The causal and temporal aspects of blood tissue specification in the chick embryo were
investigated in this study. The main focus was on the role of basic fibroblast growth factor
(bFGF) in the determination of the erythropoietic tissue, particularly in context with its
representation as a non-axial mesodermal derivative which arises in the posterior domain of
the chick embryo.
The initial strategy employed in this study was the use of agents that are known to
block the activity of bFGF, and to determine their effects on erythropoiesis. Treatment of
unincubated chick embryo explants with heparin, which binds specifically to the FGF family,
was found to inhibit primitive streak formation and erythropoiesis, and also inhibited the
formation of other mesodermal tissues. These initial findings suggested that one or more
growth factors had become bound to the heparin, and that their activity is important for the
specification of primitive streak formation and mesodermal patteming. The development of
the erythropoietic tissue was assayed by a cytochemical test for haemoglobin using 0dianisidine;
and by histological examination for blood islands and red blood cells in serial
sections of the embryos after 48 hours incubation. Microscopic examination of the embryos
at the stages of gastrulation on the first day of incubation revealed that heparin caused holes
to appear in the ventral layer; and although a primitive streak did not form, a middle layer
of mesenchymal cells were seen to accumulate between the ectodermal and ventral
"endodermal" layers. It was significant that heparin's inhibitory effect on erythropoiesis could
be reversed after the addition of a recombinant bovine bFGF to the heparin-treated embryos.
However, the exogenous bFGF did not neutralize the inhibitory effect of heparin on the
primitive streak and other mesodermal derivatives (Chapter Two).
The inhibition of erythropoiesis by heparin was also reversed by the addition of a
mesodermal-inducing factor extracted from a Xenopus embryonic cell line, namely XTC. The
XTC mesodermal-inducing factor (MIF), which belongs to the transforming growth factor-B
family and is a homologue of activin, could also reverse the inhibitory effect of heparin on
primitive streak formation; but no recognizable axial mesodermal structures subsequently
developed. Of consequence, was that both bFGF and XTC-MIF blocked heparin's effect on
the ventral layer, preventing the gaps forming. Therefore, it is suggestive that the
VI
development of an intact ventral layer is important for the determination of the erythropoietic
sequence (Chapter Three).
By taking a more specific approach using antisera to bFGF (anti-FGF) and the bFGF
receptor (anti-FGFR) on whole embryo explants, it was found that anti-FGP and anti-FGFR
were able to inhibit erythropoiesis, but not primitive streak formation. However, these
antisera caused defects in the posterior region of the embryonic axis. These embryos not only
lacked posterior blood tissue, but heart and somites were missing; whereas the anterior head
structures were well formed. These results therefore suggest that bFGF signalling is important
for the development of the posterior body plan, which includes erythropoiesis (Chapter Four).
Further evidence for the role of bFGF in the determination of the blood mesodermal
tissue line was reached in an in vitro bioassay. In this part of the investigation, specific
pieces of the blastoderm, namely pieces dissected from the posterior marginal zone (PMZ)
and inner core of the central disc (lCD) were able to form haemoglobin under particular
conditions. The PMZ components were found to have the capacity to form haemoglobin
when dissected from blastoderms of stages X to xm when cultured in serum-free medium.
This commitment to form haemoglobin could be blocked by treatment with anti-FGP at stages
X and XI, but not at the later stages of xn and XIII. The ICD components were found to
have a commitment to form haemoglobin only if this component was dissected from embryos
at stage XIT and XITI, but not before. These results suggest that a determinative event for the
haemoglobin differentiative pathway occurs between stages XI and XII. It was also found
that the stage X central disc component could be induced to form haemoglobin if a stage xm
hypoblast was added to it in tissue recombination sandwich cultures, or if bFGF (75 - 150
ng/ml) was added to the medium. These results lend further support that bFGF plays an
important role in the determination of erythropoiesis; and furthermore, suggest that the
hypoblastic tissue is the source of this induction (Chapter Four).
Finally, immunocytochemical labelling with a polyclonal antibody to bFGF has
revealed that bFGF increases significantly from stage XI in cells within the developing
hypoblast layer and in the middle mesodermal layer. These cells are located predominantly
in the posterior domain of the embryo. This polarized distribution of bFGF with the high
value of bFGF concentration in the posterior area, is presumably responsible for inducing the
overlying epiblast to form the posterior horseshoe-shaped region from which blood tissue is
seen to arise. An immunocytochemical analysis of the distribution of the FGF receptor was
vu
assessed, as an indicator of the possible competence of the cells to respond to the bFGF
signal. The bFGF receptor was found to be expressed at stage XII in cells that appeared to
be in register with those immunoreactive to the bFGF ligand; therefore suggesting an
autocrine function. It was interesting that at stage Xli an intense immunostaining with the
anti-FGFR developed in the nuclei of cells within the epiblast layer (Chapter Five).
In conclusion, this study has demonstrated that the initial determination of the
erythropoietic cell lineage in the chick is at the time when the hypoblast is in the process of
forming beneath the epiblast, Le. between stages XI and XII. Furthermore, it was found that
an induction by an FGF-like signal from the hypoblast layer (or middle mesodermal cells that
may be closely associated with the hypoblast) induces "competent" cells (Le. FGFR-positive
cells) in the epiblast to form blood tissue in the posterior domain of the chick embryo. / Thesis (Ph.D.)-University of Natal, 1994.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/4831 |
| Date | January 1994 |
| Creators | Gordon-Thomson, Clare. |
| Contributors | Fabian, B. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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