الفهرس 
Introduction & ReviewOnly 14 pages are availabe for public view
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Abstract
Limb development is an excellent model for studying how patterns of differentiated cells and tissues are generated in vertebrate embryos. The present five-fold integrative study has dealt with investigating the
development of the limb autopod in two different experimental models,
namely the chick Gallus domesticus and the tadpole Bufo regularis during
certain relevant developmental stages. To achieve the targets planned for
the study, several techniques were adopted.
The study involved several approaches which can be summarized as
follows:
- Morphological description for the developing limb autopod of the
core part of the concerned developmental stages.
- Anatomical description of the development of the skeleton including
chondrogenesis and the appearance of ossification centers during the
ontogenetic development of the limb autopod. In order to analyze the
ontogenetic patterns of skeletogenesis of limb autopods, a
combination of single and double staining transparency techniques
were applied utilizing Alcian blue and Alizarin red S stains.
A- brief histological description for the developing limb autopod in
both models in terms of skeletogenesis. This has been achieved by
using routine histological technique utilizing Mallory triple stain. In
addition, a number of semithin sections were also utilized for the
same purpose. The histological investigation was used as a guide for
determining the nature of the following and appropriate investigations.
- Investigating, for the first time, the unknown morphogenetic aspects
of the developing limb autopod in the toad Bufo regularis. The aim
was to investigate whether or not the amphibian model under consideration displays interdigital programmed cell death and if not, try to find the alternative way by which morphogenetic process of the limb autopod occurs. This has been attempted using standard immunohistochemical technique and employing caspase-3 as an indicator for the occurrence of apoptosis.
B- ased on the findings emerged from the latter approach,
immunohistochemical evaluation of cellular proliferation by
proliferating cell nuclear antigen (PCNA), an endogenous cell
replication marker, has been carried out in order to explore the way
by which limb autopod of the tadpole is developing and reaching its
specific morphogenetic shape.
D- escription for the ultrastructural characteristics of the main cellular
changes involved in the morphogenesis of the developing autopod of the chick Gallus domesticus using transmission electron microscopy.
Seven findings have been emerged from the present study and can be
summarized as follows:
1- The developing limb autopod of both models followed a general
proximo-distal progression and a postaxial polarity in digit
development which occurred in a postero-anterior direction.
However, fundamental differences had been found.
2- Although the chondrogenesis of the fore and hind limbs occurred at
the same stage in the autopod of both models (stage 54 for the
tadpole and 26 for the chick), the endochondral ossification of the
hind limb lagged behind its counterpart in the chick fore limb by one
stage (stage 35 and 34 respectively). However, the endochondral
ossification of both limbs occurred at the same developmental stage
(57) in the tadpole.
3- Based on the immunohistochemical investigation, the developing
autopod of the toad Bufo regularis as an amphibian model displayed
no signs for interdigital programmed cell death. Alternatively, the
study revealed that the morphogenetic process of the developing
autopod occurred completely by differential growth where more
cellular condensation were found in the digital area compared with
the interdigital area. Furthermore, the study pointed to the possibility
of cell migration during the morphogenesis of the developing
autopod in the toad and this was based on the gradual decrease of
cellular condensation during the development of the autopod. It can
be therefore concluded that in the developing autopod of the
tadpoles, digits appear sequentially as outgrowths from the limb
plate.
4- Analysis of the labeling index of the developing autopod of the
tadpoles revealed that the interdigital and digital labeling indices
were not the same starting from the developmental stage 53. The
ratio of interdigital labeling index to the digital labeling index in the
developing autopod suggested that during subsequent development,
the interdigital labeling index was significantly lower than the digital
counterpart. It is therefore proposed that free digits in the developing
autopod of the toad reach their specific morphogenesis, at least
partly, as a result of a decrease in the proliferation rate of the
interdigital area as compared to the digital area i.e. differential
growth.
5- Unlike the situation in the developing tadpoles which witnessed the
absence of interdigital programmed cell death, morphogenesis of the
developing autopod in the chick occurred principally by interdigital
programmed cell death during the formation of free digits. The latter
was initially detected at the most proximal region, and then more
distally as development proceeded. Clearly, the results of the present
study showed that the interdigital programmed cell death is an integral part of the formation of free digits in the chick embryo, while it is never a part of free digit formation in the developing toad. In the latter, cell migration and differential growth are the alternative processes through which digits can be freed.
6- Vascular regression occurred during mesenchymal cell condensation
and chondrogenesis in both experimental models; however, during
the freeing of the digits, this regression occurred only in the
interdigital area of the chick limb bud but not in its tadpole
counterpart. Moreover, the interdigital programmed cell death
process responsible for the freeing of the digits was accompanied
with vascular regression.
7- Fine structural investigation of the interdigital area during formation
of the digits of the chick revealed that interdigital programmed cell death was accompanied by a high deposition of collagenous material in the epithelial-mesenchyme interface, rupture of the basal lamina and detachment of ectodermal cells into the amniotic sac. Most interdigital dying cells appeared rounded and electron dense with the nucleus exhibiting a characteristic peripheral condensation of the chromatin. This was accompanied with vacuolation of most of the cytoplasmic organells. In addition, macrophages appeared gradually in the mesoderm to eliminate cellular debris and there was appearance for lysosomes. The mitochondria of the interdigital area appeared distorted and this referred to the intrinsic pathway of caspase action.
Based on the mentioned seven findings, one can conclude that the
observed differences in the mode of autopod morphogenesis in the
developing toad and chick point to fundamentally different mechanisms in both models. This in turn, reflects the fact of separate lines of creation and dismisses the notorious idea which says that the tetrapod limb follows a highly conservative developmental pattern.