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Data from gene ablation studies in mice have indicated critical roles for Lim-1, Wnt4, WT-1, and Pax-2 in the coordination and execution of kidney patterning and differentiation. However, the precise roles of these molecules, their ordering within a genetic hierarchy, and the manner in which they contribute to establishing the fates of cells of each of the components of the nephron have yet to be elucidated in any system. In this report, the temporal and spatial expression patterns of these genes within the Xenopus pronephric system were examined in detail by single- and double-probe in situ hybridization. We describe restrictions of these gene expression patterns within the pronephros which indicate a model for the partitioning of the common pronephric anlage into its three component parts--the tubules, the glomus, and the duct.
Fig. 1. Analysis of gene expression patterns in the developing
pronephros by whole-mount single-probe in situ hybridization. A
developmental series of embryos was hybridized with Xwnt-4 (A-C),
Xlim-1 (D-F), xWT-1 (G-I), and XPax-2 (J-L). In all cases, anterior is to
the right and dorsal is up. Stages shown are early tailbud (stage 23, A,
D, G, J), mid- tailbud (stage 26, B, E, H, K), and tadpole (stage 33, C, F,
I, J). Note that Xwnt-4 is expressed only in the anteriodorsalmost cells
of the anlage by stage 23. This expression corresponds to the distal tip
of the tubules at later stages (arrow in C). Xlim-1 is initially expressed
throughout the pronephric anlage including the pronephric tubules
and duct (arrowhead in F) and refines to the distal tips at late tadpole
stages (arrow in F). xWT-1 is expressed in a similar pattern to that of
Xwnt-4 at early tailbud stages. However, the two patterns are
drastically different at later stages (compare staining marked by
arrow in C-I). XPax-2 is expressed at low levels in the pronephric
anlage at early tailbud stages and maintained in the tubules and duct
(arrowhead in L) through later stages of development with strongest
expression in the distal portion of the three tubules (arrow in L).
Fig. 2. Comparison of expression patterns. Gene expression patterns
were directly compared through whole-mount and histological
examination of singly and doubly probed in situ hybridized embryos.
In frames A-I, dorsal is at top and lateral is at left. For reference, the
lateral margin of the endoderm is indicated by a black line in panels A
and E. A-D: Transverse sections through the pronephric anlage of
stage 22 embryos singly probed with Xwnt-4 (A), xWT-1 (B), Xlim-1
(C), or XPax-2 (D). E: Transverse sections through the pronephric
anlage of a stage 26 embryo doubly probed with Xwnt-4 (blue) and
xWT-1 (pink). e’: A more anterior transverse section through the same
embryo as shown in E. Note the overlap of expression domains of
Xwnt-4 and xWT-1 (arrow, purple staining). F-H: Transverse sections
of pronephric anlagen at stage 28. f’-h’: Anterior sections through the
same embryos. F, f’: Double probe with Xwnt-4 (blue) and xWT-1
(pink). G, g’: Double probe with Xlim-1 (blue) and xWT-1 (pink). H, h’:
Double probe with XPax-2 (purple) and xWT-1 (pink). I: Transverse
section through the pronephros at stage 30 doubly probed with XPax-2
(blue) and xWT-1 (pink) showing that the expression patterns are
entirely mutually exclusive at this stage. J: Lateral view of a
whole-mount stage 26 embryo probed with XPax-2 and Xlim-1. The
purple represents overlap of expression of the two genes within the
duct (blue arrow) and the tubules (black arrow). The light blue (red
arrow) represents cells that are expressing only XPax-2.
Fig. 3. Temporal expression of
genes within pronephric components.
The expression patterns of
Xlim-1, Xwnt-4, xWT-1, and XPax-2
were analyzed and compared by
whole-mount in situ hybridization
and histology over a developmental
time course. The onset of expression
in the pronephros as well as
the expression in the different layers
of the anlage and the different
components of the pronephric kidney
are shown.
Fig. 4. Summary of gene expression patterns within the pronephros.
Circles represent the pronephric anlage at various stages of
development. Dorsal is up, ventral is down, lateral is left and medial is
right. Black represents high levels of gene expression, gray represents
lower levels, and white represents no expression. The circle is divided
into the three components of the pronephros, the tubules coming from
the upper left quarter, the duct from the lower left quarter, and the
glomus from the right half. The small black triangles within the stage
38 anlage represent the distal tubules. Xlim is activated throughout
the entire anlage at stage 14. Its expression leads to the expression of
xWnt-4 within the entire pronephric anlage and xWT-1 in the medial
half ( the cells in the embryo that are adjacent to the endoderm) at
stage 18. Expression of xWT-1 represses Xlim1 and xWnt-4 and
prevents XPax-2 from ever being expressed in the medial portion of
the anlage. A late signal from the somites maintains high levels of
Wnt-4, Xlim, and XPax-2 in the distal tips of the tubules. Absence of this
signal in the more ventral part of the anlage results in a decrease in Wnt-4
expression and thus indirectly (or directly) leads to the duct fate.
