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In an expression cloning screen in Xenopus embryos, we identified a gene that when overexpressed expanded the neural plate at the expense of adjacent neural crest and epidermis. This gene, which we named geminin, had no sequence similarity to known gene families. We later discovered that geminin''s neuralizing domain was part of a bifunctional protein whose C-terminal coiled-coil domain may play a role in regulating DNA replication. We report here on the neuralizing function of geminin. The localization, effect of misexpression and activity of a dominant negative geminin suggest that the product of this gene has an essential early role in specifying neural cell fate in vertebrates. Maternal geminin mRNA is found throughout the animal hemisphere from oocyte through late blastula. At the early gastrula, however, expression is restricted to a dorsal ectodermal territory that prefigures the neural plate. Misexpression of geminin in gastrulaectoderm suppresses BMP4 expression and converts prospective epidermis into neural tissue. In ectodermal explants, geminin induces expression of the early proneural gene neurogenin-related 1 although not itself being induced by that gene. Later, embryos expressing geminin have an expanded dorsal neural territory and ventralectoderm is converted to neurons. A putative dominant negative geminin lacking the neuralizing domain suppresses neural differentiation and, when misexpressed dorsally, produces islands of epidermal gene expression within the neurectodermal territory, effects that are rescued by coexpression of the full-length molecule. Taken together, these data indicate that geminin plays an early role in establishing a neural domain during gastrulation.
Fig. 1. Ectopic neurogenesis and neural plate expansion in
embryonic ectoderm injected with geminin in one bilateral half
(oriented to the right). In situ hybridization detects cells expressing
N-tubulin (aqua in A-E, H; purple in F, G) and cells expressing
injected geminin (dark pink in A-E; H) either from an expression
plasmid (A, H) or from 15 pg injected full-length (F, G) or 250 pg
Ngem (B-E) RNAs. Endogenous geminin RNA is not visible.
Fig. 2. Geminin suppresses epidermal and expands neural gene
expression. (A) Synaptobrevin (Knecht et al., 1995; Richter et al.,
1988) expression in the olfactory placode is expanded on the
geminin-injected side of the embryo (15 pg full-length RNA
injected). (B,C) otx 2 expression on geminin-injected (B) or
uninjected (C) sides of the same embryo. In B, otx 2 expression is
expanded posteriorly and the otic vesicle and branchial arches fail to
form normally. (D-F) In situ hybridization for twist (aqua) and
injected geminin (pink). Injection of 250 pg of Ngem RNA
eliminates twist staining (D,E), whereas injection of 25 pg of Ngem
expands twist expression on the injected (right) side relative to the
control half (F). (G-I) Animal hemisphere views of late gastrulae
stained to detect epidermal keratin (aqua) and either 250 pg injected
Ngem (G,H, pink stain) or 250 pg injected GFP (I, pink stain). In G
and H, epidermal keratin staining is lost in injected cells; in I,
overlap of GFP and keratin produces a dark purple stain.
Fig. 3. Structure, intracellular localization and developmental profile of geminin expression in embryos. (A) Protein sequences of geminin. The
N-terminal domain sufficient for neuralization is overscored in red and the C-terminal coiled-coil domain is overscored in blue. Accession
numbers for geminin are AF068781 (gem L), AF067856 (gem H), AF068780 (mouse gem), and AF067855 (human gem). (B,C) Animal pole
(B) and side (C) views of a blastula embryo immunostained with a geminin antibody. Most endogenous geminin is localized to the nucleus.
(D) RT-PCR analysis of geminin RNA levels in the embryo during development. Between stage 10 and 10.25, embryos were analyzed whole
(W) or subdivided into dorsal (D) or ventral (V) halves. New (zygotic) transcription of geminin is evident at stage 9 and in the dorsal half of the
embryo at stage 10.
Fig. 4. Spatial distribution of geminin transcripts in the early embryo identified by in
situ hybridization. (A,B,E,G,I-K) Geminin staining is purple in singly stained embryos.
(C,D,F,H) Geminin expression is pink in doubly stained embryos. The expression
patterns stained in aqua are brachyury (C,D), BMP4 (F), or X-Delta-1 (H).
(L) Geminin expression detected in a section of an e15 mouse embryo. Dorsal is
oriented to the right (B-D), top (E,F) or facing out (G,H). Xenopus embryos are stages
4 (A), 10 (B), 10.25 (C, D), 12 (F), 12.5 (E), 13.5 (G), 20 (H), 28 (I) and 38 (J,K).
Fig. 5. Geminin inhibits expression of the epidermalizing growth
factor BMP4 in ventral and lateral ectoderm during gastrulation.
BMP4 expression (in aqua) occurs in lateral and ventral ectoderm of
uninjected embryos (A) but is eliminated by injection of 250 pg
Ngem RNA (pink staining B,C). A and B are lateral views; C is an
animal hemisphere view; embryos are stage 12 and dorsal is to the
right. (D) BMP4 expression is downregulated in ectoderm injected
with Ngem RNA. Expression of endogenous geminin is induced by
injected Ngem (primers recognize the 3¢ untranslated region not
included in the injected RNA). Muscle actin is not induced by
Fig. 6. BMP4 can relieve geminin’s suppression of epidermal
development. (A-F) Double in situ hybridizations with epidermal
keratin (aqua) and geminin (pink) probes at stages 10- (A), 10+ (B,
C), 12.5 (D) or 12 (E, F). A and C are dorsal views (arrowheads
mark the dorsal lip); B, E and F are animal hemisphere views.
(E) Coinjection of BMP4 and geminin; dark-purple tissue expresses
both epidermal keratin and injected geminin. (F) Coinjection of
geminin and GFP. Epidermal keratin expression is suppressed in
regions expressing injected geminin.
Fig. 7. Geminin is induced by organizer signals and activates neural
gene expression in isolated ectoderm. (A) Geminin expression in
response to inducing molecules, (B) induction of neural gene
expression by geminin, (C-F) immunostaining for N-CAM in animal
caps injected with pUASgem (C), uninjected animal caps (D) or
embryos injected in one bilateral half with pUASgem (E,F).
Fig. 8. A dominant negative geminin induces epidermis and
suppresses neural development in dorsal cells. (A-D) In situ
hybridization to detect epidermal keratin (pink) and injected geminin
(aqua; endogenous not visible); overlap produces a dark blue-purple
stain. Embryos are stage 12. (A,B) Dorsal views of embryos injected
with the geminin C-terminal domain expression plasmid (pCdim).
(C,D) Embryos injected with a plasmid encoding both Cdim and fulllength
geminin (pCdim-gem; 100 pg) and with a plasmid encoding
full-length geminin (pCMVgem; 100 pg). A remaining keratinexpressing
dorsal cell is indicated by an arrowhead. (E,F) In situ
hybridization to detect N-tubulin (dark blue in F) and ectopically
expressed geminin (pink) in transgenic embryos expressing either
pCdim (E) or pCdim-gem (F). Embryos in E, F are stage 23/24.