July 1, 1998;
A novel Xenopus mix-like gene milk involved in the control of the endomesodermal fates.
Here we describe a novel Xenopus homeobox gene, milk
, related by sequence homology and expression pattern to the vegetally expressed Mix.1
. As is the case with Mix.1
is an immediate early response gene to the mesoderm
inducer activin. milk
is expressed at the early gastrula stage
in the vegetal cells, fated to form endoderm
, and in the marginal zone
fated to form mesoderm
. During gastrulation, expression of milk
becomes progressively reduced in the involuting mesodermal cells but is retained in the endoderm
, suggesting that it may play a key role in the definition of the endo-mesodermal boundary in the embryo
. Overexpression of milk
in the marginal zone
blocks mesodermal cell
involution, represses the expression of several mesodermal genes such as Xbra
and increases the expression of the endodermal gene, endodermin
. In the dorsal marginal zone
, overexpression of milk
leads to a severe late phenotype including the absence of axial structures. Ectopic expression of milk
in the animal hemisphere
or in ectodermal explants induces a strong expression of endodermin
. Taken together, we propose that milk
plays a role in the correct patterning of the embryo
by repressing mesoderm
formation and promoting endoderm
[+] show captions
Fig. 2. Expression of milk by in situ hybridisation on whole and sectioned embryos. milk-mRNA is first detected by whole-mount in situ hybridisation following the mid-blastula transition in the marginal zone (A-D). (A) Embryo at the late blastula stage (stage 9), vegetal view, dorsal is up; (B) mid-gastrula stage (stage 11), vegetal view, dorsal is up. (C) The same embryo as in A, lateral view, dorsal is to the right. Note that milk seems to be more highly expressed in the dorsal part. (D) The same embryo as in B, lateral view, dorsal is to the right. At this stage, the dorsoventral gradient appears less pronounced. At late stage gastrula and later stages in development, milk mRNA is no longer detectable (not shown). On sectioned embryos (E-I) milk is expressed in mesendoderm at the early gastrula stage and becomes restricted to the endoderm during gastrulation. (E,F,G) Hybridisation with respectively Xbra, milk and Mix.1, on alternate sections of the same embryo at stage 10.25. Note that the expression patterns of milk and Mix.1 largely overlap that of the mesodermal marker Xbra. (H,I) Hybridisation with respectively Xbra and milk on alternate sections of an albino embryo at stage 11.5. Note that milk mRNA is excluded from the involuting mesoderm (chordomesoderm and caudal mesoderm) whereas Xbra mRNA is clearly visible. The sections in H and I were imaged under the same contrast and brightness settings and printed as negative images to reinforce contrast differences between specific and background stainings. In all panels, dorsal is to the right. Arrowhead indicates the dorsal blastoporal lip.
Fig. 4. Effect of overexpression of milk on Xenopus development. (A) milk RNA injection in the ventral marginal zone results in no marked phenotypic effect. (a) Neurula-stage embryos injected with 100 pg of milk RNA in the ventral marginal zone. Note that ventral cells have failed to involute. (b) The same embryos as in a at the tailbud stage. The two embryos have developed apparently normally. (B) milk overexpression in the dorsal marginal zone causes abnormal migration of cells. (a,c,e) Control embryos injected with 500 pg of lacZ RNA. (b,d,f) Embryos injected with 100 pg of milk RNA. (a,b) Late stage gastrula, vegetal view, dorsal is up. (c,d) Neurula stage, dorsal view. (e,f) Late stage neurula, frontal view, dorsal is up. Note that the dorsal cells have failed to involute resulting in an absence of gastrulation and neurulation. (C) Effect of the injection of different doses of milk RNA in the dorsal marginal zone. (a) The top embryo was injected with 500 pg of lacZ RNA. Below, two embryos injected with 100 pg of milk RNA at the tailbud stage show microcephaly. (b) The same embryos as in a at a later stage. (c) Different phenotypes observed in embryos injected with 1 ng of milk mRNA. (d) Dorsal view of an embryo in c; no axial structures have developed.
Fig. 5. Effect of milk injection on Xbra, goosecoid, Xvent-1 and endodermin expression. Overexpression of milk in the dorsal marginal zone results in down-regulation of the early mesodermal markers Xbra and gsc and in the stimulation of edd expression revealed by in situ hybridisation. (A) Xbra expression in an uninjected embryo at stage 11.5. (B) Xbra expression in an embryo injected dorsally with 100 pg of milk plus lacζ mRNA. The cells stained with X-gal do not express Xbra. (C) gsc expression in an uninjected embryo at stage 10.25. (D) gsc expression in an embryo injected dorsally with 100 pg of milk plus lacζ mRNA. The cells stained with X-gal do not express gsc similar to that seen with Xbra. (E) Xvent-1 expression in an uninjected embryo at stage 11.5. (F) Xvent-1 expression in an embryo injected ventrally with 100 pg of milk RNA. Note a repression of Xvent-1 hybridisation signal on the site of milk injection. (G) Embryo coinjected with 100 pg of milk plus lacζ mRNA then stained with X- gal at stage 11.5. (H) The same embryos as in G labelled by an edd- digoxigenin riboprobe. The X-gal-positive cells express edd. edd expression expands outside its normal expression domain (see control J). (I) Embryo injected with 500 pg of milk plus lacζ into one micromere at the 8-cell stage, then stained with X-gal prior to in situ hybridisation for edd at stage 12. Note a patch of edd-expressing cells within the domain of X-gal-positive cells. (J) edd expression in an uninjected embryo at stage 11.5.