Int J Dev Biol
September 1, 1998;
The expression of XIF3 in undifferentiated anterior neuroectoderm, but not in primary neurons, is induced by the neuralizing agent noggin.
The gene XIF3
encodes a neural-specific type-III intermediate filament protein whose expression in the embryo
precedes that of the neurofilaments by several hours. We now show, by in situ hybridization, that it is expressed at the neurula stage
in primary neurons and, to a lesser extent, in undifferentiated anterior neuroectoderm
. At the swimming tadpole stage
, strong expression is restricted to the midbrain-hindbrain boundary
, even-numbered rhombomeres
of the hindbrain
and the Vth and VIIth cranial ganglia
gene expression can be induced in ectodermal cells (animal caps) derived from blastula
when grown to the neurula stage
in the presence of the neuralizing agent noggin
. In agreement with the proposed ability of noggin
to neuralize, but not to promote neuronal differentiation, we find that the pattern of noggin
expression in animal caps is consistent with expression in undifferentiated anterior neuroectoderm
but not in primary neurons.
Int J Dev Biol
[+] show captions
Fig. 1. The pattern of expression of the XIF3 gene during the neurula stages. (A) At stage 15, XIF3 transcripts are found in a line either side of the midline and probably correspond to the future primary motor neurons Bilateral patches adjacent to the anterior neural plate represent expression in the placodal component of the trigeminal ganglia (arrowheads). (B) By stage 15+, XIF3 expression is also detectable in a stripe either side of the midline corresponding to the primary interneurons (arrowhead). (C) By stage 16, punctate XIF3 staining is apparent in the primary sensory neuron (Rohan-Beard cell) stripe (arrowhead). In addition there is diffuse XIF3 staining in neuroectoderm between the motor neuron and interneuron stripes and to the same anterior limit as these stripes. (D) At stage 17 there IS additional diffuse bilateral staining in the prospective midbrain (arrow), though this expression is transient. IE) By stage 18, punctate staining is seen in two patches adjacent to the anterior boundary of the neuroectoderm corresponding to neurons in the nasal placodes (arrowed). (F) At stage 20, the neural tube is almost completely folded and the primary neurons are strongly stained. Expression in the trigeminal ganglia extends around the eye.
Fig. 2. The pattern of XIF3 expression in the later embryo. (A) In the late tailbud (stage 28) expression in the Vllth cranial nerve is now apparent and within the anterior neural tube expression of XIF3 resolves into patches of high and low expression along the A-P axis. (B) Lateral view showing expression in the Vth and (arrowed) Vllth cranial ganglia and in the eye. (C) By stage 36/37 expression in the caudal primary neurons has almost completely disappeared, though staining in the cranial ganglia remains strong. Within the CNS, XIF3 is now clearly expressed in distinct domains. There is a domain in the forebrain that lies beneath the nasal pits (arrowhead) and another at the midbrain-hindbrain boundary (arrow). Within the hindbrain expression is mainly confined to rhombomeres 2,4 and 6. (D) A transverse section through a stage 36/37 embryo shows that XIF3 expression within the even rhombomeres is confined to a ventro-Iateral domain.
Fig. 3. Co-expression of XIF3 and neural specific tubulin with antibody markers for primary neurons. (A-D) Transverse sections of stage 22 embryos at two positions along the A-P axis: (A and C) embryos at stage 22 stained by in situ hybridization with an DIG-labeled antisense RNA probe to XIF3 (blue) and then by whole-mount antibody staining with anti-HNK- 1(brown). (B and 01 Sections stained by in situ hybridization with a neural specific tubulin (NST) specific antisense probe (blue) and then by whole-mount antibody staining with anti-HNK.1 (brown). At this stage the antibody stains a structure within the cell that is probablv the Goigi apparatus. Note the co-localization of the two stains in the primary neurons. In addition XIF3 staining is seen in undifferentiated neuroectoderm at more rostral levels (panel A).
Fig. 4. Noggin induces a pattern of expression of XIF3 in animal caps that is similar to that found in anterior neuroectoderm but not in primary neurons. (A) Stage 22 embryo stained by in situ hybridization with a NST specific antisense RNA probe. The anterior of the embryo (ant) is at the top. (B) Noggin injected animal caps also stained with the NST specific probe at the equivalent stage to (A) lack NST expression. (C) Stage 22 embryo stained by in situ hybridization with a XIF3 specific antisense RNA probe. (D) Noggin injected animal caps at the same stage stained with the XIF3 specific probe. Arrows indicate large patches of evenly distributed XIF3 staining. However the staining does not cover the entire animal cap. (E) Control uninjected animal caps assayed at stage 22 are negative for XIF3 expression. (F) Explant of animal cap and a small piece of dorsal mesoderm that induces the formation of primary neurons and at stage 22 results in a punctate pattern of XIF3 expression. Bar in A, 0.5 mm; in B. 0.25 mm.
Fig. 5. Further analysis of noggin injected animal caps. (A) Stage 24 embryos stained with the antl-HNK-1 monoclonal antibody which
marks primary neurons. Anterior is at the top of the panel. (B) At the same early neurula stage, noggin injected animal caps are negative
for the anti-HNK-1 epitope. (C) Uninjected animal caps are also negative with the anti-HNK-1 antibody. (D) Whole embryos at the late
tailbud stage stained with the monoclonal antibody 6F11 that recognizes an NCAM antigen and marks neural tissue. (E) Noggin injected
animal caps react positively with 6F11 across most if not all of the animal cap showing that noggin injection results in the animal cap
acquiring a neural fate. (F) Uninjected animal caps are negative with the 6F11 antibody. (G,H,I) Sections through the samples shown in
O,E and F respectively. 6F11 is seen throughout the injected animal cap explant but is strongest in the superficial cells, though this may be
due to difficulties in antibody penetration in the whole-mount sample. There is little difference in the morphology of the animal caps in noggin
injected (H) and uninjected (!) animal caps. (J) Late tailbud embryos stained with the somite marker 12/101. (K) Noggin injected animal caps
do not express the antigen recognized by 12/101 confirming that noggin can neuralize animal caps without inducing the formation of
mesoderm (LI Uninjected animal caps do not express the 12/101 antigen. Bars in A and for the other whole embryos, 1.3 mm; In B and the
other intact animal caps, 0.25 mm; in G, 0.33 mm and in H, 0.06 mm.