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We report a study on the specification of the glomus, the filtration device of the amphibian pronephric kidney, using an explant culturing strategy in Xenopus laevis. Explants of presumptive pronephric mesoderm were dissected from embryos of mid-gastrula to swimming tadpole stages. These explants were cultured within ectodermal wraps and analysed by RT-PCR for the presence of the Wilm's Tumour-1 gene, xWT1, a marker specific for the glomus at the stages analysed, together with other mesodermal markers. We show that the glomus is specified at stage 12.5, the same stage at which pronephric tubules are specified. We have previously shown that pronephric duct is specified somewhat later, at stage 14. Furthermore, we have analysed the growth factor inducibility of the glomus in the presence or absence of retinoic acid (RA) by RT-PCR. We define for the first time the conditions under which these growth factors induce glomus tissue in animal cap tissue. Activin together with high concentrations of RA can induce glomus tissue from animal cap ectoderm. Unlike the pronephric tubules, the glomus can also be induced by FGF and RA.
Fig. 1. Dissection of explants and construction of ectodermal wraps to assay for glomus specification. Explants of developing glomus/glomus anlagen/presumptive pronephric mesoderm were removed from donor embryos. The precise region of tissue explanted depended on the stage of donor embryo used, and is described in the Materials and Methods above. (Diagrams of stage 11, 13, 14 and 29 embryos adapted from Nieuwkoop and Faber, 1994.) Each explant was then placed between two stage 9 animal caps, forming an ectodermal wrap. The wrap was allowed to heal and cultured until control donor embryos reached stage 33/34. Explants were subsequently analysed by RT-PCR for the presence of glomustissue. A, anterior explant; M, middle explant; P, posterior explant.
Fig. 2. RT-PCR analysis of a developmental stage series showing the expression of markers used in this study. Expression was analysed in embryos at stages between 10. 5 (mid-gastrula) and 33/34 (late tailbud). These markers were used subsequently to analyse the differentiation of tissues within explants and growth factor-treated animal caps, or as a loading control (EF-1α). Expression patterns of all these markers are previously characterised, and the patterns of expression obtained are as predicted (see Table 1 for references). For each stage, total RNA was prepared from two embryos and subjected to RT-PCR analysis as detailed in the Materials and Methods. The following controls were performed: −RNA (no RNA was added to the cDNA synthesis, the product of which was then used in the subsequent PCR), −RT (a cDNA synthesis was performed, including RNA from a stage 33/34 embryo, in the absence of reverse transcriptase, the product of this reaction was used in the subsequent PCR), −cDNA (no cDNA synthesis was performed, water only was added to the subsequent PCR). 0.2, 0.4, 0.8, and 1.6 are controls with the implied proportions of stage 33/34 embryo cDNA added to four PCR reactions as controls to ensure that the PCR performed is in the linear region
Fig. 3. RT-PCR analysis of wraps shows that
glomus specification occurs at stage 12.5.
Explants removed from progressively earlier
stages of embryo from stage 29 down to stage 11
were cultured within ectodermal wraps and
assayed at stage 33/34 equivalent for the presence
of the glomus marker xWT1. The presence of this
glomus marker, and therefore specified glomustissue, can be seen in explants removed from all
stages from 29 to 12.5. At stages 13 and 12.5,
anterior (A) and middle (M) regions of mesoderm
explants contain xWT1 transcripts; however, the
posterior (P) explants do not, showing that in this
region of mesoderm the glomus is not specified.
No expression of xWT1 is observed in explants
dissected from stage 12 or stage 11. This data
clearly shows that glomus is specified between
stages 12 and 12.5. Absence of the expression of
xMHCα, a marker of hearttissue, in explants
removed from any stage confirms that the tissue
expressing xWT1 is indeed glomustissue. Xlim-1
expression, which marks three lineages including
the pronephros, is seen in explants of all stages
from 20 to 12.5. Expression of Xlim-1 is absent from stage 29. At this stage, it is possible to remove the developing pronephric tubules and duct before explanting the glomus. The absence of Xlim-1 transcripts therefore confirms that this dissection was performed accurately. Explants excised from stage 11 or 12 embryos did not express Xlim-1. This data concur with previous data that showed pronephric tubules are specified at stage 12. 5 (Brennan et al., 1998). Cardiac actin is expressed in explants removed at stages 11 and 12, thus proving that these ectodermal wraps did contain viable mesodermal tissue including somitetissue. The absence of xWT 1 expression in explants removed at these stages is not therefore due to the lack of viable explant within the ectodermal wraps, but to the absence of specified glomustissue. A no insert control was included consisting only of the ectodermal wrap. This sample was negative for all markers with the exception of EF-1α which is present within the animal caps that are used in the ectodermal wraps. Stage 33/34 embryos were used as a positive control. Other controls are as detailed for Fig. 2 above. For each sample, total RNA was extracted from 10 ectodermal wraps (or 2 control embryos) and subjected to RT-PCR analysis as detailed in the Materials and Methods above.
Fig. 4. Lineage-labelled orthotopic grafts from the intermediate mesoderm in the presumptive pronephric region contribute to the developing pronephros. Embryos were injected with the lineage label FLDX at the 1-cell stage and allowed to develop to stage 12.5. Grafts were taken from the middle intermediate mesoderm explant from stage 12.5 embryos, as described in Fig. 1, and inserted into host embryos of the same stage at an equivalent position. The grafted embryos were allowed to develop until stage 28 and then observed under a Leica MXFLIII UV dissecting microscope. Images were captured using a Leica DC100 video camera.
