VII.
LIMB MORPHOGENESIS
315
far these have been concentrated on techniques which eliminate limbbud ectoderm by means of chemical treatment and were initiated when
distal development occurred in some cases following versene (ethylenediamine-tetracetic acid) skinning of limb buds in the laboratories of
Bell and Saunders.* Our joint work, still not complete (Bell, Saunders
and Zwilling, 1959), has focused attention on a residual shiny layer which
is left on the mesoblast under some conditions during versene 'skinning'.
Development of distal structures was definitely associated with the
presence of the shiny layer and the conclusion was that such morphogenesis could occur in the absence of an intact apical ridge. There is
some likelihood that a 'shiny' layer may also be present after ultrasound skinning of a limb bud. We are still not certain about the nature
of the 'shiny' layer or whether the layers are identical after versene
and ultra-sound treatments. Preliminary histological examination of a
'shiny' layer left after versene treatment (Zwilling, 1959; Saunders and
Zwilling, unpublished work) indicates that it may be cellular in nature
under some circumstances and opens the possibility that at least the
functioning aspects of the ridge may be restored even though this
structure is not present as a morphological entity. Bell's observations
indicate that the 'shiny' layer seen after ultra-sound skinning of the limb
bud is not cellular and thus is probably not the same as the layer seen
after versene treatment. There is a possibility that this may be basement
membrane alone and that, under these conditions, this structure is
able to support continuing morphogenesis of the mesoblast. These possibilities are still to be resolved and have the merit of providing a basis
for hypothesis and future research.
5. Evidence for an Ectodermal Ridge Maintenance Factor
A number of direct observations as well as deductions from indirect
evidence provide the basis for a demonstration of some mesodermal
property upon which the persistence of a thickened ectodermal ridge is
dependent. The first direct evidence of this sort was provided by
Saunders (1949) when he showed that separation of the ridge from its
underlying mesoderm by means of a thin sheet of mica (30μ, thick) for
1¿ hours or more resulted in a flattening of the ridge. There were related
defects of outgrowth. In unpublished experiments of my own (mentioned
in Zwilling, 1956d) limb mesoderm was replaced by non-limb mesoderm
in an ectodermal jacket. The mesoderm was from two sources: lateral
* Kieny (1959) has reported that 18 of 50 flank grafts of versene skinned presumptive
limb-bud mesoderm from chick embryos of stage 15, 16 and 17 formed well developed
limbs. These grafts were all covered with host flank ectoderm when harvested and Kieny
believes that the 'ectoderme banal' of the host's flank is influenced by the limb mesoderm
to participate in the formation of the limb. Only one of 59 trypsin skinned mesoblasts
from the same stages developed a limb.
LIMB MORPHOGENESIS
315
far these have been concentrated on techniques which eliminate limbbud ectoderm by means of chemical treatment and were initiated when
distal development occurred in some cases following versene (ethylenediamine-tetracetic acid) skinning of limb buds in the laboratories of
Bell and Saunders.* Our joint work, still not complete (Bell, Saunders
and Zwilling, 1959), has focused attention on a residual shiny layer which
is left on the mesoblast under some conditions during versene 'skinning'.
Development of distal structures was definitely associated with the
presence of the shiny layer and the conclusion was that such morphogenesis could occur in the absence of an intact apical ridge. There is
some likelihood that a 'shiny' layer may also be present after ultrasound skinning of a limb bud. We are still not certain about the nature
of the 'shiny' layer or whether the layers are identical after versene
and ultra-sound treatments. Preliminary histological examination of a
'shiny' layer left after versene treatment (Zwilling, 1959; Saunders and
Zwilling, unpublished work) indicates that it may be cellular in nature
under some circumstances and opens the possibility that at least the
functioning aspects of the ridge may be restored even though this
structure is not present as a morphological entity. Bell's observations
indicate that the 'shiny' layer seen after ultra-sound skinning of the limb
bud is not cellular and thus is probably not the same as the layer seen
after versene treatment. There is a possibility that this may be basement
membrane alone and that, under these conditions, this structure is
able to support continuing morphogenesis of the mesoblast. These possibilities are still to be resolved and have the merit of providing a basis
for hypothesis and future research.
5. Evidence for an Ectodermal Ridge Maintenance Factor
A number of direct observations as well as deductions from indirect
evidence provide the basis for a demonstration of some mesodermal
property upon which the persistence of a thickened ectodermal ridge is
dependent. The first direct evidence of this sort was provided by
Saunders (1949) when he showed that separation of the ridge from its
underlying mesoderm by means of a thin sheet of mica (30μ, thick) for
1¿ hours or more resulted in a flattening of the ridge. There were related
defects of outgrowth. In unpublished experiments of my own (mentioned
in Zwilling, 1956d) limb mesoderm was replaced by non-limb mesoderm
in an ectodermal jacket. The mesoderm was from two sources: lateral
* Kieny (1959) has reported that 18 of 50 flank grafts of versene skinned presumptive
limb-bud mesoderm from chick embryos of stage 15, 16 and 17 formed well developed
limbs. These grafts were all covered with host flank ectoderm when harvested and Kieny
believes that the 'ectoderme banal' of the host's flank is influenced by the limb mesoderm
to participate in the formation of the limb. Only one of 59 trypsin skinned mesoblasts
from the same stages developed a limb.