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Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development

¹ Division of Stem Cell Regulation, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
² Laboratory of DNA Biology and Embryo Engineering, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
³ Department of Life Sciences, The University of Tokyo, Tokyo 153-8902, Japan
⁴ Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA
⁵ Department of Pathology, Amgen, Thousand Oaks, CA 91320, USA
^* Author for correspondence (e-mail: ryuichi{at}ims.u-tokyo.ac.jp )

View larger version (56K): [in a new window]   Fig. 1. Sequence and chromosomal localization of Sall1. (A) Amino acid sequence alignment of Sall1 and SALL1. Asterisks indicate cystine and histidine residues in zinc-finger motifs. Underline indicates a putative nuclear localization signal. Black triangles are exon-intron boundaries. (B) Sall1 maps in the central region of mouse chromosome 8. Sall1 was placed on mouse chromosome 8 by interspecific backcross analysis. The segregation patterns of Sall1 and flanking genes in 166 backcross animals that were typed for all loci are shown on the left of the figure. For individual pairs of loci, more than 166 animals were typed. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J x M. spretus) F1 parent. The shaded boxes represent the presence of a C57BL/6J allele and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 8 linkage map showing the location of Sall1 in relation to linked genes is shown on the right of the figure. Recombination distances between loci in centimorgans are shown on the left of the chromosome and the positions of loci in human chromosomes, where known, are shown on the right. GenBank Accession Number for Sall1, AB051409.

View larger version (28K): [in a new window]   Fig. 2. Generation of Sall1-deficient mice. (A) Targeting strategy of Sall1-del. Positions of zinc finger motifs are indicated as ovals. (B) Targeting strategy of Sall1-lacZ. (C) Southern blot analysis of wild-type (+/+), heterozygous (+/-), homozygous (-/-) Sall1-del mutant mice. Tail DNA was digested with BamHI and hybridized with probe B. (D) Genomic PCR of wild-type (+/+), heterozygous (+/-), homozygous (-/-) Sall1-del mutant mice. The 420 bp band was amplified from neor gene and the 200 bp band was from Sall1 genome. The positions of the PCR products are indicated as gray bars. (E) RT-PCR of Sall1 transcript in wild-type (+/+), heterozygous (+/-), homozygous (-/-) Sall1-del mutant mice.

View larger version (59K): [in a new window]   Fig. 3. Expression of Sall1 in developing embryos. (A-G,I) X-gal staining and (H,J) in situ hybridization of Sall1-lacZ heterozygous mice. (A) Lateral view of embryo at 11.5 dpc. (B) Ventral view of embryos at 14.5 dpc. (C) Otic vesicle at 10.5 dpc (ov, otic vesicle). (D) Heart at 11.5 dpc. (E) Nephrogenic primordium at 10.5 dpc (W, Wolffian duct). (F) Mesonephros at 11.5 dpc (t, mesonephric tubule). (G) Metanephros at 11.5 dpc (mm, metanephric mesenchyme; ub, ureteric bud). (H) Metanephros at 11.5 dpc (in situ hybridization). (I) Metanephros at 14.5 dpc (g, glomerulus). (J) Metanephros at 14.5 dpc (in situ hybridization) (c, comma-shaped bodies). Scale bars: 100 µm.

View larger version (111K): [in a new window]   Fig. 4. Kidney phenotypes in Sall1-deficient mice. (A) Kidneys of wild-type newborn. Urinary bladder is filled with urine. (B) Kidneys of Sall1-deficient newborn. Note kidneys are completely absent and the urinary bladder is not inflated with urine. Other organs, such as adrenal glands and testis, are normal. (C) Kidneys of another Sall1-deficient newborn with severe bilateral kidney hypoplasia. Urine is absent from the bladder. (D,E) Histological examination of kidney in wild-type mice. (F,G) Histological examination of hypoplastic kidney in Sall1-deficient mice. a, adrenal gland; bl, urinary bladder; k, kidney; t, testis. Scale bars: 100 µm

View larger version (151K): [in a new window]   Fig. 5. Kidney development in Sall1-deficient mice. (A) Metanephros in wild-type mice at 11.5 dpc. Ureteric bud (ub) branches from Wolffian duct (W) and metanephric mesenchyme (mm) are condensed around the bulging ureteric bud. (B) Metanephros in Sall1-deficient mice at 11.5 dpc. Metanephric mesenchyme is formed but reduced in size and is not invaded by the ureteric bud. (C) Metanephros in Sall1-deficient mice at 11.5 dpc. The left ureteric bud invades the mesenchyme, but does not bulge. (D) Metanephros in wild-type mice at 12.5 dpc. Branching of ureter is evident. (E) Metanephros in Sall1-deficient mice at 12.5 dpc. No kidney is detected. (F) Metanephros in Sall1-deficient mice at 12.5 dpc. Ureteric bud is observed in the left primordium, but no branching occurs. (G) Metanephros in Sall1-deficient mice at 14.5 dpc (k). Cortex and medullar regions are formed and glomeruli are observed. (H) Metanephros in Sall1-deficient mice at 14.5 dpc. Kidney is absent though gonads (g) are present. (I) Metanephros in Sall1-deficient mice at 14.5 dpc. A small kidney with poorly branched tubules and ducts is observed on the right. (J) TUNEL analysis of metanephros in wild-type mice at 11.5 dpc. TUNEL analysis of metanephros in Sall1-deficient mice at 11.5 dpc. Note the yellow apoptotic cells in the mesenchyme. Scale bars: 100 µm

View larger version (92K): [in a new window]   Fig. 6. In situ hybridization of molecular markers in 11.5 dpc metanephros of wild-type (left columns, A,C,E,G,I,K) and Sall1-deficient mice (right columns, B,D,F,H,J,L). (A,B) Pax2, (C,D) Ret, (E,F) GDNF, (G.H) BMP7, (I.J) Wnt4 and (K,L) WT-1. Scale bars: 100 µm.

View larger version (81K): [in a new window]   Fig. 7. Organ culture of Sall1-deficient metanephric mesenchyme. (A) Heterozygous metanephric rudiment cultured for 3 days. (B) Sall1-deficient metanephric rudiment cultured for 3 days. Only residual ureter is evident. (C) Sall1-deficient metanephric rudiment cultured for 3 days. A limited branching is observed. (D) Heterozygous metanephric mesenchyme cultured with spinal cord for 5 days. (E) Sall1-deficient metanephric mesenchyme cultured with heterozygous spinal cord for 5 days. sp, spinal cord; tb, renal tubules. Scale bars: 100 µm.

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