Seufert DW et al. (1999), Developmental basis of pronephric defects in Xe...

XB-ART-12059

Dev Biol 1999 Nov 15;2152:233-42. doi: 10.1006/dbio.1999.9476.

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Developmental basis of pronephric defects in Xenopus body plan phenotypes.

Seufert DW , Brennan HC , DeGuire J , Jones EA , Vize PD .

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We have used monoclonal antibodies that recognize the pronephric tubules or pronephric duct to explore the induction of the embryonic kidney in developing Xenopus embryos. Morphogenesis of the pronephros was examined in UV-ventralized and lithium-dorsalized embryos. We find that the pronephric tubules are present in all but the strongest UV-induced phenotypes, but absent from relatively moderate lithium phenotypes. Interestingly the pronephric duct, which develops from the ventroposterior portion of the pronephric anlage, is missing from more of the mild UV phenotypes than are pronephric tubules. The loss of the capacity to form pronephroi in UV-ventralized embryos is caused by the loss of tissues capable of inducing the pronephric mesoderm, as marginal zone explants from ventralized embryos are still competent to respond to pronephric-inductive signals. Explant recombination experiments indicate that the tissue responsible for both the loss of pronephroi in UV-ventralized embryos and the induction of pronephroi during normal development is the anterior somites. The absence of pronephroi in relatively mild lithium phenotypes has a developmental basis different from that of the UV phenotype, as explants from lithium-treated embryos are effective inducers of pronephroi in recombinants with competent mesoderm, even though they themselves do not form pronephroi in isolation. Together these data indicate that dorsal tissues, especially the anterior somites, are responsible for the establishment of the intermediate mesoderm and the induction of the embryonic kidneys and that even mild dorsalization destroys the capacity to form cells competent to receive this signal.

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	FIG. 1. Muscle and pronephric tubule in UV-ventralized embryos. Somite was detected with antibody 12/101 using an orange substrate and pronephros with antibody 3G8 and a dark blue substrate. In all images except G anterior is to the left and dorsal is up. The arrows indicate pronephric staining. Insets show enlargements of the pronephric tubules in the corresponding image, outlined with a dashed light blue line (A, B, D, and E). (A) Control embryo, DAI grade 5. As this is a cleared embryo, the pronephric tubules of the opposite (right) side of the embryo are also visible as a faint shadow. (B) Mild UV phenotype, DAI grade 4. (C) DAI grade 3. (D) DAI grade 2.5; note that the pronephric tubules are present below the most anterior somite. (E) DAI grade 2; the pronephros extends anterior to the somites. (F) Severe DAI grade 1. (G) DAI grade 0.
	FIG. 2. Pronephric tubule and pronephric duct development in lithium-dorsalized embryos. Stage 40 embryos were stained for pronephric tubules using 3G8 and a blue substrate and for duct using 4A6 and a purple substrate. The point at which the pronephric tubules join the pronephric duct is indicated with a white arrow in A to C. (A) Control embryo. (B) DAI grade 6, bent axis. The pronephric tubules and duct are relatively normal. (C) DAI grade 7, short axis. Both tubules and duct are much smaller than normal. (D) Slightly more severe DAI grade 7 embryo. Only a tiny piece of pronephric tissue remains (red arrow). It is not possible to distinguish between tubule and duct in this particular sample. (E) DAI grade 8, no pronephric tissue.
	FIG. 3. Scheme for generating recombinants between normal, UV-ventralized, and lithium-dorsalized explants. Three types of embryos were used, normal (left), UV-ventralized (center), or lithium-dorsalized (right). At gastrula stages the ventral half of normal embryos was removed. UV embryos were cut into two equivalent halves. Lithium embryos were cut into four equivalent quarters. Recombinants between these various explants were then generated and raised to the equivalent of stage 35. They were then stained with antibody 3G8 to detect pronephric tubules. Although not indicated here, the control experiments of recombining two normal ventral halves, or normal ventral plus UV halves, were also performed.
	FIG. 4. Pronephric tubules in UV/lithium recombinants. All samples are stage 35 equivalent. (A) UV-ventralized half-embryo stained with antibody 3G8. No pronephric tubules are present. (B) Pronephric tubules (black arrow) in a 3G8-stained recombinant between a b-galactosidase lineage-traced UV-ventralized half-embryo and an untraced lithium-dorsalized quarter-embryo. The lineage tracer was developed using a light blue/green substrate, and the border between the lineage-traced UV explant and the untraced lithium explant is marked with a dashed red line. A dark blue substrate was used to detect pronephric tubules. (C) 3G8-stained embryoid derived from a lithium quarter-embryo; no tubules are present.
	FIG. 5. Induction of pronephric tubules in tissue recombinants. Responsive mesodermal tissue, shown in light gray, corresponding to prospective lateral plate and pronephric mesoderm, was isolated from stage 11.5 embryos. This tissue was cultured either alone or in combination with various potential inducing tissues within an ectodermal sandwich made using two stage 9 animal caps. The one- (ectoderm only), two- (ectoderm plus lateral mesoderm), or three- (ectoderm, lateral mesoderm plus potential inducer) way recombinants were developed to stage 41 (with respect to the competent mesoderm) and stained with 3G8 and 4A6 to detect pronephric tubules and pronephric ducts. For additional details on dissection technique please see Materials and Methods.