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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01858

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Distinct and sequential tissue-specific activities of the LIM-class homeobox gene Lim1 for tubular morphogenesis during kidney development

¹ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030, USA
³ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
⁴ Department of Urology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA

View larger version (61K): [in a new window]   Fig. 1. Hypomorphic phenotypes in BAC transgene-rescued Lim1 mutants. (A) Schematic diagram of mouse BAC clones around the Lim1 locus. (Adapted, with permission, from Celera Discovery System.) (B) Breeding scheme to generate BAC transgene-rescued Lim1 mutants. (C) PCR genotyping of neonates. Because two different null alleles of Lim1 (Lim1neo and Lim1lz) were used, BAC transgene-rescued Lim1 mutants were easily identified by PCR genotyping. (D-Q) Phenotypes of Lim1-null (D,H), BAC transgene-rescued Lim1 mutants for line 5 (E,I,L,O) and line 7 (F,J,M,P), and wild-type (G,K,N,Q) mice at birth. (D-G) Lateral view of the head region. Insets in D and E show frontal views. Arrowheads indicate ears. (H-K) Ventral view of urogenital system of female neonates. Arrowheads indicate connective tissues that lack the uterus, which is removed and not shown in H. (L-N) Hematoxylin and Eosin-stained metanephros. (O-Q) High magnification of the cortical region of the metanephros in L-N, respectively. (R-Y) Whole-mount in situ hybridization with a Lim1 probe at E12.5 of the metanephros (R-U) and urogenital ridges (V-Y) from BAC transgene-rescued Lim1 mutants (Line 7) (R,S,V,W) and wild-type (T,U,X,Y). (S,U,W,Y) High-magnification images in R, T, V and X. a, adrenal; cd, collecting duct; cm, condensed mesenchyme; g, gonad; ge, genitalia; gl, glomerulus; k, kidney; md, Müllerian duct; np, nephron progenitor; ov, ovary; tu, tubules of nephron; ub, ureteric bud; ur, ureter; ut, uterus.

View larger version (90K): [in a new window]   Fig. 2. Nephric duct epithelium- and metanephric mesenchyme-specific Cre reporter analysis. lacZ staining of Hoxb7tg/+; R26R+/– (A,C,E,G,I,K,M,O,Q,S,U,W) and Rarb2tg/+; R26R+/– (B,D,F,H,J,L,N,P,R,T,V,X) mice. (A,B) Whole-mount embryo at E9.5. (C,D) Cross-section of mesonephric region at E9.5. (E,F) Whole-mount embryo at E10.5. Hoxb7-Cre activity was also observed in the dorsal root ganglia and spinal cord (Yu et al. 2002). (G,H) Anterior region of the nephric duct at E10.5. (I,J) Posterior region of the nephric duct at E10.5. (K,L) Sagittal section around the posterior nephric duct region at E10.5. (M,N) Dorsal view of the urogenital system at E12.5. Mosaic Hoxb7-Cre and Rarb2-Cre reporter activity was observed in the adrenal gland. (O,P) Cross-section of the metanephros at E12.5. (Q,R) Cross-section of the posterior reproductive tracts. Rarb2-Cre reporter activity was mosaic in the mesenchyme of the posterior reproductive tracts. (S,T) Whole-mount urogenital system at E14.5. (U,V) Tip of ureteric bud in the cortical region of the metanephros at E14.5. (W,X) High magnification of the glomerulus at E14.5. a, adrenal; ca, caudal mesonephros; cb, comma-shaped body; cd, collecting duct; cm, condensed mesenchyme; cr, cranial mesonephros; cs, cortical (outer) stroma; dg, dorsal root ganglia; ey, eye; fl, forelimb; gc, glomerular capillaries; gl, glomerulus; gn, gubernaculum. gu, gut; hl, hindlimb; k, kidney (metanephros); lpm, lateral plate mesoderm; md, Müllerian duct; mm, metanephric mesenchyme; ms, medullary (inner) stroma; mt, mesonephric tubule; nd, nephric duct; nm, nephric mesenchyme; np, nephron progenitor; nt, neural tube; ob, outer layer of Bowman's capsule; ov, ovary; pa, pretubular aggregate; pc, podocyte; sb, S-shaped body; tu, tubules of nephron; ub, ureteric bud; ur, ureter; wd, Wolffian duct.

View larger version (58K): [in a new window]   Fig. 3. Urogenital abnormalities in tissue-specific knockout of Lim1 in the nephric duct epithelium or nephric mesenchyme. (A-C) Lim1 expression in the metanephros of tissue-specific Lim1 mutants at E12.5. Nephric duct epithelium-specific Lim1 knockout mice, Hoxb7Cretg/+; Lim1lz/flox (B), and metanephric mesenchyme-specific Lim1 knockout mice, Rarb2Cretg/+; Lim1lz/flox (C). Lim1lz/flox (A) is shown for comparison. (D-F) Histological sections of A-C, respectively. (G-R) Phenotypes of the urogenital system of Lim1lz/flox (G,J,M,P), Hoxb7Cretg/+; Lim1lz/flox (H,K,N,Q) and Rarb2Cretg/+; Lim1lz/flox (I,L,O,R) neonates. Males (G-L) and females (M-R). (G-I) Ventral view of the urogenital system in male neonates. (J-L) Lateral view of the testicular region of male neonates. Black arrows in K indicate residual tissue of male reproductive tracts. (M-O) Ventral view of the urogenital system in female neonates. (N) Black arrow indicates hydronephrosis and the black arrowhead hydroureter. (P-R) High magnification of the anterior female reproductive tract region. Black arrows in Q indicate residual tissue of the uterus. a, adrenal; ed, epididymis; ge, genitalia; k, kidney (metanephros); np, nephron progenitor; od, oviduct; ov, ovary; t, testis; ub, ureteric bud; ur, ureter; ut, uterus; vd, vas deferens.

View larger version (35K): [in a new window]   Fig. 4. Abnormal development of the reproductive tract in nephric duct epithelium-specific Lim1 knockout mutants. (A-F) Lim1-lacZ expression in Lim1lz/+ (A,D), Lim1lz/neo (B,E) and Hoxb7Cretg/+; Lim1lz/flox (C,F) embryos at E9.5. (A-C) Lateral view of whole embryos. (D-F) Cross-sections of A-C, respectively. White arrows in B and E indicate disorganized Lim1-lacZ-positive cells in Lim1-null mutants, which form the epithelial tubule of the nephric duct in control (A,D) and Hoxb7Cretg/+; Lim1lz/flox (C,F) mice. (G-R) Lim1-lacZ expression in developing urogenital organs of Lim1lz/flox (G,I,K,M,O,Q) and Hoxb7Cretg/+; Lim1lz/flox (H,J,L,N,P,R). (G-J) E11.5. (G,H) Lateral view of the urogenital system. (I,J) Ventral view of the mesonephros. White arrows in H and J indicate the degenerating nephric (Wolffian) duct. (K-N) E12.5. (K,L) Ventral view of the urogenital system. (M,N) Ventral view of the anterior mesonephros. Black arrowheads indicate the posterior end of the Müllerian duct; white arrow and arrowheads indicate gap in the Wolffian duct epithelium that inhibits or does not inhibit Müllerian duct elongation, respectively. (O,P) Ventral view of the left half of the urogenital system at E13.5. White arrow indicates gap in the Müllerian duct epithelium. (Q,R) Lateral view of the testis region at birth. White arrows indicate residual epithelium of the male reproductive tract. ed, epididymis; g, gonad; k, kidney (metanephros); lpm, lateral plate mesoderm; m, mesonephros; md, Müllerian duct; mt, mesonephric tubule; nd, nephric duct; nm, nephric mesenchyme; sm, somite; t, testis; ub, ureteric bud; vd, vas deferens; wd, Wolffian (nephric) duct.

View larger version (68K): [in a new window]   Fig. 5. Analysis of metanephros development in nephric duct epithelium- and metanephric mesenchyme-specific Lim1 knockout mutants. Metanephros of nephric duct epithelium-specific Lim1 knockout mice, Hoxb7Cretg/+; Lim1lz/flox (B,F,J,N,R) and metanephric mesenchyme-specific Lim1 knockout mice, Rarb2Cretg/+; Lim1lz/flox (C,G,K,O,S). Lim1lz/flox (A,E,I,M,Q), Wnt4–/– (D,H) and Wnt4–/–; Lim1lz/+ (L,P,T) mice are shown for comparison. (A-P) Metanephros at birth, except for L and P (E14.5). (A-D) Hematoxylin and Eosin stained coronal sections. (E-H) High magnification of the cortical regions in A-D, respectively. (I-L) Ventral view of Lim1-lacZ-stained whole-mount metanephros. (M-P) Section of cortical region in I-L, respectively. White arrowheads in O indicate Lim1-lacZ-positive cells in degenerating renal vesicles. (Q-T) Lim1-lacZ stained whole-mount metanephros at E12.5. a, adrenal; cb, comma-shaped body; cd. collecting duct; cm, condensed mesenchyme; gl, glomerulus; m, medulla; np, nephron progenitor; pa, pretubular aggregate; rv, renal vesicle; sb, S-shaped body; st, stalk; tu, tubules of the nephron; ub, ureteric bud.

View larger version (40K): [in a new window]   Fig. 6. Molecular expression analysis of nephric duct epithelium-specific Lim1 knockout mutants. Lim1lz/flox (A,M), Hoxb7Cretg/+; Lim1flox/+ (C,E,G,I,K,O) and Hoxb7Cretg/+; Lim1lz/flox (B,D,F,H,J,L,N,P) mice. (A-J) Lateral view of posterior nephric duct. (A,B) Lim1-lacZ expression at ts6. (C-J) Whole-mount in situ hybridization at ts9 with probes for Ret (C,D), Wnt11 (E,F), Gdnf (G,H) and Pax2 (I,J). Arrows indicate ureteric bud; arrowheads indicate metanephric mesenchyme. (K-P) Dorsal view of the metanephros at ts18 with Ret (K,L), Lim1-lacZ (M,N) and Wnt11 (O,P) expression. Arrows indicate ureteric bud tips.

View larger version (55K): [in a new window]   Fig. 7. Molecular expression analysis of metanephric mesenchyme-specific Lim1 knockout mutants. Whole-mount in situ hybridization of the metanephros of Rarb2Cretg/+; Lim1flox/+ (A,C,E,G,I,K,M) and Rarb2Cretg/+; Lim1lz/flox (B,D,F,H,J,L,N) embryos at E13.5 with Wnt4 (A-D), Dll1 (E-H), Brn1 (I,J), Wnt9b (K,L) and Bf2 (M,N) probes. (A,B,E,F,K-N) Ventral view of the metanephros. (C,D,G-J) Cryosection of the cortical region of the metanephros. Broken lines indicate renal vesicle; white arrowheads in C and D indicate Wnt4-expressing pretubular aggregate cells that do not form the renal vesicle; black arrowheads in E indicate Dll1 upregulation in differentiated nephron progenitors. np, nephron progenitor; sr, stroma; ub, ureteric bud.

View larger version (84K): [in a new window]   Fig. 8. Chimera analysis of Lim1 functions in kidney development. Chimeric mice composed of wild-type and Lim1+/+; Rosa26tg/+ (A,C,E,G,I,L,O,Q) or Lim1–/–; Rosa26tg/+ (B,D,F,H,J,K,M,N,P,R,S,T) cells. (A,B) Cross-sections at E9.5. Dorsal is towards the top and ventral towards the bottom. (C-F) Sagittal sections of the mesonephros (C,D) and metanephros (E,F) regions at E10.5. Anterior is leftwards, posterior rightwards, dorsal is towards the top and ventral is towards the bottom. (G-T) Developing nephrons at E18.5. (G,H) Pretubular aggregate. (I-K) Renal vesicle. Early (J) and late (K) stages. Lim1–/– cells are excluded in a subregion of the renal vesicle (white arrow in K). (L-N) Comma-shaped body. Early (M) and late (N) stages. White arrows indicate the invaginating epithelium of the first cleft. Lim1–/– cells are also excluded from a part of the comma-shaped body at the opposite side of the epithelial invagination (white arrowhead in N). (O,P) S-shaped body. White arrow indicates invaginated epithelium originally formed; white arrowhead indicates newly formed invagination. (Q-T) Glomerulus (proximal regions of the developing nephron indicated by boxes in O and P). Early (R), middle (S) and late (Q,T) stages. White arrows indicate the developing podocyte where Lim1–/– cells are excluded; white arrowheads indicate the outer epithelium of Bowman's capsule where Lim1–/– cells can contribute. cb, comma-shaped body; cm, condensed mesenchyme; gc, glomerular capillaries; gu, gut; lpm, lateral plate mesoderm; mm, metanephric mesenchyme; mv, mesonephric vesicle; nd, nephric duct; nm, nephric mesenchyme; nt, neural tube; pa, pretubular aggregate; pc, podocyte; rv, renal vesicle; sb, S-shaped body; ub, ureteric bud.