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ACTIVITY or CERTAIN ENZYMES IN THE FORELIMB OF THE
DEVELOPING CHICK EMBRYO: A HISTOCHEMICAL STUDY
By

Martin K. Feldman

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Anatomy

1967

C%Q\50

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’2 I) "‘6

ACKNOWLEDGMENTS

I wish to acknowledge the supervision and
guidance of my advisor, Dr. James Blake Thomas,
Department of Anatomy, College of Human Medicine,
Michigan State university. Appreciation is also due
to Dre. Ester Smith, Martin Balabin, M. Lois Calhoun,
and Al Stinaon for their suggestions and help in
preparation of the final manuscript and photomicro-
graphs. Special thanks are due to Dr. Edward Fowler,
Department of Veterinary Pathology at the Ohio State
university, for his invaluable help with the histo-
chemical techniques.

I offer final thanks to the graduate students
in the Anatomy Department for their suggestions and

encouragement throughout the course of the research.

11

TABLE OF CONTENTS
Page
INTRODUCTION . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . 4
MATERIAI.AND METHODS
Preliminary Histochemical Survey . . . . . . . l6

AcP,.ATPase, and LDH.Activities in Forelimb
Development . . . . . . . . . . . . . . . . 18

RESULTS
Acid Phosphatase . . . . . . . . . . . . . . . 21
Adenosine Triphosphatase . . . . . . . . . . . 23
Lactic Dehydrogenase . . . . . . . . . . . . 24
DISCUSSION’AND CONCLUSIONS

Functions and Distribution of AcP,.ATPase,
ludLDH..............o.. 30

Possible Implications of Enzymatic Activity . 32
General Considerations . . . . . . . . . . . . 41
SWRY O I C O I O Q Q C O O O C O O O O O O O C 46

LIERATURE CITED I O O O O O O O I O O O O O O O ‘8

iii

LIST OF TABLES

Table Page

I. Comparison between Hamilton and Ham-
burger (1951) and Saunders' (1948)
methods of classifying forelimb de-
velopment in the chick . . . . . . . . . . . 5

II. Acid phosphatase activity in de-
veloping chick forelimb . . . . . . . . . . 27

III. Adenosine triphosphatase activity in
developing chick forelimb . . . . . . . . . 28

IV. Lactic dehydrogenase activity in de-
veloping chick forelimb . . . . . . . . . . 29

iv

LIST OF FIGURES

Figure Page
1. Forelimb bud. Stage 19. H. a E. . . . . . . 55
2. Forelimb bud. Stage 21. H. S E. . . . . . . 55
3. Forelimb bud. Stage 24. H. 5 E. . . . . . . 57
4. Forelimb. Stage 27. H. & E. . . . . . . . 57
5. Forelimb. Stage 31. R. &‘E. . . . . . . . 59
6. Forelimb bud. Stage l9..AcP activity . . . 61
7. Forelimb bud. Stage 21. AcP activity . . . 63
8. AER. Stage 21. AcP activity . . . . . . . 63
9. AER. Stage 21. AcP activity . . . . . . . 65

10. AER. Stage 24. AcP activity . . . . . . . 67

ll. Porelimb bud. Stage 24. AcP activity . . . 67

12. Proximal portion of forelimb. Stage 27.
AcP activity . . . . . . . . . . . . . . . 69

13. Distal tip of forelimb. Stage 27. AcP
act1v1ty O O O O O O O O O O O O O O O O O 71

14. Skin of the forelimb. Stage 27. AcP
‘CC1v1ty C O O O O O C O I O O O O C O O O 71

15. Forelimb. Stage 31. AcP activity . . . . 73
16. Forelimb bud. Stage 19. ATPase activity . 75

17. Forelimb bud. Stage 21. ATPase activity . 75

Figure

18.
19.
20.

21.
22.
23.
24.
25.
26.
27.
28.

29.

Forelimb bud. Stage 24. ATPase activity .
Porelimb. Stage 27. ATPase activity . . .

Developing bone in cross section. Stage 31.
ATPase activity . . . . . . . . . . . . . .

Developing bone. Stage 31. ATPase activity.
Forelimb bud. Stage 19. UDH activity . . .
Forelimb bud. Stage 21. LDH activity . . .
AER. Stage 24. LDH activity . . . . . . .

Axillary region. Stage 24. LDH activity

0

Forelimb. Stage 27. LDH activity . . . . .
Axillary region. Stage 27. LDH activity .

Bone formation in cross section. Stage 31.
LDH activity . . . . . . . . . . . . . . . .

Developing long bone. Stage 31. LDH
actiVity . 0 O 0 C O O O I O O O O I O 0

vi

79
81
83
83
85
85
87

89

89

91

INTRODUCTION

Histologic and experimental data have, for a
number of years, suggested the importance of certain
areas or structures within the devalOping chick fore-
limb to normal morphogenetic activity. Only recently
and to a limited extent has the biochemical analysis
of these processes been attempted. Findings to data
indicate that certain areas vhich appear histologically
homogenous actually exhibit distinct enzymatic and
molecular patterns of develOpment and growth. Either
as a result or cause of induction, biochemical activ-
ity may increase or decrease, supplying or denying
tissues of the limb molecular components which may be
necessary to maintain or deveIOp specific structures
at the desired time. It is the purpose of this study,
therefore, to determine the distribution and develop-
nent of several important enzymes during critical
phases of limb formation in the chick.

By determining the level of enzyme activity, it

can be hypothesised that certain biochemical processes

2

are active in any given tissue. Enzyme histochemistry
is one method used to determine this activity. It is
true that these techniques introduce problems of inter-
pretation due to the subjective nature of the data and
difficulties in diffusibilities of various dyes used.
They do, however, enable the investigator to qualitate
enzyme activity in individual elements of forelimb
tissue and, when differences are sufficiently marked,
to make rough quantitative comparisons of this activ-
ity. Histochemistry represents the most sensitive
ensyme technique available since it can localize
activity within a single cell. Biochemical analysis,
on the other hand, usually involves homogenation of
the entire structure. Obtaining sufficient and pure
quantities of any one element becomes almost impos-
sible in non-homogenous structures like the limb bud.
For these reasons, the histochemical approach was
selected for this study.

The objectives of this investigation are to:
(1) survey histochemically a series of enzymes to
determine which are active during stage 27, (2) ana-
lyse the chick limb bud during stages 19, 21, and 24
for various enzymes found active in the survey, (3)

determine histochemically the location and activity

3
of selected enzymes present during stages 27 and 31
in an attempt to show activity during chondrogenesis
and osteogenesis, respectively, and (4) show via hema-
toxylin and eosin staining of similar tissues any
relationships which exist between histologic and bio-
chemical phenomena.

It is hoped that this research will supply in-
formation about the molecular activity of forelimb
growth and provide an understanding of an induction
system of primary importance, setting the stage for
future chemical, hormonal, and regeneration studies

on the avian limb.

LITERATURE REVIEW

The development of the chick forelimb has been
studied for many years and has involved three distinct
disciplines: (1) gross and histological, (2) experi-
mental, and (3) biochemical morphogenesis. Saunders
(1948) followed by Hamburger and Hamilton (1951)
chronologically classified the various stages involved
in limb morphogenesis. A comparison of these two
systems is illustrated by Table I. The Hamburger and
Hamilton method.will be used throughout the remainder
of this paper.

Zwilling (1961) reviewed in detail the morpho-
logical process involved in early limb deveIOpment.
Initial thickening of somatopleure lateral to somites
17-20 called Welff's crest occurs between stages 15
and 17, followed by a stage of growth and elongation
between stages 18 and 23. Stage 18 is essentially
that growth occurring as somites 30-36 develop and
stage 19 as somites 37-40 develop. During stages 18-20

a distal thickening of ectoderm develops and protrudes

Table I. Comparison between Hamilton and
Hamburger (1951), and Saunders'
(1948) methods of classifying
forelimb deve10pment in the chick.

 

 

Time in Hamilton and Saunders‘
hours Somites Hamburger stages stages
50-55 24-27 15 1
51-56 26-28 16 2
52-64 29-32 17 3
65-69 30-36 18 4
68-72 37-40 19 5
70-72 40-43 20 6

84 43-44 21 7

34 22 8-9
84-96 23 10

96 24

120 27

168 31

6
as a nipple-shaped structure in cross section termed
the apical ectodermal ridge (AER). Stage 21 is char-
acterized by the formation of blood vessels and a
pronounced ventral bending of the limb bud with rapid
elongation of the posterior half. Stage 22 contains
a marked mesodermal condensation which will eventually
form the limb's skeletomuscular system, and is typi-
fied by a large flat paddle-shaped limb directed ven-
trally and posteriorly. Stage 23 is defined as a
further elongation with marked swelling at the base
of the forelimb. Romanoff (1960) described the for-
mation of cartilage commencing with stage 27 and ossi-
fication with stage 31.

Histologically, Zwilling (1961) showed that two
distinct germ layers are involved, the outer ectoderm
and the inner mesoderm. After initial formation of
the limb bud, rapid proliferation of the mesoderm
causes significant elongation and marked ventral bend-
ing from stages 18-21. The mesoderm, itself, is spongy
and traversed by a number of vessels forming an exten-
sive plexus just under the ectoderm. This plexus is
supplied totally by the axial artery as it branches
off the most distal portion of the elongated limb bud

just under the AER.which shunts blood back to the

7
general circulation via the anterior cardinal vein as
reported by Romanoff (1960).

Electron microscopic studies by Jurand (1965)
indicate that mesodermal cells during this early stage
lack complete cell membranes, and cytOplasmic conti-
nuity exists between neighboring cells. They possess
an extensive endoplasmic reticulum which appears to be
associated with the mitochondria. Golgi bodies are
scarce. Jurand found that during this early stage,
the ectoderm is a fairly uniform, single layer of
cuboidal cells with numerous microvilli along the dis-
tal surface and many folds, processes, and microvilli
between the cells. In the proximal portion of the
bud, the ventral ectoderm is conspicuously thickened
and columnar. The cells of the newly formed AER.con-
tain numerous elongated mitochondria, many Golgi
bodies, and an elaborate endoplasmic reticulum. These
organelles are evidently not associated with the high
mitotic activity alone, since the mesoblast has a far
greater mitotic activity and fewer intracellular struc-
tures than the cells of the AER. The basement membrane,
an scellular structure approximately 300-400 angstroms
thick, exists around the entire bud between the meso-

derm and ectoderm.

8

From stage 21-25, maximal AER.devclopment occurs.
According to Jurand (1965), the outer and intermediate
ridge cells begin to show numerous lysosomes and neo-
rotic centers in the cytoplasm. Degenerating cells
appear to be phagocytized by surrounding cells. The
basement membrane remains as described previously with
the exception of definite discontinuities just under
the AER. Romanoff (1960) reported that the mesoderm
begins to differentiate slightly with the formation
of more distal vessels. Saunders (1966a) noted that
a massive zone of necrosis called the posterior nec-
rotic zone (PNZ) appears in the ventral proximal meso-
derm and is an essential step in the separation of the
upper arm.from the body wall. Necrotic centers similar
to the PNZ develop within the interdigitsl tissue and
are related to separation of the digits.

Fell (1925) first noted that by stage 27 the
central mesenchyme condenses to form long cylinder-
shaped structures in a process called chondrogenesis.
Precartilage, cells which have not yet taken on the
histologic characteristics of cartilage, develop into
secreting chondrocytes. Once the cartilaginous struc-
tures are formed, a layer of fibroblasts develops

around the shaft. This is soon replaced by a layer

9
of osteoblastic cells in the form of a periosteal
collar. Romanoff (1960) reports that the exact time
at which these changes occur varies considerably with
the different regions of the forelimb.

The phenomenon of a proximal-distal sequence of
development is aptly demonstrable in forelimb growth.
Through a series of experiments, Saunders (1948) was
able to mark various parts of the presumptive fore-
limb tissue with carbon particles and trace their final
destination in the fully developed limF. Very early
‘wing buds were found to contribute to the entire limb,
but as elongation proceeded, the apex of the bud first
contributed only to the forearm and later only to the
extreme distal end of the wing. This same sequence is
followed with cartilage, bone, and feather formation
indicating conclusively a proximal-distal sequence of
limb formation. We can assume from this study that
sometime during elongation the cells forming various
parts of the adult limb become fully differentiated
and determined.

Zwilling (1961) tried to relate the gross and
histologic sequence of events with a theory of devel-
opmental induction. He hypothesised that initial limb

development is dependent upon activation of the

10
mesoderm by some as yet unexplained mechanism followed
by an ectodermal induction by a mesodermal ”factor"
supplied by the activated mesoderm allowing formation
of the AER.and the proximal ventral ectodermal thicken-
ing. At this stage an interdependence is established
between the two layers. Jurand (1965) similarly clas-
sified limb development as consisting of three distinct
periods. They are: (l) the period of initiation of
activity within the somatopleura, (2) the period of
interaction between the mesoblast and the epiblast,
and (3) the period of formation of definite mesodermal
components within the limb mesoblast.

The importance of the ectodermal component can-
not be minimized and has been the object of much ex-
perimentation. Saunders (1948) reported that carbon
particles placed on the AERMvere found dispersed
throughout the entire limb as development proceeded,
suggesting that the AER.plays a role in the formation
of the entire limb rather than one discrete part. If
the AER.is removed, only a partial limb develops or
reduced distal components form from the remaining
stump. Bell, gg‘gl., (1962) and Goetinck and.Abbot
(1963) found that if some of the ectodermal cells

remained, the limbs proceeded to develop normally.

11

According to Zwilling (19563,b), if the ectoderm is
rotated 900 leaving the mesoderm untouched, limb out-
growth appears reoriented to the ectodermal component.
TranSplantation of two AER's to one cut surface of a
limb produces partial or complete limb reduplication.

Evidence of an interaction between the mesoderm
and ectoderm is probably best supported by studies of
the formation of the basement membrane. Speculation
by Edds (1958) as to the origin of the membrane led
to several series of experiments. Bell, 55 gl., (1959)
noted that the basement membrane formed around limb
mesoderm implants in the flanks of chick embryos only
when covered by a layer of ectoderm. Hay and Revel
(1963) injected tritiated proline into Ambygtoma lar-
vae and observed autoradiographically that it accumu-
lated first within the mesoderm and ectoderm of the
skin and later in the material of the basement mem-
brane, suggesting a possible transfer of products from
both layers during the formation of the basement mem-
brane. Praline is found within collagen, a substance
existing in large amounts within the basement membrane
according to studies by Harkness (1961). Kallman and
Grobstein (1965) reported that radioactive proline,

when supplied to selected mesenchyme i3 vitro, passes

12

through a filter and accumulates along the epithelial
side. This materiel has been called trapocollagen,
and is thought to be the soluble monomer of collagen.
The epithelium at this point, according to Pierce
(1966), catalyses the polymerization of the tropocol-
lagen into recognizable collagen fibrils. Since
presence or absence of the basement membrane may sig-
nificantly control normal limb development, inter-
actions between these two layers with regard to this
important structure need considerably more study.

To support the theory that a mesodermal factor
is involved in ectodermal induction, Saunders (1949)
placed a small mica plate between the mesoderm and
ectoderm. Both AER.formation and elongation failed
to occur. In 1963, he was able to demonstrate that
reversed wing bud stumps, if placed anterioposteriorly
and dorsoventrally, will allow for duplicate digit
formation, presumably due to the redistribution of the
maintenance factor within the postaxial area of the
mesoderm. If, however, a small Mylar sheet is placed
between the reoriented stump and the postaxial‘meso-
dorm, reduplication fails to occur. This evidence
suggests that postaxial tissues supply the necessary

factor for normal morphogenesis. Zwilling (1964)

13
supported the conclusion that an interaction does
occur, for when limb ectoderm or mesoderm is grown
alone, or when limb mesoderm.is grown with non-limb
ectoderm, only random, unrecognizable structures
develop. Limb mesoderm and limb ectoderm together,
however, form recognisable limb structures with some
exhibiting marked digit formation.

Biochemically, relatively little has been
attempted to elucidate the mechanisms involved in limb
development and growth. Milaire (1962), Moog (1944),
and Jurand (1965) noted the histologic development of
acid phosphatase (Ac?) activity in limbs of several
vertebrates including the chick. During stages 18-22,
Milaire observed.AcP activity in the ventral ectoderm
and.AER. Ultrastructurally, Jurand noted that the
lysosomes, extra-nuclear necrotic centers, and Golgi
bodies within the AER.demonstrated high activity during
stage 2h. Hoog found that the skin lost most of its
activity by stage 29, after*which time all layers be-
came negative except for slight reactivity in the
nuclei. Mesenchyme generally retained its activity
until differentiation occurred, and cartilage, when it
first appeared, was negative. Alkaline phosphatase

activity was also studied histochemically by Hileire

14

(1952) and Moog (1944) and biochemically by HtWhinnie
and Saunders (1966). Alkaline phosphatase activity
appeared strongly in the AER, weak in the ventral
ectoderm, and non-existent in the dorsal ectoderm in
most vertebrates during stages 18-22. By stage 29,
the activity of the ectoderm was lost, while mesen-
chymal activity remained until differentiation, dis-
appearing in developing cartilage.

Milaire (1962) observed ribonucleic acid (RNA)
in the proximal postaxial ectoderm.during stages 18-22.
The ventral mmsoderm exhibited high RNA content just
under the ventral ectoderm. Hinrichaen (1956) noted
that the highest concentration existed in the AER.

Neel (1948) reported that ascorbic acid accu-
mulated as a ring around the base of the limb bud from
stage 24-27, disappearing as develOpment proceeded. It
was found to collect between the developing digits and
in myogenic areas prior to histologic differentiation.
He also found that fat accumulated in higher concen-
trations in mesenchymal cells than in chondroblasts.

Reel (1948), Prichard (1952), and Milaire (1962)
studied glycogen accumulation in the developing fore-
limb. Weel observed glycogen only in muscle after

histologic differentiation. Milaire noted that it was

15
present in preosteoblasts and cartilage, but negli-
gible in periosteal fibroblasts. All of this activity
substantiates our previous knowledge that an inter-
action exists between the ectoderm and mesoderm, es-
pecially in the ventral and distal areas of the

elongated bud.

MATERIAL.AND METHODS

Preliminary Histochemical Survey

Fifteen enzymatic systems were studied in an
attempt to determine which of these enzymes were
active within the limb bud of a stage 27 embryo.

Frmm these data the most informative enzymes were
selected and studied in detail over longer periods of
limb development. Fertilized eggs from‘White Leghorn
chickens were incubated at 100°? for approximately 5
days. Five limbs were obtained for each study, pre-
pared and sectioned at 12 microns, and stained accord-
ing to the selected histochemical technique. A known
positive rat tissue, usually liver or kidney, was
always included in each staining procedure serving as
the false negative. In addition, identical tissue was
processed in incubation media which contained no sub-
strate; this served as the false positive control.

The following is a list of the enzymes studied

and the techniques used:

16

10.

ll.

12.

13.

14.

15.

17

Acid phosphatase - simultaneous coupling
azo dye technique. (Barks, 1960)

Acetylcholinesterase - acetylthiocholine
method. (Gomori, 1952)

Adenosine triphOSphatase - lead nitrate
method. (Wachstein and Meisel, 1957)

Aldolase - tetrazolium salt method. (Abe
and Shimiger, 1964)

Alkaline phosphatase - simultaneous coup-
ling azo dye technique. (Burstone,
1958)

Cytochrome oxidase - Burstone technique.
(Burstone, 1959)

DPN-diaphorase - tetrazolium salt tech-
nique. (Scarpelli, gg‘gl., 1958)

Glucose-6—ph05phate dehydrogenase . nitro-
BI method. (Nachlas, 25 31., 1958)

B-glucuronidase - naphthol AB-BI glucuroni-
dide method. (Hayashi, £5 31..., 1964)

Lactic dchydrogenase - nitro-BT method.
(Nachlas, 35 31., 1958)

Leucine aminopeptidase - Nachlas technique.
(Nachlas, gg‘gl., 1957a)

Malic dehydrogenase - nitro-BT method.
(Nachlas, $5.51., 1958)

unnamine oxidase - tetrasolium.salt tech-
nique. (Glenner, gg‘gl., 1957)

Succinic dehydrogenase- nitro-BT method.
(Nachlas, £5 5;, 1957b)

IPN-diaphorase ~ tetrazolium salt tech-
nique. (Scarpelli, gg‘gl., 1958)

18

The results indicated that three enzymes ex-
hibit moderate to intense activity at this stage of
development. These were: (1) acid phosphatase (AcP),
(2) adenosine triphoaphataae (ATPaee), and (3) lactic
dehydrogenase (LDH). In addition, the two diaphorase
eyeteme, DPN-d and TPN-d, were found to be strongly
positive. Enzymes found to give weak positive reac-
tions included alkaline phosphatase, laucine amino-
peptidase, monamine oxidase, and aldolase. Enzyme
activity was found negative for succinic dehydrogenase,
malic dehydrogenase, cytochrome oxidase, glucoae-é-
phosphate dehydrogenase, s-glucuronidase, and acetyl-

cholinesterase.

AcP,.ATPaae, and LDH.Aetivities in Forelimb Development

Three enzymes exhibiting moderate to intense
activities in the preliminary survey were examined fur-
ther in forelimb buds from stage 19. 21, 24, 27, and 31
embryos. These enzymea were AcP,‘ATPase, and LDH.
White Leghorn chicken eggs were obtained and incubated
routinely at 100°? for the desired period of time. The
stage of embryonic development was determined from
gross examination of the embryo and histological

analysis of forelimb structures as outlined by

19
Hamburger and Hamilton (1951). At least 20 limbs were
examined for each stage selected. Serial sections
were prepared.

AcP-activity was studied using Barka‘s (1960)
simultaneous aso dye technique. Limbs were obtained
and placed for approximately 12 hours in a cold 4%
neutral formol calcium solution and then transferred
to 3 .88M sucrose solution. Tissues were frozen and
sectioned on a cryostat at 12 microns. Rat kidney
sections served as false negative and positive con-
trols. Within each embryonic section, the liver and
Spinal cord always gave intense positive reactions.

nATPase activity was examined using wachstein
and Meieel's (1957) lead nitrate method. The method
requires fresh frozen sections. All tissues were
once again cut at 12 microns, incubated and stained
as required. Rat liver served as false negative and
positive controls. Within each section of limb and
associated body structures was found some liver tissue
which always exhibited an intensely positive reaction
and served as an intraembryonic control.

LDH was studied further using the Nachlas,

35 51.. (1958) nitro-BT method, which also required

fresh frozen tissue. During each staining trial or

20

run, a rat kidney section served as the false nega-
tive and positive control. Liver again acted as an
intraembryonic control since it always stained in-
tensely for LDH. Since the final transfer of electrons
in the conversion of lactate to pyruvate is dependent
upon the functional state of the DPN and TPN diaphorase
systems, and since our tetrazolium salt technique also
depends on these systems to form the final colored pre-
cipitate, it was necessary to determine the activity of
the two diaphorascs. Using the Scarpelli, gt gl.,
(1958) technique, strong activity was found to exist
from day 3 on, and would not, therefore, interfere
with the LDH determinations.

In an effort to compare normal histologic
structures with enzymatic activity present in limb
morphogenesis, every tissue stained histochemically

was also stained with hematoxylin and eosin.

RESULIS

Enzyme activity was noted according to the
following empirical scale: 0, negative; 1, slight;
2, moderate; and 3, intense. Tables 11, III, and
IV illustrate average histochemical values observed
in the various structures of the forelimb. The re-
sults were statistically analyzed according to the
Kenn-Whitney two-tailed U test (Siegel, 1956). Fig-
ures 1-5 represent hematoxylin and eosin stained
sections of normal forelimb bud from stage 19, 21,

24, 27, and 31 embryos.

Acid Phosphatase

During stage 19, the limb bud exhibited a
1.0-1.1 activity throughout the ectoderm and meso-
derm. However, it was observed that slightly more
activity persisted in the newly-formed AER.(Fig. 6).
By stage 21, the AER developed a 2.4 activity con-
centrated within a central core and bordering on the

underlying meanderm (Figs. 8 and 9). The axillary

21

22

ectoderm exhibited a significantly increased 1.9 ac-
tivity (Fig. 7). All other areas of the mesoderm and
ectoderm retained a low 1.0-1.3 activity (Fig. 7). By
stage 24, the Ac? activity of the AER.developed into
two or more 3.0 sites of activity. One was located in
the central core as described in the previous stage,
while the others were found at the extreme tip of the
IAER bordering on the amnionic cavity (Fig. 10). The
activity of the axillary region decreased considerably
by this stage, while the rest of the ventral ectoderm
maintained its 1.9 activity (Fig. 11).

By stage 27, the AcP activity of the ectoderm
around the entire limb increased markedly giving a
2.6-2.7 reaction. Enzyme activity within a majority
of cells observed was localized along the apical por.
tion of these ectodermal cells due primarily to dis-
placement of the cytoplasm by the nuclei which line
the basal border (Fig. 14). The ectoderm'which pre-
viously represented the AER still exhibited a 3.0
activity, but decreased considerably in size to
approximately one or two cells thick (Fig. 13). The
'mesodema still gave a law 1.5 activity with the ex-
ception of an almost total lack of activity in those

cells condensing to form cartilage (Fig. 12).

23

The relatively intense Ac? activity noted in
the ectoderm during stage 27 remained high by stage
31, but gave some indication of a slight decrease in
activity. Although not significantly different from
the previous stage, the differentiating mesoderm ex-
hibited a slight increase in activity. The cartilage
gave a markedly increased 1.6 activity. Moderate 2.2
activity was observed within the periosteal collar

around the cartilage (Fig. 15).

Adenosine Triphosphatase

During stage 19, a 1.0.ATPase activity appeared
evenly distributed throughout the entire limb bud
(Fig. 16). The first sign of a significant increase
in activity was seen in the ventral half of the nose-
derm.in stage 21 embryos (Fig. 17). By stage 25, the
ventral half of the meanderntmaintained this increased
activity. while the cells of the AER tended to show a
slight increase in activity. All other structures
demonstrated only slight 1.0 activity (Fig. 18). The
activity in the ventral.mesoderm.tapered off'vithin
the ventral body wall near the arilla (Fig. 18).

The enzyme activity during stage 27 varied con.

siderably fron.that of stage 24. The AER, itself, now

24

disappeared, and the ectoderm only exhibited a slight
1.0 activity. The newly-formed condensation of carti-
laginous cells indicated a moderate 2.0 activity. The
ventral mesenchyme maintained.its moderate activity.
A distal reactive center of nesenchyme gave 2.0 reac-
tivity (Fig. 19).

By stage 31. the entire meander: exhibited a
1.8 activity'with the exception of a 2.8 reaction in
the cartilaginous calls and 3.0 activity within the
periosteal collar. The activity surrounding the car-
tilage tapered off on both ends forming a cylinder
around the midshaft region. The ectodera.retained its

1.0 activity (Figs. 20 and 21).

Lactic‘Dehydrogenase

A 1.0 EDH activity was evenly distributed
throughout the limb bud during stage 19 (Fig. 22). By
stage 21, a significantly increased 1.8 activity was
seen in the area of the AER.and in the ventral aeso~
dermal condensation (Fig. 23). During stage 26, 1.6
activity remained in the All (Fig. 23). The ventral
nesodera exhibited similar 2.2 activity which extended
into the body cavity as two branches. One followed the

axillary area into the mesodenn of the ventral body

23

'wall, while the other extended upward into the ease-
darn.of the dorsal body wall and into the mesodara.of
the limb, itself. Intense 3.0 activity localised in
a single row of mesodermal cells just under the ecto-
dermal layer along the ventral meaodermal condensation
(Fig. 25). Another site of intense 3.0 activity was
observable in the mesodermal cells lining the ventral
surface of the AER (Fig. 24).

The mesodermal activity during stage 27 was
similar to the above. The intense 3.0 activity in the
row of mesodermal cells along the ventral ectoderm*was
still present (Fig. 27). The more moderate 2.1 activ-
ity of the ventral mesodermal condensation extended
into the ventral body cavity and appeared distinct from
the 1.0 activity within the dorsal proximal mesoderm
(Fig. 26). The cartilage stained moderately. The ecto-
derm during this stage of development exhibited only a
1.1-1.2 activity.

By stage 31, the activity within the ectoderm
increased significantly to 2.0 around the entire limb.
The activity of the dorsal mesoderm increased to 1.6,
while the ventral mesoderm maintained its already in-

creased l.8 activity. The cartilage cells indicated

26
an intense 3.0 reaction (Fig. 28). Only a slight .9
reaction could be seen surrounding the cartilage in

the cells of the periosteal collar (Fig. 29).

 

27

Table 11. Acid phosphatase activity in deve10ping
chick forelimb.

 

 

 

 

Ectoderm Mesoderm
Stage DE VF. AER 'DM vn c B
19 1.0 1.1 1.1 1.0 1.0 - -
21 1.3 1.9Mr 2.4“ 1.0 1.1 - -
24 1.3 1.9 3.0** 1.0 1.1 - -

 

 

 

27 2.6***| 2.7**‘1 - 1.5 1.5 .2 -

 

 

 

 

 

 

31 2e3 |2e3 J " log log 1a6*** 2.2

 

 

DE Dorsal ectoderm

VB Ventral ectoderm

AER. Apical ectodermal ridge
DM Dorsal mesoderm

VM Ventral mesoderm

C Cartilage

B Bone

Negative

Slight activity
Moderate activity
Intense activity

eee
00°

1

. UNHO

P(u$x)- .05 as compared to the previous stage
** P(ugx)- .02 as compared to the previous stage
*** P(ugx)- .002 as compared to the previous stage

1Mann-Whitney U Teat; Two-tailed Test (Siegel,
1956)

 

Table III. Adenosine triphosphatase activity in
developing chick forelimb.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ectoderm Mesoderm

Stage DE v2 AER 'DM VM 0 B
19 1.0 1.0 1.0 1.0 1.0 " -
21 1.0 1.0 1.3 1.0 1.8** - -
24 1.0 1.0 1.3 1.0 2.0 - -
27 1.0 1.0 - 1.0 1e6 2.0 ’
31 1.0 1.0 . 1.8“ 1.8 Zea“ 3e0

DE Dorsal ectoderm

VB Ventral ectoderm

AER. Apical ectodermal ridge

BM Dorsal mesoderm

VM Ventral mesoderm

C Cartilage

B Bone

0 Negative

1.0 Slight activity

2.0 Moderate activity

3.0 Intense activity

* P(u5x)- .05 as compared to the previous stagei

** P(ugx)- .02 as compared to the previous stage

oeee

‘P(ugx)- .002 as compared to the previous stage

1Mann-Whitney 0 Test; Two-tailed Test (Siegel,‘
1956)

29

Table IV. Lactic dehydrogenase activity in develop-
ing chick forelimb.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. Ectoderm Mesodenm
Stage DE v1: AER .DM vs C n
19 1.0 1.0 1.0 1.0 1.0 - '-
21 1.0 1.0 1.8*** 1.0 1.8” - -
24 1.0 1.2 1.6 1.2 2.2 - -
27 1.2 1.1 - 1.0 2.1 2.0 -
31 2.0**12.0‘*+ - 1.6" 1.8 3.0% .9

 

DE Dorsal ectoderm

VE Ventral ectoderm

AER. Apical ectodermal ridge
Dorsal mesoderm
Ventral mesoderm
Cartilage

Bone

E?

Negative

Slight activity
Moderate activity
Intense activity

000

P(ugx)- .05 as compared to the previous stagei‘
P(ugx)- .02 as compared to the previous stage
P(ugx)- .002 as compared to the previous stage

w

1Mann-Whitney U Test; Two-tailed Test (Siegel,
1956)

 

DISCUSSION AND CONCLUSIONS

Functions and Distribution of AcP, ATPase, and LDH

AcP is believed to be concerned with the hydroly-
sis of phosphoric acid esters and, in particular, phos-
phomonesters. According to Schmidt and Laskowski
(1961), this results in the formation of an ortho-
phosphate. Schmidt (1961) noted that At? is active in
areas of high cellular activity such as in the cells
of the prostate gland, liver, kidney, and mucosa of
the small intestine. Brechet (1960), Cone and Eschen-
berg (1966), and Clever (1966) reported high activity
‘within cells undergoing rapid degradation. AcP appears
to be concentrated in the Golgi bodies (Jurand, 1965;
Sobel and Avrin, 1965), lysosomes (Jurand, 1965;
Brachet, 1960; Clever, 1966), and cytolysomes (Jurand,
1965). In addition, the nuclei and ce11.membranes of
some cells show moderate activity (Jurand, 1965). AcP
is not an homogenous enzyme according to Pearse (1960),
but made up of many different ones which share a sim-

ilar pH optimum of approximately 5.0. Differentiation

30

31
of these specific enzymes requires biochemical analysis
of pH activity curves, quantitative response to various
substrates, and inactivation or suppression by selected
inhibitors (Schmidt, 1961).

Kielley (1961) described ATPase as an essential
enzyme in the release of energy from adenosine tri-
phosphate. It is found in all living cells, and, in
general, exists in association with formed intracele
lular structures. Several different types of enzymes
exist. One is located.within the structure of the
myofibrils of myosin and is presumably related to mus-
cular contraction. 0thers are found associated with
the endoplasmic reticulum, cell membranes, and mito-
chondria (Essner, g3 21., 1965; Kielley, 1961). Mbog
(1947) and Robinson (1952) analyzed the content of
ATPase in deve10ping chick.musc1e. The enzyme activity
was found to precede histologic and functional differ-
entiation. In early developing cells, the sarcoplasmic
elements appeared to be highest in activity. Later,
this activity diminished and was replaced by a high
content of myofibrillar ATPase. In developing rat
muscle, Herrman and Nicholas (1948) and de Villafranca
(1954) showed that activity increased with functional

differentiation and contractility.

32

According to Barks and Anderson (1963), EDH
catalyzes the reversible conversion of pyruvate to lac-
tste. Its presence indicates activity of the glycolytic
cycle in the production of lactate from glucose, and is
found in all animal tissues, but mostly in muscle and
liver cells (Schwert and Winer, 1961). Lindsay (1963)
described five isozymic structures of LDH. Unfortunately,
they cannot be distinguished via standard histochemical

procedures.

Possible Implications of Enzymatic Activity

Enzymes during Jurand's "period of initiation”
exhibit only slight ubiquitous activities. The absence
of pattern reflects the absence of differentiated
tissue. The slight activity of the enzymes fails,
however, to correlate with an increase in the prolifer-
ation rate of either the mesoderm or ectoderm, which
may seem somewhat unusual in view of the high energy
requirements of embryonic tissues.

Starting with stage 21, a series of structures
begin to differentiate from the original germ layers.
This change is reflected by enzymatic activity prior
to and during histologic differentiation. One of the

first recognisable structures is the ventral mesodermal

33
condensation in the postaxial mesoderm. Its rapid
mitotic rate and high RNA content bear a possible rela-
tionship to its inductive capacity as described by
Saunders and Gasseling (1963). The present study in-
dicates that moderate ATPase and LDH activity reflect
this possible initiation of differentiation as the cells
begin to utilize energy via the glycolytic cycle. It is
precisely at this time that the ectoderm begins to
thicken in the formation of the AER in response to an
inductive activity which is unique to the mesoderm.of
the limb bud. Whether or not this or any other bio~
chemical activity is absent in non-limb mesoderm has
yet to be studied.

The AER deserves particular attention because of
its almost certain role in the distal outgrowth of the
normal forelimb. It appears histologically by stage 18,
reaching maximum development between stages 22 and 26.
Any interaction between the AER.and the underlying meso-
derm would most certainly occur at this time, for during
stage 27, a rapid series of intracellular changes occur
in the ridge, both structurally and biochemically, which
lead to its complete destruction.

Numerous previously reported experimental pro-

cedures attest to the importance of stages 22.26. For

34
example, carbon, placed within the limb mesoderm prior
to stage 21, could not be located by Saunders (1948)
in the newly formed digits. Searls (1965) noted that
radioactive sulfate uptake by precartilage cells begins
during stage 22 and not before. According to Saunders,
Cairns, and Gasseling (1957), leg mesoderm from.embryos
in stages 24 and 25, when grafted into proximal levels
of forelimb mesoderm, forms toes, scales, and claws
typical of hindlimb development, while earlier meso-
dermal grafts do not.’ Saunders and Gasseling (1957)
showed that by stage 26 the properties of the prospec-
tive humeral feather tracts are fixed and resist
changes following the reorientation of presumptive com-
ponent mesoderm and ectoderm. Stage 26 hind limb meso-
derm loses its ability to form bony elements if in-
serted under the AER of the forelimb (Saunders, 35;g;.,
1959). Finally, Searls (1965) found that radioactive
sulfate disappeared from the basement membrane and was
no longer absorbed by the mesoderm by stage 26. All of
these studies tend to support the hypothesis that in-
ductive interaction begins on or around stage 21, and
is progressively completed in the various elements of
the forelimb by stage 26.

Balinsky (1956) and Coetinck (1966) suggested

35
that the presence of the basement membrane between the
mesodermhand ectoderm during this important period pre-
vents any possible interaction, while its absence
allows for an interaction between cells of different
basic type. Balinsky (1936, 1957) noted that as super-
numary limbs are induced in amphibians, the basement
membrane is reduced or absent. Also, if the amputation
surface of a severed amphibian limb is covered with
epidermis and dermis, it fails to regenerate (Balinsky,
1956). Grobstein (1962) showed that mesenchyme £2.2i552
reacts‘with pancreatic rudiments to form recognisable
acini across a Millipora filter as long as the pore size
‘III large enough for certain molecules to pass through
and induce adjacent tissues.

Pierce (1966) observed that the basement mem-
brane may act in a similar‘uay and filter out many of
the undesirable molecules. Finally, Goetinck and.Abbot
(1963) were able to show that ectodermless limb buds of
the chick, if exposed to collagenase and then recombined
with the ectoderm, form outgrowths if implanted into the
flank area. Since this treatment results in destruction
of the basement membrane, it can be said that the mem-
brane is not essential for limb outgrowth to occur.

Jurand (1965) offered visual evidence that direct

36

contact occurs between the AER and the mesoderm. From
stage 22-26, "discrete discontinuities" were observed
in the basement membrane at the base of the AER. The
study presented here suggests possible biochemical
events which precede and coincide with this phenomenon.
Moderate or intense AcP activity from stage 21-24 may
indicate destruction of the membrane, since this enzyme
is often released from lysosomes with accompanying pro-
teolytic agents (Tappel, 25 g;.,l963). The intense LDH
activity observable in the mesoderm under the AER.dur-
ing stage 24 suggests rapid glycolytic activity, in an
attempt, perhaps, to rebuild the membrane, utilise pro-
ducts of the destructive process, and/or participate
directly or indirectly in an interaction between the
AEI.and the mesoderm. It would be unwise to speculate
further or more specifically on this point considering
the sparse data available from this study alone.

Non-ridge ectoderm during this phase of develOpo
‘ment gave practically no evidence of the underlying
activity within the mesoderm*with the exception of
moderate Ac? activity in the ventral ectoderm as ob-
served by Moog (1944) and later verified by this study.
A peculiar single layer of intense LDH activity within

the mesoderm is difficult to explain except as a

37
reflection of the active mesodermal tissues below.

Data suggest two essentially different processes
at'uork‘vithin the.AER. One is the process character.
ized by cells undergoing rapid cellular activity. It
is exemplified by the presence of numerous elongated
mitochondria (Jurand, 1965), frequent endoplasmic re-
ticulum (Jurand, 1965), and high RNA concentration
(Hinrichson, 1966). The study presented here reveals,
in addition, high amounts of ATPase and LDH«activity
'within the ridge. These changes are consistent'vith
rapid protein synthesis and accompanying cytodiffersn-
tiation and inductive capacities occurring from stage
21-26. The second process is that of cellular destruc-
tion and autolysis. By stage 23, the appearance of
numerous lysosomes, Golgi bodies, and necrotic centers
called "cytolysomes" precedes this phenomenon. Uithin
a short time these structures begin to break down, re-
leasing large quantities of proteolytic enzymes into the
cytoplasm.and thus, destroying the cell. AcP, the
enzyme found in highest concentrations within the lyso~
somes, seems to reflect more than any other entity the
destruction of these cells culminating in the disappear-
ance of the AER by stage.27. The data presented here

vividly depict the so? activity localised in the ridge

38
during this phase of development.

Electron micrographs of these cells by Jurand
(1965) indicate that those changes originate within the
cytoplasm of the cells and only involve the nucleus
secondarily. Moreover, the initiation of this destruc-
tion appears to be spontaneous and originates from
within the cells themselves. This process is similar
to the destruction of other necrotic centers of the
forelimb as described by Saunders (1966a). The role
of genetic or hormonal factors in this phenomenon have
yet to be determined.

In this study, the PNZ could not be demonstrated
as possessing high AcP activity. Saunders (1966a) has,
however, been able to demonstrate activity within these
cells. During stage 27, a distal necrotic center ex-
hibited moderate ATPase activity, indicating high energy
utilization needed for digit formation.

Indications suggest that muscle begins to
differentiate as early as day 4. MOscona and Moscona
(1952) showed that isolated stage 24 myogenic and
chondrogenic cells when grown in tissue culture
developed along divergent paths. Spontaneous move-
ment was observed to originate on day 7 (Balaban,

1967). Although some‘ATPase and LDH activity preceded

39

functional differentiation, the most significant in-
crease was noted in the dorsal mesoderm at precisely
the time that movement was first observed, according
to the present study. In addition, AcP activity showed
some signs of increasing. The results suggest that
enzymatic activity in the dorsal mesoderm, at least,
coincides with functional differentiation.

Histologically, the first suggestion of car~
tilage formation occurs at approximately stage 27.
However, it appears that cells are biochemically unique
as long as three days before histologic differentiation.
Radioactive sulfate was first detected by Amprino
(1955) in cells during stage 24. Later, Searls (1965)
found that precartilage accumulated sulfate as early as
stage 22. Deuchar (1963) noted that proline, a pre-
cursor of collagen, was observed in the perichondrial
layer before collagen became visible histologically in
connective tissue using stains such as Malloy's and
Mhson's procedures or by standard biochemical tests.
Similarly, developing cartilage in Ambystoma limbs
undergoing regeneration, accumulated proline readily
according to Revel and Hay (1963). In the study pre-
sented here, Moog's (1944) results were confirmed in

that.scP activity noticeably disappeared from

40
differentiating cartilage, leading to the assumption
that this enzyme does not play a role in chondrogenesis.
Likewise, no evidence can be found to suggest that
ATPase or LDH were involved.

Once the cartilaginous cells differentiated, an
entirely different picture appeared. All enzymes
studied became moderately to intensely active. The
only explanation that seems plausible is that once csr~
tilage is formed, it immediately begins the process of
decay and degeneration as described by Fell (1925).
Rapid cellular growth and chemical synthesis may occur
in these cells just prior to destructive processes as
discussed previously in the AER. The rapid prolifsr~
ation may, instead of being a separate entity, bring
about or initiate the destruction which follows. The
two may in certain embryological situations be insepar-
ably linked. The cells may, in a sense, "burn them-
selves out."

_Bone formation in stage 31 embryos is most read-
ily studied within the cells of the periosteal collar.
Pritchard (1952) discovered that glycogen exists in
these cells and is presumably hydrolyzed by the phos-
phatases, accounting for the increase in AC? activity

observed in the present study. This process liberates

bl
numerous free phosphate ions, which are now capable of
combining with calcium in the formation of an inorganic
matrix during the process of osteogenesis. Energy re-
quirements would appear to be immense as depicted by
the intense ATPase reactivity of the cells.

As the epidermis develops,.AcP activity increases
uniformly, levels off during stage 27, and then begins
to decline. This same phenomenon was observed earlier
by Moog (1946). The results of the study presented
here indicate that LDH activity increased significantly
during stage 31. The reason for this activity during

these stages remains undetermined at this time.

General Considerations

The above study has involved primarily changes
in biochemical patterns, both spatially within different
tissues at a given time, and temporally between similar
structures at progressively increasing intervals. As
far as possible, the data presented here and elsewhere
link gross morphological, histologic, and electron
microscopic data with enzymatic activity occurring as
structures appear and develop. In certain instances,
disappearance of structures has been correlated with

decreased enzyme activity; i.e. disappearance of the

42
AER coincides with the disappearance of AcP‘activity
within the ridge cells and with LDH activity in the
mesoderm just under the ridge. Ultimately, of course,
the biochemical changes which occur prior to and dur-
ing cytological differentiation should be determined.
Unfortunately, in this study, only a few of these
changes could he demonstrated.

Histochemical procedures, such as those used
here, demonstrate cellular localization of enzyme ac-
tivity within the limits of the techniques selected.
The results are subject to problems of diffusion,
errors in quantitation, and losses in enzyme activity
between sectioning and incubation of tissues. Al-
though somewhat academic, it is not known if increases
within any particular tissue or structure come from
increased production of the enzyme which is probably
genetically controlled, or from an increase in the
activation of preformed enzymes as controlled by the
cytoplasm or the environment in general. The tech-
niques used were developed initially for laboratory
animals, such as rats and mice, and for adult tissues
which react somewhat differently than embryonic limb
buds. Also, there is little evidence to support the

fact that an enzyme found within adult tissues plays

43
any role in early limb morphogenesis that cannot be
supplied by completely different molecular structures.
Finally, staining of enzyme activity involves placing
the tissue in optimal temperatures, supplying neces-
sary coenzymes and cofactors, and allowing them to in-
cubate for the proper length of time, a process which
establishes an environment much different than the con-
ditions found within the normal developing limb. It
has often been said that results are only as good as
the methods used. Unfortunately, these are the best
techniques available at the moment and their short-
comings should be realised.

Many of the enzymes studied exhibited only
slight activities. Small residual levels of activity
may actually be very important to the maintenance and
growth of cells during these phases of deveIOpment. It
may be that they are simply being manufactured and stored
in a latent formwwaiting to be triggered by the internal
or external environment of the cell. A second possi-
bility is that the cellular enzymes may not play any

part in metabolism until a certain threshold is reached.

The exact role of this early activity is not known.
Future studies should involve the use of radio-

actively-labeled molecules that form.necessary

44
components of various substrates, macromolecules, and
enzymes. Accumulation of these structures are, in fact,
more likely to occur before enzyme activity can be ob-
served. An excellent example of this phenomenon is the
induction of specific enzymes by their substrates, a
process whereby external or internal molecular regu-
lators “turn on" the genetic apparatus controlling
specific enzyme production (Waddington, 1966). Histo-
chemistry and electron microscopy will be used together
by future developmental scientists to demonstrate enzyme
activity within the smallest organelles. Ultimately,
however, biochemical embryology will be concerned with
the normal as it compares to the experimental variable.
For example, by supplying selected inhibitors or limit-
ing the amount of available cofactors, enzyme activity
in a tissue or structure can be controlled or inhibited
and the effect studied. Experiments concerned with
interactions between the two germ layers could involve
the use of various size Millipore filters and mica or
Mylar sheets placed between the cells. Fractionaliza-
tion and analysis of limb mesoderm and ectoderm may
result in the purification of the hypothesized meso-

dermal or ectodermal "factors" which could be studied

45
biochemically and histochemically in vivo and in vitro.
All these things and.more are needed to elucidate the

problems of forelimb morphogenesis in the chick.

SUMMARY

The development of acid phosphatase, adenosine
triphosphatase, and lactic dehydrogenase activity was
studied histochemically in the chick forelimb from
stage 19 through 31 (ca. day 3 to day 7). Development
of the apical ectodermal ridge, mesenchyme, non-ridge
ectoderm, muscle, cartilage and bone was observed. Acid
phophatase activity was highest in the apical ecto-
dermal ridge, epidermis, and periosteal collar sur-
rounding the developing long bones. Adenosine tri-
phosphatase activity was found intensely only within the
cells of the periosteal collar and cartilage. Lactic
dehydrogenase activity was concentrated mainly in meso-
dermal components such as the ventral mesodermal conden-
sation, the row of mesodermal cells immediately under
the ventral ectoderm, and in the cartilage. The appear-
ance of enzyme activity reflected the differentiation of
the mesoderm.and ectoderm.as the various structures and

tissues form. The hypothesized interaction between the

46

47
:esoderm and ectoderm was particularly evident in
activities located within the AER, the row of ventral

mesodermal cells, and ventral ectoderm.

LITERATURE CITED

Abe, T. and N. Shimiger. 1964. Histochemical method
for demonstrating eldoleee. Histochemie 4:209.

Amprino, R. 1955. Autorediographic research on the
S35 sulfate metabolism.in cartilage and bone
differentiation and growth. Acta Anet. 24:121.

Baleban, M. 1967. Personal communication. Michigan
State University; East Lansing, Michigan.

Belinsky, B. 1956. A new theory of limb induction.
Proc. Nat. Acad. Sci. 42:781.

Balineky, B. 1957. New experiment! on the mode of
action of the limb inducer. J. Exp. 2001.
134:239.

Berke, T. 1960. A simple azo-dye method for histo-
chemicel demonstration of ecid phoephetele.
Nature 187:248.

Berke. T. and J. Anderson. 1963. Histochemiatry.
Harper end Row, Inc.. New York.

Bell, 2., M. Canceling, and E. Zwilling. 1962. On
the role of ectoderm in limb development.
Develop. Biol. 4:177.

Bell, 5., M. Kaighn, and L. Feseenden. 1959. The role
of meaodermel end ectodermal components in the
development of the chick limb. Develop. Biol.
1:101.

Brechet, J. 1960. The Biochemistry of Development.
Pergemon Press, London.

48

49

Burstone, M. 1958. Histochemical comparison of
naphthol-AS substrates using frozen-dried
tissues. J. Histochem. Cytochem. 6:87.

Burstone, M. 1959. New histochemical techniques for
the demonstration of tissue oxidase (cytochrome
oxidase). J. Histochem. Cytochem. 7:112.

Clever, U. 1966. Gene activity patterns and cellular
differentiation. Amer. 2001. 6:33.

Cone, M. and K. Eschenberg. 1966. Histochemical
localization of acid phosphatase in the ovary
of Carrie remigis. J. Exp. 2001. 161:337.

Deuchar, E. 1963. Sites of earliest collagen forma-
tion in the chick embryo as indicated by uptake
of tritiated proline. Exp. Cell Res. 30:528.

de Villafranca, G. 1954. Adenoainetriphoaphatase ac-
tivity in developing rat muscle. J. Exp. 2001.
127:367.

Edda, Hg, Jr. 1958. Development of collagen in the
frog embryo. Proc. Nat. Acad. Sci. 44:296.

Esaner, E., J. Fogh, and A. Fabrizio. 1965. Local-
ization of mitochondrial adenoaine triphoapha-
tase activity in cultured human cells. J. Histo-
Chem. CyCOChem. 13 : 647 e

Fell, H. 1925. The histogenesia of cartilage and bone
in the long bones of the embryonic fowl. J. Mor-
phol. Physiol. 40:417.

Glenner, 6., H. Burtner, and G. Brown. 1957. The his-
tochemical demonstration of monamine oxidase ac-
tivity by tetrazolium salts. J. Hiatochem. Cyto~
chem. 5:591.

Goetinck, P. 1966. Genetic aspects of skin and limb
development. In: Current Topics 13 Develop-
mental Biolo , Egl.‘1. Academic Press, New York.

50

Goetinck, P. and U. Abbot. 1963. Tissue interaction
in the acaleless mutant and the use of scaleless
as an ectodermal marker in studies of normal limb
differentiation. J. Exp. 2001. 154:7.

Gomori, G. 1952. Microscopic Histology. University
Press, Chicago. Pp. 203-212.

Grobstein, C. 1962. Interactive processes in cyto-
differentiation. J. Cellular Comp. Physiol.
Suppl. 1. 60:35.

Hamburger, V. and H. Hamilton. 1951. A series of
normal stages in the development of the chick
embryo. J. Morphol. 88:49.

Harkness, R. 1961. Biological functions of collagen.
Biol. Rev. Cambridge Phil. Soc. 36:399.

Hay, E. and J. Revel. 1963. Autoradiographic studies
of the origin of the basement lamella in
Ambystoma. Develop. Biol. 7:152.

Hayashi, M., Y. Nakajima, and.W. Fishman. 1964. The
cytological demonstration of B-glucuronidase em-
ploying naphthol AS-BI glucuronidide and hexa-
zonium pararosanilin: a preliminary report.

J. Histochem. Cytochem. 12:293.

Herrmann, H. and J. Nicholas. 1948. Enzymatic liber-
ation of inorganic phOSphate from adenosinetri-
phosphate in developing rat muscle. J. Exp. 2001.
107:177.

Hinrichsen, K. 1956. Die Bedeutung der epithelialen
Randleiste fur die Extremitatenentwicklung.
Z. Anat. Entw. Gesch. 119:350.

Jurand, A. 1965. Ultrastructural aspects of early
development of the fore-limb buds in the chick
and the mouse. Proc. Roy. Soc. London Ser. A.
162(988):387.

Kallman, F. and C. Grobstein. 1965. Sources of col-
lagen at epitheliomesenchymal interfaces during
inductive interaction. Develop. Biol. 11:169.

51

Kielley, W. 1961. Acid anhydride hydrolysins. In:
:35 Enzymes edited by P. Boyer, H. Lardy, and
K. Myrback. Academic Press, New York. Pp. 149-
157.

Lindsay, D. 1963. Isozyme patterns and properties of
lactic dehydrogenase from developing tissues of
the chick. J. Exp. 2001. 152:75.

McWhinnie, D. and J. Saunders. 1966. Developmental
patterns and specificities of alkaline phospha-
taae in the embryonic chick limb. Develop.
Biol. 14:169.

Milaire, J. 1962. Histochemical aspects of limb
morphogenesis in vertebrates. Advan. Morpho-
genesis 2:183.

Moog,.F. 1944. Localizaticns of alkaline and acid
phosphatases in the early embryogenesis of the
chick. Biol. Bull. 86:51.

Moog, P. 1947. Adenylpyrophosphatase in brain, liver,
heart, and muscle of chick embryos and hatched
chicks. J. Exp. 2001. 105:209.

Moscona, A. and H. Moscona. 1952. The dissociation
and aggregation of cells from organ rudiments of
the early chick embryo. J. Anat. 86:287.

Nachlas, M., D. Crawford, and A. Seligman. 1957a.
Histochemical demonstration of leucine amino-
peptidase. J. Histchem. Cytochem. 5:264.

Nachlas, M., K. Tsou, E. DeSouza, E. Cheng, and A.
Seligman. 1957b. Cytochemical demonstration of
succinic dehydrogenase by the use of a new
p-nitrophenyl substituted ditetrazole. J. Histo-
chem. Cytochem. 5:420.

Nachlas, H1, D. Walker. and A. Seligman. 1958. Histo-
chemical method for the demonstration of diphos-
phOpyridine nucleotide diaphorase. J. Biophys.
Biochem. Cytol. 4:29.

52

Pearse, A. 1960. Bistochemistry. Brown, Little, and
Co., Boston.

Pierce, G. 1966. The development of basement membranes
of the mouse embryo. Develop. Biol. 13:231.

Pritchard, J. 1952. A cytological and histochemical
study of bone and cartilage formation in the rat.
J. Anat. 86:259.

Revel, J. and E. Hay. 1963. An autoradiagraphic and
electron microscopic study of collagen synthesis
in differentiating cartilage. Z. Zellforach.
61:110.

Robinson, D. 1952. A study of the adenosinetriphos-
phatase activity of developing chick muscle.
Biochem. J. 52:633.

Romanoff, A. 1960. The Avian Embryo. Macmillan Co.,
New York.

Saunders, J. 1948. The proximo-distal sequence of
origin of the parts of the chick wing and the
role of the ectoderm. J. Exp. 2001. 108:363.

Saunders, J. 1949. An analysis of the role of the
apical ridge of ectoderm in the development of
the limb bud in the chick. Anat. Rec. 105:567.

Saunders, J. 1966a. Death in embryonic systems.
Science 154:604.

Saunders, J. 1966b. Personal communication. State
University of New York at Albany: Albany, New
York.

Saunders, J., J. Cairns, and M. Gasseling. 1957. The
role of the apical ridge of ectoderm in the
differentiation of the morphological structure and
inductive specificity of limb parts in the chick.
J. Morphol. 101:57.

Saunders, J. and M. Gasseling. 1957. The origin of
pattern and feather germ specificity. J. Exp.
2001. 135:503.

53

Saunders, J., M; Canceling, and J. Cairns. 1959. The
differentiation of prospective thigh mesoderm
grafted beneath the apical ectodermal ridge of
the wing bud in the chick embryo. Develop.
Biol. 1:281.

Saunders, J. and M. Gasseling. 1963. Trans-filter
propagation of apical ectoderm maintenance factor
in the chick embryo wing bud. Develop. Biol.
7:640

Scarpelli, D., R. Hess, and A. Pearce. 1958. The
cytochemical localization of oxidative enzymes.
1. Diphosphopyridine nucleotide diaphorase and
triphosphopyridine nucleotide diaphorase. J.
Biophys. Biochem. Cytol. 4:767.

Schmidt, 6. 1961. Nonspecific acid phosphomonester-
ases. In: lbs Enzymes edited by P. Boyer,
H. Lardy and K. Myrback. Academic Press, New
York. Pp. 37-47.

Schmidt, G. and M. Laskowski. 1961. Phosphate ester
cleavage. In: The Enzymes edited by P. Boyer,
R. Lardy and K. Myrback. Academic Press, New
Yorke Ppe 3’359

Schwert, G. and.A. Diner. 1961. Lactic dehydrogenase.
In: 132 Enzymes edited by P. Boyer, H. Lardy and
K. Myrback. Academic Press, New York. Pp. 127-
148.

Searla, A. 1965. An autoradiographic study of the
uptake of 835-sulfate during the differentiation
of limb bud cartilage. Develop. Biol. 11:155.

Siegel, S. 1956. Nonparametric Statistics for the
Behavioral Sciences. McGraw-Hill Book Co., New
York a

Sobel, H. and E. Avrin. 1965. Localization of acid
phosphatase activity in rat pancreatic acinar

cells: a light microscopic study. J. Histochem.
Cytochem. 13:301.

54

Tappel, A., P. Sawant, and S. Shihko. 1963. Lysosomes:
distribution in animals, hydrolytic capacity and
other properties. In: tLysosomes edited by
A. deReuck and M. Cameron. J. and A. Churchill,
Ltd., London. Pp. 78~ll3.

Wachstein, M. and E. Meisel. 1957. Histochemistry of
hepatic phDSphatases at a physiologic pH. .Amer.
J. Clin. Path. 27:13. '

'waddington, C. 1966. Principles of DeveIOpment and
Differentiation. Macmillan Co., New York.

Neel, P. 1948. Histophysiology of the limb bud of the
fowl during its early development. J. Anat.
82:49.

Zwilling, E. 1956a. Interaction between limb bud
ectoderm and mesoderm in the chick embryo. 1.
Axis establishment. J. Exp. 2001. 132:157.

Zwilling, E. 1956b. Interaction between lflmb bud
ectoderm and mesoderm in the chick embryo. 11.
Experimental limb duplication. J. Exp. 2001.
132:173.

Zwilling, E. 1961. Limb morphogenesis. Advan.
Morphogenesis 1:301.

Zwilling, E. 1966. Development of fragmented and
dissociated limb bud mesoderm. Amer. 2001.
9:20.

55

Figure 1. Forelimb bud. Stage 19. Note the meso-
derm (mes) and ectoderm (ect) of the newly-formed
limb bud. H. & E. X140.

Figure 2. Forelimb bud. Stage 21. The ectoderm is
composed of the apical ectodermal ridge (AER), the
dorsal ectoderm (de), and the ventral ectoderm (ve).
The ectoderm of the limb is continuous with that of
the amnionic sac (as). many blood vessels (bv) appear.
The mesoderm has undergone marked condensation.

H. 5: E. X140.

57

Figure 3. Forelimb bud. Stage 24. This stage is
characterised by further elongation of the bud, a
ventral mesodermal condensation (vmc), a large
axillary artery (as), and a venous plexus (vp)
just under the AER. H. & E. X140.

Figure 4. Forelimb. Stage 27. Mesenchyme has con-
densed to form cartilage (cart). Numerous arteries
(a) and nerves (n) are seen entering the limb.

H. & E. X46.

.r

“we“.

«.3.»

watt;

a

 

 

Figure 5. Forelimb. Stage 31. A periosteal collar
has formed around the shaft of the cartilaginous
long bones. Note the scapula (sc), humerus (hu),
radius (re), and ulna (ul). H. &.E. X40.

...
O...
max: oe.’ a.
3.4.... 3...... ......st R).-.“
\0 Band.
Irv...0¢ea.}\u .u‘o. f... .. 0/.» ..o.\\

. are.“ ...
fir.- v b , .-
I ! 9‘s \o.{ es.“
...”...rr. .a v... 7 .. .. o
1... ...... ...... .. ...Ps . .

 

 

 

61

Figure 6. Forelimb bud. Stage 19. Activity is
slight in the mesoderm (mes), ectoderm (act), and
amnionic sac (as). Azo dye technique for AcP.

a. X140.

b. X560.

 

 

‘3
V

Figure 7. Forelimb bud. Stage 21. Note the moderate
activity in the AER and in the axillary ectoderm (ae).
Azo dye technique for AcP. X140.

Figure 8. AER. Stage 21. Cells of the ridge radiate
out from a central core as identified by the arrow.
Azo dye technique for.AcP. X350.

 

2a ..

its

9‘. p

   

 

65

Figure 9. AER. Stage 21. Note the intense reaction
in the core of the AER. Azo dye technique for AcP.
X700.

 

67

Figure 10. AER. Stage 24. Two centers of activity
have deve10ped, one in the central core and one in
the periphery. Azo dye technique for AcP. X360.

Figure 11. Forelimb bud. Stage 24. Activity in the
axillary ectoderm (ac) has decreased considerably.
Azo dye technique for'AcP. X140.

 

Figure 12. Proximal portion of forelimb. Stage 27.
The mesoderm, which is condensing to form cartilage,
shows an almost total lack of activity. The ecto-
derm around the limb shows an intense activity in
almost all cells. Azo dye technique for AcP. X110.

 

V

v
s

31...”. sue. .. H ..
*u/ a ...w...-.:..

 

 

71

Figure 13. Distal tip of forelimb. Stage 27. An in-
tense activity remains in the ectoderm.which once
represented the AER, as indicated by the arrow. The
size of the ridge is now markedly reduced to only one
or two layers thick. Azo dye technique for AcP. x225.

Figure 14. Skin of the forelimb. Stage 27. Activity
appears to be more concentrated along the apical
portion of the cells due primarily to the presence of
the nucleus (nu) along the proximal border. Azo dye
technique for AcP. x710.

 

73

“C

Figure 15. Forelimb. Stage 31. A20 dye technique
for AcP.

a. Moderate activity is observed in the ectoderm
around the forelimb. Slight activity is seen
in the undifferentiated mesoderm. x24.

b. The cartilage (cart) exhibits moderate
activity as does the periosteal collar (pc)
around it. X140.

.0.

 

Figure 16. Forelimb bud. Stage 19. Slight activity
is evenly distributed throughout the ectoderm (ect)
and mesoderm (mes). Lead nitrate method for ATPase.

X225.

Figure 17. Forelimb bud. Stage 21. The ventral
mesoderm (vm) exhibits moderate activity. Lead
nitrate method for ATPase. X56.

   

 

77

Figure 18. Forelimb bud. Stage 24. The ventral meso-
derm (vm) shows moderate activity. The AER has become
moderately active. Lead nitrate method for ATPase.
X112.

 

 

79

Figure 19. Forelimb. Stage 27. Developing carti-
lage (cart) and a distal reactive center (drc) ex-

hibit moderate activity. Note the AER.has now dis-
appeared. Lead nitrate method for ATPase. X36.

 

 

Figure 20. Developing bone in cross section. Stage 31.
Note the intense reaction in the cartilage (cart) and
in the periosteal collar (pc). Lead nitrate method for
ATPase. X140.

 

 

 

 

 

61

Figure 21. Developing bone. Stage 31. Intense perio-
steal collar activity appears only in the midshaft
region of the diaphysis. Lead nitrate method for
ATPase. x112.

 

 

 

 

Figure 22. Forelimb bud. Stage 19. Note the slight
activity evenly distributed throughout the mesoderm

(mes) and ectoderm (ect) of the bud. Nitro-BT method
for LDH. X225.

Figure 23. Forelimb bud. Stage 21. The AER now
gives a moderate reaction (arrow). Moderate ac-

tivity is also observed in the ventral mesodermal
condensation (vmc). Nitro-BT method for UDH. X56.

 

 

 

85

Figure 24. AER. Stage 24. Moderate activity is
observed in the AER. The meaodermal cells just under

the AER exhibit an intense reaction (arrow). Nitro-BT
method for LDH. X360.

 

Figure 25. Axillary region. Stage 24. Intense
enzyme activity is seen in the ventral meaodermal cells
just under the ventral proximal ectoderm (arrow). The

activity extends into the body cavity. Nitro-BT method
for LDH. X360.

 

 

 

 

87

Figure 26. Forelimb. Stage 27. Two distinct centers
of mesodermal activity exist. The ventral mesodermal
condensation exhibits an intense reaction in the pro:-
imal portion just under the ventral ectoderm (l) and
extends into the ventral body wall (vbw). Another
site of moderate activity is in the dorsal proximal
mesoderm (2), which extends into the dorsal body wall
(dbw). Nitro-BT method for LDH. X112.

 

Figure 27. Axillary region. Stage 27. Note the in-
tense activity in the row of mesodermal cells border-
ing the ventral ectoderm of the limb and extending

into the ventral body wall (arrows). Nitro-BT method
for EDH. X300.

 

 

Figure 28. Bone formation in cross section. Stage 31.
The cartilage (cart) exhibits an intense reaction and

the mesoderm (mes) gives only a moderate reaction.
Nitro-BT method for mu. X150.

 

 

91

 

 

Figure 29. Developing long bone. Stage 31. The
periosteal collar (pc) exhibits only a slight re-
action. fiitro-BT method for LDH. X280.

 

 

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