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Morphogenesis of the carpal elements in the regenerating forelimb of adult Notophthalmus viridescens viridescens.

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Morphogenesis of the Carpal Elements in the
Regenerating Forelimb of Adult Nofophthcrlmus
viridescens viridescens '
MARY M. BENZO, CAMILLO A. BENZO AND HAROLD W. MANNER
Department of Anatomy, State University of New York, Upstate Medical
Center, Syracuse, New Y o r k 13210 and Department of Biology,
Loyola University, Chicago, Illinois 60626
ABSTRACT
This study is concerned with the morphogenesis of the carpal
elements in the regenerating forelimb of the adult newt. Blastema cells surrounding the remnant bony stumps begin to differentiate into cartilage on the twentieth post-amputation day. Subsequently, masses of cartilage build up from the
radial and ulnar stumps. The radial mass is larger and differentiates more
rapidly than the ulnar mass. By the fifty-fifth post-amputation day, the eight
basic carpal elements are formed, with fusion of two of the units, intermedium
with ulnare, occurring by the seventieth day. The completed regenerate possesses the seven carpal elements characteristic of the normal adult limb. The
present results show that during limb regeneration in the adult newt the carpal
elements are restored to their original number and position and that the pattern
of such carpal djfferentiation proceeds in a proximodistal direction influenced
by the stump remnants of the radius and ulna.
Regeneration, as defined by Thornton
('68) is that pattern of morphogenetic
events which restore the normal structure
of an organism following the loss of
part(s) of it. The sequence of events occurring in the regeneration of limbs in
the adult salamander has been divided
into three relatively distinct phases;
wound healing, blastema formation and
blastema differentiation (Manner, '53;
Iten and Bryant, '73). The last period,
blastema differentiation, involves the transformation of blastema cells into the fullyformed tissue elements of the regenerated
limb. Although various aspects of limb
regeneration in urodele amphibians have
been extensively explored, some basic
questions remain unanswered. For example, the number and position of the carpal
elements in the adult salamander forelimb have long been known (Francis, '34),
yet there is no information available on
the regeneration of the specific carpal elements from the blastema. However, it is
known (Manner, '53) that the original
blastema cells begin to differentiate into
cartilage on the twentieth post-amputaANAT. REC., 183: 421-430.
tion day (late bud stage; Iten and Bryant,
' 7 3 ) . Thus, in the present study, we have
followed the development of the carpal
elements from the beginning of cartilage
differentiation through the formation of
the completed carpal structure in the regenerating newt forelimb in an attempt
to determine the morphogenetic events
involved in the restoration of the normal
carpal pattern in the adult salamander.
MATERIALS AND METHODS
One hundred adult newts, Notophthdrnus viridescens viridescens, selected on
the basis of size uniformity (mean snout
to tail length t standard deviation: 7.74
rt_ 0.44 cm), were kept in fresh spring
water maintained at 23.5 i 1 ° C and given
daily feedings of brine shrimp. Following
anesthetization in a solution of 0.05%
tricaine methanesulfonate (MS-222), the
right forelimb of each animal was amputated through the distal one-third of the
Received Jan. 13, '75. Accepted May 9, '75.
1 Work supported in part by Grant 011-7124A from
The Research Foundation of State University of New
York and by Grant RR05402 (Oll-EOP3C) from
USPHS, General Research Support Branch.
42 1
422
M. M. BENZO, C. A. BENZO AND H. W. MANNER
radius and ulna. On the tenth post-amputation day (Iten-Bryant late dedifferentiation stage) and on every fifth day thereafter, through the seventieth day of regeneration, the right forelimb of each of
five animals was reamputated through
the mid-humerus and prepared for either
histological or gross examination.
For histological study, two of the limbs
from each post-amputation stage were
fixed for five days in cobalt calcium formal
solution (McManus and Mowey, '46), dehydrated overnight (approximately 15
hours) in methylene glycol monoethyl
ether (Cellosolve), and cleared for one
hour in toluene. The limbs were embedded
in paraffin and serially sectioned at 10 p in
the frontal plane. Sections were stained
with Hughesdon's modification (Carleton
and Drury, '57) of Mallory's collagen stain.
The three remaining limbs were prepared for gross examination. Whole limbs
were fixed for one week in a solution of
5% formalin in 70% ethanol, cleared for
24 hours in 4% KOH and stained with
alizarin red for 20-24 hours. To avoid
shrinkage and other distortion artifacts
common with initial storage in 100%
glycerine, the limbs first were passed
through a graded series of solutions consisting of decreasing proportions of 4%
KOH and glass-distilled water and increasing concentrations of 100% glycerine
according to a modification of the procedure of Davis and Gore ('47). The limbs
were then stored in 100% glycerine.
Regenerating carpal morphology
Twentieth to thirty-fifth post-amputation
days
New cartilage originates as early as the
twentieth post-amputation day (late bud
stage) between the periosteum and the
bone in the radius and ulna. This thin
layer, composed primarily of chondroblasts, surrounds the shafts of the bony
stumps, and by the thirtieth post-amputation day (early digits stage) thin cartilage
capsules at the end of the stumps join with
the cartilage encasing the bony shafts to
form a continuous casing. The growth
occurring during this plase is heterogeneous, the rate of the radial mass being
greater than that exhibited by the ulnar
mass. Subsequent observations show that
the radial mass contains the precursors of
the distal end of the radius plus the following carpal elements : radiale, centrale,
pre-pollicis and the intermedium unit. The
ulnar mass contains the distal end of the
ulna plus the precursors of the ulnare unit
and basale 1 through basale 4 (fig. 2).
Thirty-fifth to forty-fifth post-amputation
days
During this period (late digits stage),
the development of new cartilage can be
separated into three phases: (1 ) The carpal units begin to take shape and position
which are characteristic of the units in
the mature, normal limb. There is also
a separation of units by the formation of
grooves (indicated by stippling in the illustrations) which later sever the units from
RESULTS
each other. The environmental factors or
the cellular processes responsible for such
Normal carpal morphology
separation are unknown; ( 2 ) The cartiAccording to Francis ('34), the carpus lagenous caps surrounding the distal ends
of the adult salamander consists of seven of the radius and ulna begin to elongate,
elements (fig. 1). These include the pre- moving the carpal complex distally; ( 3 )
pollicis, a common basale 1 2 unit which Although not specifically discussed here,
serves the first two digits, a basale 3 for the digits are elongating and new joints
the third digit and a basale 4 for the are being added to them. It should be
fourth. The centrale articulates with all noted that phalangeal differentiation was
the other carpal elements. The radiale observed to occur after carpal differentiaarticulates proximally with the radius, dis- tion had begun; the inclusion of the four
tally with the pre-pollicis and medially metacarpals in each illustration is for
with the fused intermedium
ulnare. The position reference only.
intermedium
ulnare unit is the largest
At 35 days, the radial mass has sepain the carpus and articulates with the rated into three distinct units; the radiale,
ulna, radius, radiale, centrale and basale 4. pre-pollicis and a common intermedium 4-
+
+
+
CARPAL REGENERATION IN ADULT NOTOPHTHALMUS
centrale. The ulnar mass, however, remains as a single unit although the lines
of future separation are evident. By 40
days, these presumptive divisions become
a reality, and two large cartilagenous
masses can be seen; a basale 1
2 element and a common basale 3
basale 4
ulnare unit (fig. 3 ) .
+
+
+
Forty-fifth t o seventieth post-amputation
days
In this phase of regeneration there occurs further separation and positional
change in the carpal complex and considerable elongation of the radial and ulnar
extensions. At 45 days, the intermedium
centrale unit has differentiated into the
definitive intermedium element located
between the distal ends of the radius and
ulna, and centrale located distal to the
intermedium (fig. 4 ) . This completes the
basic morphogenesis of the radial mass.
The data show that differentiation as well
as growth are more rapid in the radial
element. At this stage, the ulnar mass is
now composed of three elements; a basale
1 2 unit, a basale 3
4 unit and ulnare
(fig. 4). By 55 days, the basic morphogenesis of the ulnar mass is completed
with the differentiation of the basale 3
4
unit into basale 3, located distal to centrale,
and basale 4 which is distal to ulnare.
Basale 1 2 remains as a common element (fig. 5).
The present study was terminated on
the seventieth post-amputation day. At
this time, bone had begun replacing cartilage, and the digits, the last elements to
differentiate, undergo further development
and elongation. The terminal cartilagenous
portions of the radius and ulna have elongated distally, moving the carpal complex
ahead of them. The radius was observed
to elongate more rapidly than the ulna;
such differential growth might explain, in
part, the subsequent fusion of the intermedium with the ulnare unit (fig. 6 ) , resulting in seven carpal elements characteristic of the normal adult forelimb. The
line of carpal development from the beginning of cartilage differentiation on the
twentieth post-amputation day to the complete, regenerated carpal complex as seen
on the seventieth day is outlined in figure
7. For consistency, chronological time is
+
+
+
+
+
423
used in this scheme since the Iten-Bryant
series ends with the thirty-fourth postamputation day (late digits stage).
DISCUSSION
The morphogenesis of the carpal complex in the regenerating forelimb of the
adult newt has been reported. The original
blastema cells begin to differentiate into
cartilage on the twentieth post-amputation
day. Shortly after, masses of cartilage
build up from the radial and ulnar stumps.
The radial mass is larger and develops
more rapidly than the ulnar, such that by
45 days the radial mass has differentiated
into the pre-pollicis, radiale, centrale and
intermedium, while the ulnar mass requires 55 days to form the three basale
and ulnare elements. Thus, eight basic
carpal elements are formed by the fiftyfifth post-amputation day, with subsequent
fusion of the intermedium with ulnare into
a common unit occurring by the seventieth
day. The completed regenerate possesses
the seven carpal elements characteristic
of the normal adult limb.
The present results indicate that during
limb regeneration in the adult newt the
carpal elements are restored to their original number and position. In addition, our
results show that the pattern of carpal
differentiation in the regenerating limb
proceeds in a proximodistal direction influenced by the stump remnants of the
radius and ulna, the process recapitulating
the normal developmental pattern (Strasser, 1879). A proximodistal sequence of
differentiation is supported by: (1) There
is direct contact between the radial and
ulnar stump remnants and newly-forming
cartilage in the regenerate; ( 2 ) The future
carpal elements arise from extensions of
the radial and ulnar cartilagenous masses;
( 3 ) The individual carpal units subsequently form distal to the radius and ulna;
( 4 ) The digits are the last elements to
develop and to differentiate. Although
such development tends to support the
view that the regional organization of the
skeletal system is in a proximodistal direction (Michael and Faber, ’71), we have
no evidence to exclude the possibility that
determination or “differentiation tendencies” may occur in a distoproximal direction (de Both, ’70). In the proximodistal
424
M. M. BENZO, C. A. BENZO AND H. W. MANNER
scheme, the primary differentiation of
blastema mesenchymal cells in the stump
area results in the formation of proximal
structures (carpals) and the distal structures (digits) differentiate from those
relatively few cells in direct contact with
the apical epidermis (Michael and Faber,
’71).
In the present study, we have determined
the morphological sequence of events
occurring during regeneration of the carpal complex in the adult newt. As yet
unknown are the mechanisms underlying
the sequential differentiation and eventual
restoration of the specific carpal elements
from the cartilage masses to their original
number and position in the completed
regenerate. It is hoped that the present
work might serve as a descriptive base for
subsequent investigations into the chemical and mechanical factors involved in this
aspect of limb differentiation and regenertion. Such study not only would increase
our knowledge of the mechanism of outgrowth, but also would add to our understanding of the nature of “limbness”
(Zwilling, ’72).
ACKNOWLEDGMENT
The authors wish to thank Dr. Chester
L. Yntema and Dr. Philip B. Armstrong
for their helpful comments in the preparation of this manuscript.
LITERATURE CITED
Carlton, H. M., and R. A. B. Drury 1957 Histological Technique. Third ed. Oxford University Press, London, p. 107.
Davis, D. D., and U. R. Gore 1947 Clearing
and staining skeletons of small vertebrates.
Fieldiana: Technique No. 4, pp. 3-16.
de Both, N. J. 1970 The developmental potencies of the regeneration blastema of the axolotl limb. Wilhelin Roux’ Archiv., 265: 242-273.
Francis, E. T. B. 1934 The Anatomy of the
Salamander. Oxford University Press, London,
pp. 40-44.
Iten, L. E., and S. V. Bryant 1973 Forelimb
regeneration from different levels of amputation i n the newt, Notothalmus viridescens:
Length, rate, and stages. Wilhelm Roux’ Archiv., 173: 263-282.
Manner, H. W. 1953 The origin of the blastema and of new tissues i n regenerating forelimbs of adult Triturus viridescens viridescens
(Rafinesque). J. Exp. Zool., 122: 229-257.
McManus, J. F. A,, and R. W. Mowey
1946
Staining Methods Histological and Histochemical Harper and Brothers, New York, p. 23.
Michael, M. I., and J. Faber 1971 Morphogenesis of mesenchyme from regeneration blastemas
in the absence of di5it formation in A m b y s t o m a
mexicanum. Wilhelm Roux’ Archiv., 168: 174180.
Strasser, H. 1879 Zur Entwicklung der Extremitatenknorpel bei Salamandern und Tritonen.
Morph. Jahrb., 5: 240-315.
Thornton, C. S. 1968 Amphibian Limb Regeneration. In: Advances in Morphogenesis. Vol. 7.
M. Abercrombie, J. Brachet and T. King, eds.
Academic Press, New York, pp. 205-249.
Zwilling, E. 1972 Limb morphogenesis. Develop. Biol., 28: 12-17.
PLATES
Abbreviations
B1-B4, Basale carpal
elements one
through four
C, Centrale
I, Intermedium
M1-M4, Metacarpals one
through four
PP, Pre-pollicis
R, Radiale
Rad, Radius
U, Ulnare
U1, Ulna
PLATE 1
EXPLANATION OF FlGURES
Figures 1-6 are semi-diagramatic representations of the morphogensis
of the carpal complex i n the adult newt forelimb based on camera lucida
reconstructions. The plane of initial amputation and the four metacarpals
are included as reference points.
1 Dorsal view of right carpus of a normal adult newt
2
Thirtieth post-amputation day stage showing the chondrifying centers of the carpal complex.
3
Fortieth post-amputation day stage depicting two cartilagenous masses
originating from the ulna and three from the radius.
4 Forty-fifth post-amputation day stage showing the completed morphogenesis of the radial mass into four carpal elements. There are three
ulnar derivatives at this stage.
426
CARPAL REGENERATION IN ADULT NOTOPHTHALMUS
M. M. Benzo, C. A. Benzo and H. W. Manner
PLATE 1
427
PLATE 2
EXPLANATION OF F I G U R E S
428
5
Fifty-fifth post-amputation day stage showing the completed devdopment of the ulnar mass. The carpal complex now consists of eight
elements.
6
Seventieth post-amputation day stage depicting the completed regenerate possessing the seven characteristic carpal elements. The intermedium has fused with uliiare forming a common element.
7
Line of carpal development from the beginning of cartilage differentiation to the completed carpal complex in the regenerating newt
forelimb.
CARPAL REGENERATION IN ADULT NOTOPHTHALMUS
M. M. Benzo, C. A. Benzo and H. W. Manner
PLATE 2
n
POST-AMPUTATION
DAYS
20
BLASTEMA CELL
I
I
I
R A D I A L MASS
U L N A R MASS
45
55
429
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adults, elements, viridescens, notophthalmus, regenerative, forelimb, morphogenesis, carpa
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