Lecture 14 Notes on Xenopus gastrulation, epiboly, tissue separation and convergence extension March 9, 2011.doc

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Cell and Systems Biology
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Maurice Ringuette

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Lecture 14 Notes March 9, 2011 Early Amphibian Development- continued from the lecture 13 notes Verbatim from Developmental Biology, 7 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 1 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 (Brackets 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. 1BCR 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 90 , 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 ind
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