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An antibody that recognizes tyrosine hydroxylase can be used as a marker for hatching gland cells in Xenopus embryos. Using this marker, we have shown that hatching gland cells are induced at the end of gastrulation and that presumptive hatching gland cells are localized to the anterior neural folds in Xenopus. The movements of neurulation bring the hatching gland cells together to form a characteristic Y pattern on the dorsoanterior surface of the head. The Y pattern delineates several zones of surface ectoderm which can be visualized by the presence or absence of ciliated cells. As development proceeds the hatching gland pattern is altered, demonstrating the active changes involved in forming the face. Lithium, UV irradiation and retinoic acid can be used to alter the hatching gland pattern in specific ways which help to understand the underlying mechanisms of ectodermal patterning.
Fig. 1. Staining of Xenopus embryos with an anti-TH
antibody. (A) Whole mount of a stage 28 Xenopus embryo
showing the inverted Y pattern of the hatching gland. The
embryo is flattened to show the face of the embryo in the
same focal plane as the dorsal midline. Anterior is at the
bottom, posterior at the top. The anterior arms of the Y
end at the cement gland (eg). The cells of the anterior
arms appear stretched as compared to cells of the central
part of the pattern. The hatching gland cells continued up
the dorsal midline to the level of the otic capsule.
(B) Staining of specific cells, indicated by arrows, in the
retina. Lens (1). (C) Staining of periodically arranged
neurons in the ventral floor of the neural tube. Neural tube
(nt), notochord (n). (D) Ventralbrain region near the eye
showing staining of specific neurons. Scale bars=50um.
Fig. 2. Section through a stage 28 Xenopus embryo
showing hatching gland cells. (A) Epifluorescent view of
cells stained using the anti-TH antibody. (B) The same
section viewed in bright field showing that the cells
recognized by the anti-TH antibody, located between the
arrows, have a clear apical cytoplasm and pigmented basal
cytoplasm. This morphology matches that of hatching gland
cells. The anti-TH antibody staining was always in the
apical cytoplasm and the fluorescent cells were always on
the surface. Scale bar=50um.
Fig. 3. Example of explant experiment designed to localize presumptive hatching gland cells. (A) Cross section of two
explants fixed prior to curling showing that the surface explants were a single cell layer, relatively free of contaminating
deep cells. Scale bar=100^m. (B) An example of a TH-positive explant removed from the anterior neural folds at stage
14/15 and cultured in MBS until stage 28. Scale bar=50/zm. (C) An embryo stained for hatching gland cells, after removal
of surface ectoderm from one anterior neural fold and culturing to stage 28. It is clear that there is substantial loss of
hatching gland cells from the operated side of the embryo since one arm of the Y is missing. Scale bar=100um.
Fig. 4. Presumptive hatching gland cells are located on the surface of the anterior neural folds. (A) A schematic drawing
of stage 14/15 neural folds showing the positions where surface ectoderm explants were taken. The areas examined were:
(1) the transverse neural fold; (2) the anterior neural fold; (3) the posterior neural fold. (B) After the operation, explant
and embryo were cultured to stage 28. Explants were examined for hatching gland cells and embryos were assayed for
damage to the hatching gland pattern. Schematics represent the degTee of damage seen in embryos, from no damage
(symmetrical pattern) to partial, or total loss of one arm of the Y, from the operated side. No operation refers to control
embryos and MBS refers to embryos raised in MBS, as were explants, to see if this altered the hatching gland pattern. The
stippled ovals represent the cement gland.
Fig. 5. Embryos that failed to close their anterior neural
folds had hatching gland cells on either side of the open
neural tube. (A) A cross section through an embryo raised
in Danilchik's medium showing the failure of the neural
tube (nt) to close and of the epidermis (e) to seal over the
neural tissue. (B) Whole mount of a stage 28 embryo,
stained for hatching gland cells, after a large portion of the
anterior neural tube failed to close. The hatching gland
pattern is split over the entire pattern and hatching gland
cells are found at the edge of the exposed neural tissue.
Scale bar=100um.
Fig. 6. The relationship of the hatching gland to other
surface ectodermal zones defined by the presence or
absence of ciliated cells. (A) A stage 28 embryo stained for
both /3-tubulin and TH. Ciliated cells are seen as evenly
spaced cells with tufts of cilia. A non-ciliated zone is
clearly circumscribed by hatching gland cells except at the
most anterior tip which is bounded by the cement gland
(eg). The embryo is mounted face forward with the cement
gland down. (B) Another non-ciliated zone has been
described down the dorsal midline by Chu and
Klymkowsky (1989) and can be seen in this whole mount
double stained for /J-tubulin and TH. This non-ciliated
zone is the hatching gland cells anteriorly, and posteriorly
(between the arrows) appears to be normal epidermis.
Anterior is down, and posterior up. Scale bar=100um.
