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Pre-pattern in the pronephric kidney field of zebrafish

Cardiovascular Research Center and Developmental Biology Laboratory, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02129, USA

View larger version (92K): [in a new window]   Fig. 1. Expression of wt1 in the developing kidney. wt1 first appears as bilateral stripes in the intermediate mesoderm at the two- to three-somite stage. (A) An eight-somite stage embryo showing wt1 expression in the anterior intermediate mesoderm. (B) Co-staining for alpha-tropomyosin indicates that the anterior border lies at the level of the first somite and extends caudally to the level of the fourth somite. (C) Two-color in situ hybridization with wt1 (orange) and pax2.1 (purple) labeling the intermediate mesoderm; the anterior-most cells express only wt1 (arrow). Posteriorly, we find a cell population that expresses both wt1 and pax2.1. (D) At the 11-somite stage, wt1 expression medial to the two original stripes is observed (arrow). (E,F) wt1 staining in pronephric cell aggregates ventral to the somites at the 23 somite stage in (E, arrow) and shown in cross-section in F. (G,H) Expression of wt1 is observed in the paired pronephric primordia at the 36 hpf stage (G), shown in cross-section in H. (I) wt1 (orange) localizes to the glomerulus while pax2.1 (purple) marks the pronephric tubule and anterior duct at the 48 hpf stage, a higher magnification of the same photograph is shown in J. (K) Cross-sections of a 48 hpf embryo with midline glomerular wt1 staining ventral to the dorsal aorta. (L) Co-staining with wt1 (orange) and pax2.1 (purple) at 76 hpf reveals that while wt1 still localizes to the glomerulus, pax2.1 expression is only observed in the tubules, a higher magnification of the same photograph is shown in M. (N) wt1-stained 76 hpf embryo shown in cross-section, note the absence of wt1 transcripts in the pronephric tubule and the restriction to the glomerulus. Anterior is towards the left in all panels except D, where it is at the top of the panel. (A,B,D,E,G) Dorsal views; (C) Lateral view; (I,J,L,M) Dorsolateral views. g, glomerulus; pp, pronephric primordia; pt, pronephric tubule. Arrowhead in E indicates where the wt1 stripes begin to fade.

View larger version (80K): [in a new window]   Fig. 2. Nephric expression of sim1. (A) Dorsal view showing sim1 expression in the intermediate mesoderm. This is first evident at the two-somite stage. (B) Double in situ hybridization using sim1 and wt1 probes indicates that the posterior limit of wt1 expression coincides with the anterior limit of sim1 expression (arrow). (C) The anterior limit of sim1 expression lies at the level of somite four or five. Similar labeling experiments with pax2.1 (D) and pax8 (E) reveal that their anterior limit lies at the level of somite two. (F) sim1 continues to be expressed in the intermediate mesoderm and the ventrolateral portion of the somite throughout the segmentation period. (A,C,D,E) Dorsal views; (B) Lateral view; (F) Dorsolateral view. Anterior is towards the left in all panels. im, intermediate mesoderm; s, somite.

View larger version (58K): [in a new window]   Fig. 3. Origin of glomerular and tubular precursors. (A-D) Time lapse analysis of a single embryo labeled at the eight-somite stage at the level of the second somite. (A) A phase image; (B) the corresponding fluorescence image – arrows indicate the labeled patch. (C) Fluorescence image indicating that cells have started to move in by the 12-somite stage. However, by the 18-somite stage, the cells have become difficult to visualize using fluorescence microscopy (D). In order to detect these cells in older embryos, we have used an anti-fluorescein antibody coupled to a peroxidase and stained with DAB. (E) Whole-mount DAB-stained 48 hpf embryo showing that cells labeled at the level of the first somite give rise to the glomerular segment of the pronephros. (F) Cells labeled at the level of the third somite can give rise to the pronephric tubule. Anterior is towards the left in all panels. hb, hindbrain; g, glomerulus; pt, pronephric tubule; ov, otic vesicle.

View larger version (134K): [in a new window]   Fig. 4. Histological analysis of labeled embryos. All panels are cross-sections of DAB-stained embryos. (A,B) The intermediate mesoderm lateral to the first somite contains progenitors of the glomerulus (A), shown in higher magnification in (B). (C,D) Labeled cells lateral to the second somite can give rise to tubular cells (C), shown in higher magnification in (D). g, glomerulus; gt, gut; n, notochord; nt, neural tube; pt, pronephric tubule; s, somite.

View larger version (77K): [in a new window]   Fig. 5. The nephric duct forms by in situ differentiation of intermediate mesoderm cells. (A) Phase image of an 11-somite stage embryos, the arrow indicates the intermediate mesoderm adjacent to the somites. (B) Fluorescence image of the same embryo in A after the labeling of a patch of cells at the level of somite 9. Embryos at the 48 hpf stage where cells were labeled in the undifferentiated IM (C) or the growing duct (D) at the 10-somite stage, give rise to the nephric duct. Shown here are two typical examples of labeled duct segments, note that the label is continuous, and is the width is never greater than the width of one somite. (E) Cross-section of an embryo with a labeled patch in the duct. Anterior is towards the left in A-D. n, notochord; nt, neural tube; pd, pronephric duct; s, somite.

View larger version (99K): [in a new window]   Fig. 6. Regulative development of the zebrafish pronephros. Glomerular and tubular precursors in the intermediate mesoderm were ablated (arrow in A). Embryos were fixed immediately after unilateral (left side) ablation and processed for in situ hybridization with a probe for wt1 (B). All of the wt1 stripe is missing from the operated side (arrow). After 2 hours of recovery time, wt1 expression returns to normal (arrow in C). (D) Histology of a 48 hpf embryo that had glomerular progenitor cells unilaterally ablated at the 9-somite stage. A normal midline glomerulus and tubules are clearly visible. (E) A higher magnification of the section in (D). (F) Unoperated control side of the same embryo in (D,E). Anterior is towards the left in A-C; E-G are cross-sections. g, glomerulus; gt, gut; n, notochord; nt, neural tube; pt, pronephric tubule; s, somite.

View larger version (24K): [in a new window]   Fig. 7. Pre-patterning in the intermediate mesoderm directs pronephric kidney cell fate. (A) Expression patterns of wt1 (orange), pax2.1 (blue) and sim1 (brown) at the eight- to ten-somite stage. The notochord (N) and somites (S) are shown as axial references. Glomerular progenitors, bordered by yellow lines, lie in the anterior-most nephrogenic mesoderm and express wt1; tubule progenitors, bordered by green lines, are found caudally in a zone that expresses both wt1 and pax2.1; and duct cells, horizontal lines, develop from cells that express pax2.1 and sim1. (B) Diagram of the mature pronephric kidney (unilateral) indicating the final position of the segment. The midline glomerulus shown in yellow, the tubule drawn in green and the nephric duct diagrammed using horizontal lines. Anterior is at the top of the figure.