CSB331H1 Lecture Notes - Lecture 14: Inverted Microscope, Chordin, Platelet-Derived Growth Factor

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4 Apr 2012
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Lecture 14 Notes March 9, 2011
Early Amphibian Development- continued from the lecture 13 notes
Verbatim from Developmental Biology, 7th Ed, Gilbert S, Sinauer, Chapter 10 (with some minor
modifications and information unrelated to the lecture removed)
Morphogenetic processes associated with Xenopus gastrulation
During gastrulation in Xenopus, internalization of the mesoderm and endoderm occurs simultaneously. Mesoderm
internalization occurs by involution; whereas endoderm internalization is promoted in part by rotation of the
endoderm cells and association with the migrating mesoderm. As illustrated by a slide focused on epiboly,
mesoderm involution begins dorsally, spreading laterally and ventrally to form a ring-like blastopore enveloping
the yolk plug. Once internalized, the mesoendoderm (a combination of mesoderm and endodermal cells at the
leading edge) moves as a coherent unit towards the animal pole, using the blastocoel roof (BCR) as a migratory
surface.
Fibronectin and epiboly in Xenopus
Fibronectin is expressed early during embryonic development, however, it precise biological functions were until
recently unknown. Using a combination of confocal and digital time-lapse microscopy, they analyzed cell
behaviors in Xenopus gastrulae that were injected with an anti-FN neutralizing monoclonal antibody. The
monoclonal antibody used was directed against the integrin-binding domain of FN, hence when bound to FN
dimers, binding of FN to cell surface integrins is blocked. This in turn inhibits receptor-mediated FN
fibrillogenesis. In another set of experiments, one-cell embryos were also injected with an mRNA coding for a
dominant-negative construct of β1 integrin subunit. Immunohistochemical analyses show that these two reagents
were effective in inhibiting the assembly of FN fibrils on the inner surface of the blastocoel roof. Among the
defects observed by the absence of FN was a failure by ectodermal cells underlying the outer epithelial cells to
undergo radial intercalation. As a result, there was no thinning of the blastocoel roof, which in turn inhibited
gastrulation movements. Confocal analysis of the BCR reveals that FN is required for mitotic spindle orientation.
In the presence of FN, the mitotic spindles are orientated within the plane of the BCR epithelium. In the absence
of FN, mitotic spindle orientation is randomized, indicating that FN plays an important role in promoting cell
polarity, a perquisite for radial interaction.
Histological analysis reveals that a stable interface (Bracket’s cleft) in maintained between the
BCR and involuting mesoendoderm – an interface essential for the movement of two tissues past each other.
Hence, a fundamental question is what prevents surface ectodermal and mesoendoderm cells from mixing with
each other during gastrulation? Since the BCR is covered with a network of fibronectin fibrils, one hypothesis is
that the FN network coating the inner surface of the blastocoel roof serves both as an adhesive substratum to
promote cell migration and as a barrier to prevent the mixing of cells from different layers. While promoting the
formation of lamellipodia by involuting cells, the FN networks is not dense or thick enough to prevent mixing and
fusion of these two tissues. As you are aware, basal laminae have barrier capacity, but no basal laminae lines the
BCR. Moreover, similar cadherins are expressed by the BCR and underlying mesoendoderm. To address this
issue, Wacker et al. used a BCR assay to examine when separation behavior is initiated and how it is regulated
(Wacker et al. 2000. Development and control of tissue separation at Gastrulation in Xenopus. Devel. Biol.
224:428-439). The senior author of this manuscript is Prof. Rudi Winklbauer, a faculty member in our
Department.
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BCR assay
BCR explants are taken from stage 10-10.5 Xenopus embryos are place with the BCR facing up on
BSA-saturated glass-cover slips. In other words, the FN-coated inner surface of the BCR is exposed. Without
anchoring, the BCR explants roles up, preventing further experimentation. Cellular aggregates or single cells are
placed on the surface of the BCR explants and monitored over time to determine if the cells remain on the surface
or integrate into the roof. To monitor if separation behavior is expressed, cellular aggregated are taken from
embryos injected with the fluorescent dye Lucifer yellow dextran. Note that when cellular aggregates isolated
from involuting prospective anterior mesoderm are placed on a BCR explant (also called an animal cap explant),
the cells remains on the surface, hence expressing separation behavior. In contrast, cell aggregates derived from
animal caps gradually became integrated into the blastocoel roof.
Tissue Separation: separation behavior by cell aggregates from different regions and stages
A histogram was used to demonstrate that at stage 9, the entire prospective involuting marginal
zone (fated to form mesoderm) reintegrates into the BCR. However, below the IMZ showed separation behavior,
suggesting a sharp boundary exits the animal and vegetal poles. As gastrulation progresses, and the lip region
rotates 90O, separation behavior spreads to the internalized cells (anterior, intermediate and eventually to the
posterior mesoderm). A conclusion reached was that separation behavior is acquired by IMZ cells just prior to the
beginning of their involution. The acronyms are defined at the bottom of the slide.
In another set of experiments, Dr. Winklbauer demonstrated animal cap cell aggregates exposed to
the potent mesoderm inducing factor activin acquire separation behavior, whereas bFGF alone had no effect.
However, the separation behavior by activin was negated if embryos were injected with mRNA coding for a
dominant negative FGF receptor construct (XFD), indicating activin induction is upstream of FGF signaling. I
indicated in the slide that bFGF induces posterior and ventrolateral mesoderm, whereas activin is a potent dorsal
and anterior mesoderm inducer to highlight that they contribute to the development of distinct mesoderm domains.
However, the data also reflect that they have distinct and overlapping effects on mesoderm, such as regulating
separation behavior. You do not need to known the downstream effectors of these signaling pathways, only the
results obtained and that the BCR assay allows analysis of the molecular mechanisms regulating separation
behavior.
To gain further insight into the mechanism of tissue separation, embryos were injected with
mRNA coding for dominant negative EP/C or XB/U cadherins and the explanted cells analyzed for separation
behavior. Dominant negative constructs were generated by deleting the cytoplasmic tail (i.e. EP∆C). The data
presented on a slide was obtained with EP∆C cadherin. The results were the same when XB∆C was injected,
reflecting that these two closely related cadherins have overlapping distributions and functions. The data indicate
that lowering cadherin function appears to be sufficient to induce separation behavior. Hence, differential
adhesion between the involuting mesoderm cells and overlying BCR may play a critical role in determining
separation. Reduced adhesiveness has been shown in other organisms to play an important role in promoting
tissue separation.
Mesoderm migration assay
Data derived from a publication by Prof. Rudi Winklbauer’s laboratory (Nagel et al. 2004. Devel
131:2727-2736) entitled “Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling”.
In vertebrates, PDGF-A and its cognate receptor PDGFR-α, are expressed early during development.
Mutations in this signaling pathway in Xenopus causes aberrant mesoderm involution during gastrulation, leading to
compromised anterior development. Prof. Winklbauer’s laboratory used an in vitro mesoderm migration assay to
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