October 1, 1999;
Synergism between Pax-8 and lim-1 in embryonic kidney development.
Pax genes encode a family of highly conserved DNA-binding transcription factors. These proteins play key roles in regulating a number of vertebrate and invertebrate developmental processes. Mutations in Pax-6 result in eye
defects in flies, mice, and humans, and ectopic expression of this gene can trigger the development of ectopic compound eyes
in flies. Likewise, mutation of other Pax genes in vertebrates results in the failure of specific differentiation programs-Pax-1 causes skeletal defects; Pax-2, kidney
defects; Pax-3 or Pax-7, neural crest defects; Pax-4, pancreatic beta-cell defects; Pax-5, B-cell defects; Pax-8, thyroid
defects; and Pax-9, tooth
defects. Although this class of genes is obviously required for the normal differentiation of a number of distinct organ systems, they have not previously been demonstrated to be capable of directing the embryonic development of organs in vertebrates. In this report, it is demonstrated that Pax-8 plays such a role in the establishment of the Xenopus embryonic kidney
, the pronephros
. However, in order to efficiently direct cells to form pronephric kidneys, XPax-8 requires cofactors, one of which may be the homeobox transcription factor Xlim-1. These two genes are initially expressed in overlapping domains in late gastrulae, and cells expressing both genes will go on to form the kidney
. Ectopic expression of either gene alone has a moderate effect on pronephric patterning, while coexpression of XPax-8 plus Xlim-1 results in the development of embryonic kidneys of up to five times normal complexity and also leads to the development of ectopic pronephric tubules. This effect was synergistic rather than additive. XPax-2 can also synergize with Xlim-1, but the expression profile of this gene indicates that it normally functions later in pronephric development than does XPax-8. Together these data indicate that the interaction between XPax-8 and Xlim-1 is a key early step in the establishment of the pronephric primordium
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FIG. 2. XPax-8 and Xlim-1 expression in the presumptive embryonic kidney. Expression of Pax-8 as revealed by whole-mount in situ hybridization of Xenopus embryos of various developmental stages (samples on the left). Xlim-1 (Taira et al., 1994a) expression at similar stages is illustrated on the right. The two distinct
patterns of pronephric expression converge until they are indistinguishable by stage 25. A, stage14, XPax-8. B, stage 14, Xlim-1. C,
stage 20, XPax-8. D, stage 20, Xlim-1. E, stage 24, XPax-8. F, stage 23, Xlim-1. G, double staining of stage 16 embryo with XPax-8 in dark brown and Xlim-1 in pink/red. Computer-generated false
coloring was used to enhance the difference between the two
immunohistochemical substrates. H, graphical interpretation of data shown in G. Green represents XPax-8 only expression domains in the presumptive otic vesicle and possibly in the posterior pronephric region. Red represents the Xlim-1 only domain in the ventral mesoderm belt. Blue represents the area of overlap. I, graphical representation of data in E and F; both XPax-8 and Xlim-1 are expressed in overlapping domains throughout the presumptive pronephros (blue). ot, otic vesicle; pn, pronephros. Anterior is to the
left, and dorsal up, in all samples.
FIG. 3. Ectopic expression of Pax-8 plus Xlim-1 leads to the development of abnormally large pronephroi and ectopic pronephroi. All samples were stained with antibody 3G8. A reproduced at same magnification, as are G and H (100-mmscale bars are in E and H). Anterior
is to the left, dorsal is up. (A) Normal stage 36 pronephric tubules. Red arrows indicate the three normal dorsal branches. Faintly stained nephrostomes can be seen extending from the two left branches. (B, G) Enlarged pronephroi in XPax-8 plus Xlim-1- (1:1 ratio) injected embryos. In B the tubules to the left are slightly distended, probably due to osmotic pressure, and this thickness probably represents distortion rather than enlargement. However, the right side of this same pronephros contains many additional tubule branches that are all
of normal thickness. In C, all tubules are of normal width. In D the pronephros (red arrow) is of only slightly greater than normal size, but it is adjacent to an ectopic pronephros (green arrow) which is almost as large. In F, two additional ectopic pronephroi (green arrows) are obvious, dorsal and posterior to the normal position of the organ (white arrow). (G) Control stage 39 embryo stained for pronephric tubules
using 3G8 and a dark blue substrate and pronephric duct using 4A6 and a light blue substrate. Note that G and H are at a later stage of development than A, and the scale is also slightly different. (H) XPax-8 plus Xlim-1-injected embryo, stage 39, stained as in G. Note the enlarged region of nonmigratory duct staining (light blue) in the vicinity of the pronephric tubules and also the presence of small ectopic pronephric tubules (dark blue stain, green arrows). Anterior is to the left, and dorsal is up in all panels.
FIG. 4. Histological analysis of enlarged and ectopic pronephroi. Dorsal is up in all samples. (A) Control. Transverse section through a normal pronephros, pronephric tubules stained with 3G8 in dark blue and pronephric duct stained with 4A6 in light blue. (B) Enlarged pronephros. The pronephros on the injected side of the same embryo as shown in A. This sample developed from an embryo injected with
XPax-8, Xlim-1, and b-galactosidase mRNA. The lineage tracer was developed with a red substrate and can be observed in the epithelia of the enlarged pronephric tubules. Once again, tubules are stained dark blue, and duct stained light blue. The red tracer stain can be clearly visualized in tubules due to the restriction of the 3G8 epitope to the apical surface of tubule epithelia. (C) Ectopic pronephric tubule. The ectopic tubules (arrowhead) are stained with 3G8 and a dark blue substrate and contain the b-galactosidase lineage tracer (red). Somites were stained with 12/101 and a light blue substrate. Note the reduction in size of the somites on the left side below the ectopic tubules. (D)
Ectopic pronephric tubule. A sample processed in the same manner as that shown in C. Both normal (b-galactosidase negative) and ectopic (b-galactosidase positive, arrowhead) pronephric tubules are visible on the left side. Once again, the somites are smaller on the side with
the ectopic pronephric tubules. S, somite; N, notochord.
FIG. 5. XPax-2 may supersede XPax-8 function later in pronephric development. In situ hybridization of stage 36 embryos with antisense
mRNA probes for XPax-8 (A), XPax-2 (B), and Xlim-1 (C). Note the even levels of expression throughout the tubules for XPax-8 compared
to the restriction of high levels of expression to the dorsal tips of the tubules for XPax-2 and Xlim-1. Also note that XPax-2 and Xlim-1 are
expressed in the nephric duct while XPax-8 is not. Embryos coinjected with XPax-2 and Xlim-1 mRNA show similar phenotypes to those
of embryos injected with XPax-8 and Xlim-1. D shows the injected side of a XPax-2 plus Xlim-1 coinjected embryo stained with the 3G8 antibody. Note the enlarged pronephros (boxed) compared to the uninjected side shown in E, indicating that XPax-2 and XPax-8 are functionally equivalent in this assay.