways (see Fig. 2) and how many
were located at bronchioloalveolar junctions, which were
not defined until day 15 of gestation.
Airway maps prepared from the three fetal infracar~
diac lobes were compared directly with the 7 0 cardboard reconstruction of the adult lobe that served as the
The reconstructed adult infracardiac lobar bronchus
consists of 15 unit airways and gives rise to 14 welldefined lateral divisional bronchioles, each the origin of
a branching subsystem ending in terminal bronchioles.
Of these, the proximal are larger and more highly arborized than the distal, and a 15th, most distal lateral
branch of the lobar bronchus is simply a terminal bronchiole. The final segment of the lobar bronchus ends in
a spray of terminal bronchioles and can itself be considered a peripheral airway subsystem.
Examination of Figure 4 shows that on fetal day 13,
the infracardiac lobar bronchus has 15 lateral branches
and ends in a spray of fine airways. An organized layer
of mesoderm, harbinger of smooth muscle differentiation, invests virtually the entire length of the lobar
bronchus and the proximal reaches of its lateral
branches. At this stage, only eight unit airways can be
distinguished along the lobar bronchial stem owing to
the fact that the lateral airways tend to arise in pairs a t
what appear to be airway trifurcations. As the lobar
bronchus elongates over the next 48 hr, however, the
origins of its lateral branches are gradually separated,
so that by day 15 each is distinct, as in the adult.
Arborization of nonalveolated airways is at its height
on day 14, by which time it is clear that proximal lateral
airway systems are much more highly branched than
those arising further out along the bronchial stem. A
complete, organized layer of mesoderm now invests even
the terminal ramification of the lobar bronchus and has
spread peripherally along several additional orders of
branches in each of the lateral, divisional bronchiolar
systems.
The organized mesodermal sleeve makes only a slight
peripheral advance during the next 24 hr of gestation.
Distal to the sleeve, the previously nonalveolated
reaches of the airway now begin to transform into respiratory saccules. This high water mark of substantial
mesodermal investment at fetal day 15 corresponds with
very little alteration to the extent of the adult conduct-
Fig. 5. Rough maps of nonalveolated airways in 13-day (top), 14-day
(middle), and 15-day (bottom) fetal hamster left lungs (not to scale),
showing spread of small-granule cell clusters (black dots) and peripheral progression of developing smooth muscle (dashes). Positions of
ciliated cells are not marked on this figure. Developmental events
resemble those in the infracardiac lobe (Fig. 4).In the 15-day sketch,
the bronchial axis has been compressed to fit the page.
HAMSTER SMALL-GRANULE CELLS AND AIRWAY GROWTH
415
416
S.N. SARIKAS, R.F. HOYT, JR.,AND S.P. SOROKIN
ing airway, considered as numbers of specific unit airways and not as airway length. The only apparent
differences between the pattern seen in the 15-day airway map and the adult reconstruction can be accounted
for by perinatal or postnatal alveolarization of scattered
unit airways a t the periphery of the lateral branching
systems and in the terminal segment of the lobar
bronchus.
Thus it appears that although a few peripheral airways destined to become terminal bronchioles may have
been laid down by day 13, these cannot reliably be
distinguished from presumptive distributing bronchioles, because not all of the latter have yet acquired
an investment by organizing myoblasts. In 14- and 15day lobes, however, unit airways in the lateral branching systems can be divided into three main groups: 1)
those corresponding to the hierarchy of distributing
bronchioles in the adult, which are well invested by
smooth muscle; 2) those corresponding to terminal bronchioles, which are also invested by smooth muscle; and
3) those likely to be converted into alveolated airways of
the respiratory zone, without a definite mesodermal investment. At 14 days, it is still difficult to discriminate
accurately whether a few peripheral airways are to become distributing or terminal bronchioles, but by 15
days the distinction is a simple one.
Number and Distribution of Endocrine Cell Loci in the Fetal
Hamster Lung
The basic centrifugal patterns of airway formation,
mesodermal condensation associated with smooth muscle differentiation, and small-granule cell cluster formation were similar in all major subdivisions of the
lung pair, as exemplified in the drawn-to-scale airway
diagrams of the infracardiac lobe (Fig. 4) and the roughly
scaled maps of the left lung (Fig. 5).
Small-granule APUD cells and their clear-cell precursors first appeared in lobar bronchi and divisional distributing bronchioles on fetal day 13, and they
subsequently developed in a centrifugal wave, spreading
into more peripheral unit airways of the lateral and
terminal bronchial branching subsystems. Almost without exception, the leading edge of their advance did not
exceed that of the organizing myoblasts investing the
airway. Both were coterminous on day 13, but smallgranule cell clusters trailed the muscle by 3-4 generations on day 14 only to become coterminous again a t day
15 (Figs. 4,551, when a few clusters had reached bronchioloalveolar junctions. Small-granule cell differentiation
preceded that of ciliated epithelial cells by many generations of branches and a full 24-48 hrs (Fig. 4).
Counts of small-granulecell loci in fetal lungs (Table 1)
In the three age groups studied, small-granule cell
clusters and single cells were identified at a total of
2,368 loci. Of this total, only 14 were single clear cells,
representing 1.7% of all loci in the 13-day, 1.0% in the
lCday, and 0.2% in the 15-day fetal lung.
Over half (54%) of all loci occurring in the the 13-day
fetal lung pair consisted of cell clusters found along the
lobar bronchi. Although the actual number of clusters
in lobar bronchi had increased by 139 at day 14 and by
another 130 a t day 15, the rise was exceeded by the
differentiation of clusters in more recently established
peripheral airway generations. Thus, the lobar bronchi
contained 40% of all endocrine cell loci on day 14, and
only 26% on day 15.
This pattern of small-granule cell development was
common to all major subdivisions of the lung pair. Close
examination of Table 1reveals a steady increase in the
number of small-granule cell clusters in each individual
lobar bronchus, followed and ultimately exceeded by a
TABLE 1. Total number of small-granulecell clusters and solitary clear cells in 13-, 14-,and 15-day fetal hamster lungs
No. of
No. of
Fetal
small-granule cell clusters
solitary clear cells
Total No. of
Lobar
Peripheral
Lobar
Peripheral
clusters and
age
(days)
Sample
bronchus
bronchioles
Total
bronchus
bronchioles
Total
clear cells
3
82
1
2
34
79
45
13
Left lung
1
58
0
16
57
1
41
Right lower lobe
0
26
0
0
9
26
17
Right middle lobe
0
18
0
0
I1
18
Right upper lobe
7
0
41
0
0
29
41
12
Infracardiac lobe
4
225
1
99
221
3
122
Total
(1.7)
(100)
(0.4)
(1.3)
(54)
(44)
(98)
(%)
3
194
2
1
191
115
76
14
Left lung
2
2 17
0
2
137
215
78
Right lower lobe
0
99
0
0
99
52
47
Right middle lobe
0
65
0
0
65
23
42
Right upper lobe
2
74
1
1
72
32
40
Infracardiac lobe
7
649
5
642
2
261
381
Total
(100)
(0.7)
(1.0)
(59)
(99)
(0.3)
(40)
(%)
496
0
1
1
497
120
376
Left lung
15
477
1
1
2
479
93
384
Right lower lobe
0
183
0
0
183
71
112
Right middle lobe
151
0
0
0
151
37
114
Right upper lobe
0
184
0
184
0
70
114
Infracardiac lobe
3
1,494
2
1
1,491
391
1,100
Total
(0.07)
(0.13)
(99.8)
(0.20)
(100)
(74)
(26)
(%I
HAMSTER SMALL-GRANULE CELLS AND AIRWAY GROWTH
rise in the number of clusters in more peripheral airways as these younger generations are populated
through formation and maturation of clear-cell clusters.
This sequence of events is seen somewhat earlier in the
larger left lung (Fig. 5) and right lower lobe and somewhat later in the smaller right middle lobe. In the right
upper and infracardiac (Fig. 4) lobes, which are smaller
still, the lobar bronchi are short and contain few smallgranule cell clusters. In these two lobes, on day 13 the
majority of endocrine cell loci in the wall of the lobar
bronchus are found close to branch points rather than
in the internodes, but the pattern of development is the
same as in the larger lobes: An increase in loci in the
lobar bronchus during days 13-15 is exceeded by the
formation of cell clusters in younger peripheral airways.
From examination of the airway diagrams and Table
1, it appears that any single lobe would serve as a
reasonable model for further quantitative studies of
small-granule cell formation in fetal hamster lung. The
infracardiac lobe has been chosen in the present instance because comparison can be made with data already available regarding the number and distribution
of small-granule cells in the infracardiac lobe of the
adult hamster lung published previously by Hoyt et al.
(1982a,b). In the following description, all references to
the adult hamster pertain to these two studies.
417
As a consequence of these differential growth patterns
during fetal and postnatal life, the relative composition
of the bronchiaI tree changes. Of total conducting airway length, the lobar bronchus accounted for 23% on
day 13,13% on days 14 and 15, and only 7% in the adult.
Similarly, the proportion provided by distributing bronchioles decreased continually as the lobe matured, from
77% at 13 days, to only 48% at 15 days and 46% in the
adult. Conversely, terminal bronchioles, which could not
be identified reliably on day 13, accounted for 33% of
total airway length a t 14 days, 39% at 15 days, and
finally 47% in the reconstructed adult lobe.
Population density and distribution of small-granule cell
clusters in the infracardiac lobe (Table 3)
Twelve small-granule cell clusters were located in the
lobar bronchus of the 13-day infracardiac lobe examined.
By day 14 and again by day 15, the number of clusters
had more than doubled to 32 and 70, respectively, but
there was only a slight further increase noted betwen 15
days and the adult. This was reflected by a steady increase in the density of clusters (clusters per millimeter
along the airway long axis) during fetal development,
when the lobar bronchus was growing slowly. The population density was lower in the adult, however, owing
to perinatal and postnatal lengthening of the unit airways in the lobar bronchus.
In distributing bronchioles, there was a reduction in
Quantitative Investigation of Small-Granule Cell Cluster
the population density of small-granule cell clusters beFormation in the Fetal Hamster lnfracardiac Lobe
tween day 13 and day 14 of gestation. This could be
Dimensions of the conducting airways in the infracardiac
attributed to a n increase of 45 new unit airways (Table
lobe (Table 2)
2) but only six clusters during this period. By day 15,
The conducting airway system of the infracardiac lobe with a reduction of 12 unit airways (Table 2) and a
increased in total length from 4.5 mm on day 13, to 12.1 nearIy threefold increase in the number of small-granmm on day 14, and 18.3 mm on day 15, as compared ule cell clusters, the population density had more than
with 59.5 mm in the adult. This growth may be divided doubled. The population of small-granule cell clusters in
into two phases. During the first phase, the number of the adult distributing bronchioles was more than 2.5
identifiable conducting airways increases substantially times that a t 15 days, but the density remained nearly
as terminal buds divide to form additional generations constant because of the threefold increase in total length
of branches and a s the sleeve of developing muscle ad- of these airways (Table 2).
vances peripherally. There is relatively little elongation
Cell clusters were not common in terminal bronchioles
of, say, proximal elements among distributing bron- of the developing infracardiac lobe. Only five were found
chioles, and that which does occur is offset by addition in the 14-day and nine in the 15-day fetuses. In the
of younger and shorter peripheral generations; conse- adult, however, 256 clusters, or 41% of the total populaquently, mean length of unit airways remains nearly tion, occurred in terminal bronchioles.
constant.
Small-granule cell clusters were widely distributed
This process comes to a n end between day 14 and day throughout the fetal infracardiac lobar bronchus. In the
15 with the appearance of primitive respiratory saccules 13-day lung, 75% of the lobar bronchial unit airways
beyond the leading edge of the muscle coat, signaling contained such clusters; a t 14 and 15 days, the figure
the beginning of alveolarization in the distalmost divi- increased to about 90%. In contrast, most peripheral
sions of the bronchial tree.
bronchioles were devoid of clusters. In the 15-day lung
During the second, perinatal and postnatal phase, only 53% of distributing bronchiolar unit airways and
growth of the conducting airway system is accomplished 8%of terminal bronchioles contained cell clusters, comexclusively through elongation of units previously laid pared with 85% and 83%, respectively, in the adult lobe.
down. This phase overlaps the first to some extent and
begins in older, proximal airways. For example, the Relationship of small-granule cell clusters to airway
increase in lobar bronchial unit airways during days intersections and bronchioloalveolar junctions (Table 4)
13-15 seems due largely to relative separation of the
Examination of the serial sections and maps revealed
origins of previously formed lateral branches brought that over 25% of all clusters were Iocated on junctional
about a s a result of elongation of the bronchus itself. rings (Fig. 2) a t airway intersections in the 13-day and
Furthermore, by day 15 the lobar bronchus has attained 14-day infracardiac lobes. However, by 15 days, the numroughly half of its adult length, compared with one-third ber of clusters had increased mainly along regions of the
in the case of the more peripheral distributing bron- larger airways between branch points (Fig. 4), so that
chioles and only one-quarter for terminal bronchioles, only 11%of all clusters appeared to be located at airway
each of which consists of a single unit airway.
intersections. In the adult, where loci were marked on a
Adult'
13
Day
Distributing
bronchioles
14
15
Day Day Adult'
~
-
-
13
Day
0.05
0.02
3.9
33
76
0.06
0.04
7.2
39
120
55
13
Day
180
219
All airways
14
15
Day
Day
21
32
44
92
12
12
29
75
32
14
100
38
90
71
87
70
29
19
8
13
Day
29
'Data from Hoyt et al. (1982a,b).
'Measured along the airway long axis.
31ncludes six cell clusters located in the respiratory zone.
Small-granule
cell clusters/mm2
Total number
of clusters
% of all
clusters in the lobe
Unit airwavs
with clusters
(% total)
13
Day
Lobar
bronchus
14
15
Day Day Adult'
23
49
35
5
44
57
85
272
105
53
10
12
Distributing
bronchioles
14
15
Day Day Adult'
-
13
Day
5
7
8
9
5
7
1
1
83
41
256
9
Terminal
bronchioles
14
15
Day Day Adult'
38
100
41
3
13
Day
21
100
72
2
31
100
184
11
85
100
6213
10
All airways
14
15
Day Day Adult'
TABLE 3. Distribution and density of small-granule cell clusters in the infracardiac lobe of 13-, 14-, and 15-day fetal
and adult hamster lungs
209
Adult'
0.25
0.08
0.07
0.08
0.28
0.19
0.04 0.04
0.06
0.17
4.5
28.0
12.1
18.3
59.5
100
100
100
100
47
112
Terminal
bronchioles
14
15
Day Day
Adult'
'Data from Hoyt et al. (1982b).
'Of 15 unit airways making up the lobar bronchus in the adult, the two most proximal were not included in the reconstruction.
47
92
80
84
No. of
8
12
19
15'
unit airways
Length (mm) of unit airways
0.34 0.07 0.07 0.11 0.33
0.13 0.13 0.14
Mean
0.18
0.04 0.04 0.06
f SD
0.05 0.05 0.07
0.14
8.7 27.0
3.5
6.6
1.6
2.4
4.5
Total
1.0
77
54
48
46
13
7
%Total of
23
13
all airways
13
Day
Lobar
bronchus
14
15
Day Day
TABLE 2. Dimensions of the airways in the infracardiac lobe of 13-, 14-, and 15-day fetal and adult hamster lungs.
419
HAMSTER SMALL-GRANULE CELLS AND AIRWAY GROWTH
TABLE 4. Number of small-granule cell clusters at airway intersections and
bronchioloalveolar junctions in the infracardiac lobe of 13-, 14-, and 15-day fetal and adult
hamster lungs
Small-granulecell clusters,
actual number (9% of total)
Position
Airway intersections
Junctional rings
Carinal points
Total
Bronchioloalveolar
junctions
Neither intersections
nor junctions
Total
13 day
14 day
15 day
Adult'
6 (15)
5 (12)
11(27)
11(15)
8 (11)
19 (26)
-
8 (4)
12 (7)
20 (11)
7 (4)
99 (16)
25 (4)
124 (20)
160 (26)
30 (73)
53 (74)
157 (85)
337 (54)
41 (100)
72 (100)
184 (100)
621 (100)
-
'Data from Hoyt et al. (1982a).
Patterns of Development in the Pulmonary Airways
scale-model reconstruction of this lobe, 20% were found
on junctional rings a t intersections. Very few clusters
By 13 days of gestation in hamsters, the main scaffold
populated the small distributing and terminal bron- of the bronchial tree is already present with all the main
chioles of the 15-day infracardiac lobe (Fig. 41, and only subdivisions of the lobes defined. Each lobar bronchus
4% were located at the recently formed bronchioloalveo- forms a much more distinct axis than is the case in
lar junctions, compared with 26% in the adult.
human beings (Wells and Boyden, 19541, and proximal
branches are more extensively subdivided than distal
DISCUSSION
ones, so that the lobes are approximately pyramidal in
The results of this quantitative, cartographic study on configuration. Divisional distributing bronchioles furthe developing small-granule cell system of hamster nish a less distinct axis for their subdivisions and are
lungs, based on limited sampling, bears out the main less pyramidal overall. These configurations are reconclusions reached in the associated morphological tained in subsequent developmental stages to maturity.
study (Sarikas et al., 1985), based on extensive samAll major segments of the future conducting airways
pling. Both studies indicate that the system is not fully can be identified in all lobes of 14-day lungs by the
established by late fetal life, although it undergoes rapid presence of a n investment of differentiating smooth
development during the last quarter of gestation.
muscle and connective tissue cells about them. By 15
Specific maps necessarily are based on individual ani- days these airways have been completely defined as a
mal specimens, and because of the time required to result of further advance of the muscle sleeve to cover
prepare them accurately, it is a practical necessity to terminal bronchioles. At the same time, alveolarization
limit their number. In answering anticipated criticism begins in earnest. Further development of the purely
that the value of the quantitative study is diminished conducting passages is mainly in length and diameter
because measurements were taken from only one ex- of the unit airways and in the differentiation of their
ample for each of the three fetal stages, we would re- constituent cells.
spond as follows: 1)The single examples were selected
Taking the infracardiac lobe as a model, and after
as representative from a wealth of material used in the comparing 15-day prenatal with adult stages, it appears
morphological work and were completely rather than that very few muscle-invested airways are subjected to
statistically studied by serial sections passing through retrograde alveolarization as a mechanism to increase
the entire lung pair a t each stage, so that neither airway the respiratory surface, since only 10 unit airways are
branches nor small-granule cell clusters were over- lost in the interval (Table 2). In hamsters, unlike dogs
looked. 2) When maps drawn a t 13, 14, and 15 days of or human beings (Boyden and Tompsett, 1961, 1965;
gestation for any one of the five lung lobes were laid Boyden, 1967), alveolarization appears virtually conside by side for comparison, it was at once seen that a fined t o present and future branches of the 15-day fetal
characteristic branching pattern for the particular lobe lung distal to the muscle coat, and this may help to
was recognizable at all three stages, and when arranged explain why transitional, alveolated airways like respiin ascending order of gestational age they formed a ratory bronchioles are either short or absent in this
coherent developmental sequence for the airways of that species (Tyler, 1983).
lobe. Had any stage been grossly aberrant, it would have
confuted the series. 3) In the case of the infracardiac
Population Densities of Small-Granule Cells in Developing
lobe, it was further evident from comparison with our
and Adult Lungs
scale model of this lobe in adult lung that the branching
As
seen
in
data
from
the infracardiac lobe (Table 3,
pattern seen in fetal life is similar to the pattern present
at maturity and that the four stages illustrate the devel- Fig. 4), the overall population density of small-granule
cell clusters is very low on days 13 andl4 (ca. 3 clusters
opmental history of this lobe.
420
S X . SARIKAS, R.F. HOYT, JR., AND S.P. SOROKIN
per millimeter airway length) but rises sharply to 11per
millimeter on day 15 and remains about the same (10
per millimeter) in the adult. This could be taken to mean
that the population density attained in late fetal life is
maintained a t this level by uniform addition of new cell
clusters during perinatal and postnatal elongation of
airways laid down by day 15, but examination of the
breakdown by airway classes shows this to be incorrect.
Inasmuch as the lobar bronchus contains 80% of its
adult complement of cell clusters by day 15, the distributing bronchioles 39%, and the terminal bronchioles
only 3.5%, the data uphold the conclusion of a proximalto-distal progression of small-granule cell development
drawn in the morphological part of this study (Sarikas
et al., 1985).
Between days 13 and 14 the small-granule cell population increases by a factor of 1.8; between days 14 and
15, it increases by 2.4 times; and between day 15 and
adulthood, it rises by 3.4 times. A growth spurt in the
last day before birth conceivably could account for much
of the latter, more than threefold increase in smallgranule cell clusters, but we have no counts for the day
of birth (day 16) to determine if this is so. Nevertheless,
our finding of a mitotic figure in a neonatal small-granule cell cluster, and the evident immaturity of many
small-granule cells in early postnatal lungs as reported
in the accompanying paper, both argue that neoformation of clusters continues on into postnatal life.
Our data do show that the increase in the population
between day 15 and adult life is largely accounted for by
the appearance of clusters in previously unpopulated
unit airways and only in a minor way by formation of
new clusters to fill in between older established clusters
in elongating airways. In the lobar bronchus, all but
two, or 90%, of the unit airways contained cell clusters
at day 15, whereas all possessed them in the adult. In
distributing bronchioles, 53% of unit airways had clusters on day 15, compared to 85% in the adult, and in
terminal bronchioles, 8% had clusters on day 15 compared to 83% in the adult. Thus, the greater part of
small-granule cell neoformation after 15 days occurs in
unit airways of the more distal muscle-invested portions
of the bronchial tree.
Relationship Between Small-Granule Cell and Other
Differentiations in the Lungs
From the inspection of the developmental maps (Figs.
4, 51, spatial and temporal association Lippears to exist
mainly between the appearance of new clusters of smallgranule cells and the peripheral advance of the sleeve of
differentiating smooth muscle along the airway. In contrast, branch points have already been selected and
branching has occurred before clear-cell precursors of
the small-granule cells become manifest at these locations, and other cellular differentiations in the pulmonary endoderm such as ciliation lag many generations
of branching behind the wave of small-granule cell
formation.
During fetal development, precursors of small-granule
cells almost without exception do not appear ahead of
the muscle coat but either advance with its leading edge
of lag behind it by a few generations of branching. Indeed, in the adult infracardiac lobe only 6 of 621 clusters
were located in alveolar ducts. bevond the muscle coat.
Whether a causal, perhaps inductive interaction occurs
between special elements in the investment and the
epithelium to initiate formation of small-granule cells
awaits further investigation.
Because formation of the respiratory zone is already
well under way before small-granule cells significantly
populate terminal bronchioles, no case can be made in
hamsters that the clusters stimulate subepithelial capillary growth in preparation for alveolarization, as recently proposed by Stahlman et al. (19851, based on
observations in human lungs. Although most of the clusters occupying the junction between the conducting airway and respiratory zone in hamsters are unusual in
being closely invested by capillaries (Hoyt et al.,
1982a,b), they appear too late in development to have
served such a trophic purpose. Once established in the
terminal bronchioles, if these cell clusters exert any
influence over formation of the respiratory zone, it must
be a n inhibitory one, possibly acting to prevent excessive retrograde alveolarization.
CONCLUSIONS
Taken together, the morphological and quantitative
studies on the development of small-granule cells in
hamsters show that prenatal stages are compressed into
a shorter period, both in terms of days and proportion of
the gestational period (4 days, 25%) than is true of either
the rat (8 days, 36%), rabbit (14 days, 44%), or man (30
weeks, 79%, according to Stahlman and Gray, 19841, yet
development has not been compressed sufficiently to
provide a system largely populated by mature cells a t
the time of birth.
ACKNOWLEDGMENTS
This study was supported in part by NIH Research
Grant HL-19379; a major portion of this work was submitted by Dr. Sarikas in partial fulfillment of the requirements for the Ph.D. degree a t Boston University.
LITERATURE CITED
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and childhood. Am. J. Anat., 121:749-762.
Boyden, E.A., and D.H.Tompsett (1961) The postnatal growth of the
lung in the dog. Acta Anat., 47t185-215.
Boyden, E.A., and D.H.Tompsett (1965) The changing patterns in the
developing lungs of infants. Acta Anat., 61:164-192.
Hoyt, R.F., Jr., H.Feldman, and S.P. Sorokin (1982a) Neuroepithelial
bodies (NEB) and solitary endocrine cells in the hamster lung. Exp.
Lung Res., 3.299-311.
Hoyt, R.F., Jr., S.P. Sorokin, and H. Feldman (1982b) Small-granule
(neuro) endocrine cells in the infracardiac lobe of a hamster lung.
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Sarikas, S.N., R.F. Hoyt, Jr., and S.P. Sorokin (1985) Ontogeny of
small-granule APUD cells in hamster lung. A morphological study.
Anat. Rec., 213t396-409.
Sorokin, S.P., and R.F. Hoyt, Jr. (1978) PAS-lead hematoxylin as a
stain for small granule endocrine cell populations in the lungs,
other pharyngeal derivatives and the gut. Anat. Rec., 192:245-260.
Stahlman, M.T., and M.E. Gray (1984) Ontogeny of neuroendocrine
cells in human fetal lung. I. An electron microscopic study. Lab.
Invest., 51:449-463.
Stahlman, M.T., A.G. Kasselberg, D.N. Orth, and M.E. Gray (1985)
Ontogeny of neuroendocrine-cells in human fetal lung.- 11. An
immunohistochemical study. Lab. Invest., 5252-60.
Tyler, W.S. (1983) Comparative subgross anatomy of lungs. Pleura,
interlobular septa, and distal airways. Am. Rev. Resp. Dis.,
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