spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 30 May 2007
doi: 10.1242/dev.005033

Development 134, 2501-2509 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Linton, J. M.
Right arrow Articles by Reichardt, L. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Linton, J. M.
Right arrow Articles by Reichardt, L. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The ECM protein nephronectin promotes kidney development via integrin {alpha}8ß1-mediated stimulation of Gdnf expression

James M. Linton1, Gail R. Martin2 and Louis F. Reichardt1,3,*

1 Department of Physiology, 1550 Fourth Street, University of California, San Francisco, San Francisco, CA 94143, USA.
2 Department of Anatomy and 1550 Fourth Street, University of California, San Francisco, San Francisco, CA 94143, USA.
3 Howard Hughes Medical Institute, 1550 Fourth Street, University of California, San Francisco, San Francisco, CA 94143, USA.


Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 1. Generation of a Npnt-null allele. (A-F) Targeting strategy for generating Npnt mutant alleles using a BAC containing part of the Npnt locus. (A) Representation of the modified BAC (273P10) used for targeting, showing the first 11 exons (vertical bars) of the Npnt gene present in this BAC. Boxed region spans exons 1 and 2. (B,C) Illustration of the modifications that were made to the BAC DNA, including insertion of loxP sites in the introns 5' and 3' to the first exon, an insertion of a neomycin expression cassette flanked by frt sites, and restriction sites (asterisk). (D) Representation of the Npntfloxneo allele, produced following homologous recombination between the modified BAC and the Npnt locus in ES cells. (E) Mice carrying Npntfloxneo were crossed to mice expressing CRE recombinase under the ß-actin promoter (Lewandoski et al., 1997Go) to create the Npnt{Delta}ex1 allele. Note that this allele still contains the neo cassette. (F) Southern blot of DNA from two ES cell clones, one heterozygous for the Npntfloxneo allele and the other wild-type at the Npnt locus. An EcoRI digest produces an 8 kb wild-type and a 4 kb mutant band. Each clone is represented by a series of three fourfold dilutions (left to right). (G) RT-PCR for Npnt and Gapdh expression in Npnt+/+, Npnt+/- and Npnt-/- mice. Total RNA was extracted from spleens of newborn mice using the RNeasy mini kit (Qiagen Inc., Valencia, CA), and reverse transcribed using Superscript II and oligo(dT)12-18 Primer (Invitrogen Corp., Carlsbad, CA). PCR was performed using forward and reverse primers that recognize sequences in Npnt exons 4 and 8, respectively, and primers that recognize a sequence in Gapdh exon 3. Control reactions without reverse transcriptase were negative for both PCR reactions (not shown). (H) Immunostain for nephronectin in kidneys from wild-type and Npnt{Delta}ex1 homozygous (null) newborn mice using an anti-nephronectin antibody that recognizes sequences in the C-terminal region of the protein (Brandenberger et al., 2001Go). Primers for wild-type allele, NpntWT, NN-1A: 5'-AGTCCATCCTGATCACTGGCT-3' and NN-1C: 5'-GCAACCTTCAGCGTCCC-3', band size 279 bp. Primers for mutant allele, Npnt{Delta}ex1, NN-1B: 5'-TATGGCTTCTGAGGCGGAAAGAAC-3' and NN-1F: 5'-AAGTGGAGCTTCAGGACACAG-3', band size 509 bp.

 

Figure 2
View larger version (88K):
[in this window]
[in a new window]

  		Fig. 2. Renal agenesis in Npnt-null mice. (A-C) Urogenital tracts from newborn female littermates, shown in wholemount. (A) Npnt+/+ urogenital tract including kidneys with adrenals, ureters, bladder and uterine horns. (B) Npnt-/- urogenital tract with unilateral kidney agenesis. Note that other than the absence of the right kidney and ureter (asterisk), the urogenital tract appears normal. The adrenal gland on the right is attached to the dorsal mesentery, and part of the dorsal aorta is present. (C) Npnt-/- urogenital tract with bilateral kidney agenesis (asterisks). Again, the rest of the urogenital tract appears normal. (D,E) Medial sections of Npnt+/+ and Npnt-/- newborn kidneys (scale bar, 1 mm). Insets show regions containing glomeruli (arrows) at higher magnification (scale bar, 100 µm), demonstrating that kidney development, including nephron formation, occurs in Npnt-/- kidneys. (F) Percentage of Npnt heterozygous and homozygous animals with two, one or no kidneys. The percentage agenesis was determined by dividing the number of kidneys [expected (2 per animal)-observed] by the number of kidneys expected. Ad, adrenal gland; Bl, bladder; DA, dorsal arota; Ki, kidney; Ur, ureter; Ut, uterus.

 

Figure 3
View larger version (80K):
[in this window]
[in a new window]

  		Fig. 3. Developmental origin of renal agenesis in Npnt-null mice. (A-G) Embryos at the stages indicated, immunostained in wholemount for Calbindin. (A) In the Npnt+/+ embryo at E11.0 the ureteric bud (arrowhead) has invaded the MM. (B) In the Npnt-/- embryo the UB (arrowhead) is similar to that in the wild-type embryo. (C) In the Npnt+/+ embryo at E11.5 the UB has branched (open arrowheads). (D) In the Npnt-/- embryo the UB (arrowhead) has not extended into the MM (asterisk) or branched. (E) In the Npnt+/+ embryo at E12.5 the UB has undergone several rounds of branching (arrowheads). (F,G) Npnt-/- embryos, showing the variable extent of branching at E12.5. (H-J) Transverse sections through E13.5 Npnt+/+ and Npnt-/- kidneys. (I,J) Left and right kidneys from one embryo. Note that metanephric fields have been invaded by the UB and nephron development is occurring, but the kidneys are smaller than normal in the Npnt-/- embryo. Nephrogenesis is occurring, but is less advanced than in the wild-type littermate. (K) Transverse section of an E13.5 Npnt-/- embryo through the region in which the kidney would normally develop. Note the bilateral kidney agenesis (arrows). (K') Boxed region in K is shown at higher magnification. Broken line demarcates the MM. Scale bars, 100 µm. Go, gonad; In, intestine; MM, metanephric mesenchyme; UB, ureteric bud.

 

Figure 4
View larger version (53K):
[in this window]
[in a new window]

  		Fig. 4. The basement membrane is normal in Npnt-null embryos during kidney development. (A-F) Transverse sections through E11.5 Npnt+/+ and Npnt-/- embryos stained with antibodies against laminin (LN) or Collagen IV (COL IV). (G-L) Transverse sections through E13.5 Npnt+/+ and Npnt-/- embryos stained with antibodies against LN, COL IV or fibronectin (FN). Note the similar staining patterns in mutant and wild-type embryos. Scale bars, 50 µm. ND, nephric duct; UB, ureteric bud.

 

Figure 5
View larger version (122K):
[in this window]
[in a new window]

  		Fig. 5. Gdnf expression is reduced in the Npnt-null embryonic kidney at E11.5 but is normal at E10.5 and E13.5. (A-P) Transverse sections through Npnt+/+ and Npnt-/- embryos. (A-H) Expression at E11.5 of the genes indicated, as detected (A-F) by in situ hybridization or (G,H) by immunostaining. (A,B) Note the apparent absence of Gdnf RNA in the MM of the mutant (demarcated by dotted circles), whereas the level of Gdnf expression appears comparable in the adjacent limb bud (arrows) in mutant and wild-type embryos. (C-F) Note that expression of the Eya1 and Six2 transcription factor genes is similar in mutant and wild-type MM. (G,H) PAX2 protein is detected in both the UB (solid arrowhead) and its branches (open arrowhead), as well as in the MM. Note the lack of invasion of the UB into the MM of the Npnt mutant (asterisk). (I-L) Expression at E10.5 and (M-P) at E13.5 of the genes indicated, as detected by in situ hybridization. Note that Gdnf and Eya1 expression appears comparable in Npnt+/+ and Npnt-/- embryos at these stages, although the mutant embryonic kidneys are smaller than normal at E13.5. Scale bars, 100 µm. Abbreviations as in previous figures.

 

Figure 6
View larger version (105K):
[in this window]
[in a new window]

  		Fig. 6. Gdnf expression is reduced in the Itga8-null embryonic kidney at E11.5 but is normal at E10.5 and E13.5. (A-L) Transverse sections through Itga8+/- and Itga8-/- embryos, showing gene expression as detected by in situ hybridization. (A,B) Note the apparent absence at E11.5 of Gdnf RNA in the MM of the Itga8-/- mutant (demarcated by dotted circles), whereas the level of Gdnf expression appears comparable in the adjacent limb buds (arrows) in Itga8+/- and Itga8-/- embryos. (C,D) Pax2 is expressed in both the MM and the UB (arrowhead). Note the lack of UB invasion in the Itga8-/- MM (asterisk). (E-L) Expression at E10.5 (E-H) and at E13.5 (I-L) of the genes indicated. Note that expression of Gdnf, Pax2 and Six2 is similar in Itga8+/- and Itga8-/- MM at these stages. Open arrowheads point to UB branches in the MM at E13.5. Scale bars: 100 µm. Abbreviations as in previous figures.

 

Figure 7
View larger version (58K):
[in this window]
[in a new window]

  		Fig. 7. Reducing Spry1 dosage in Itga8-null embryos rescues kidney development. (A) Graph illustrating the average number of kidneys per animal at birth in animals of the genotypes indicated. Note that the percent agenesis is significantly reduced in Itga8-/-;Spry1+/- versus Itga8-/-;Spry1+/+ animals (P<0.005; see legend to Table 1). The rescue of kidney development was complete in Itga8-/-;Spry1-/- animals. However, we found the proportion of Itga8-/-;Spry1-/- animals that demonstrated a duplicated ureter phenotype did not appear to differ from the proportion of their Spry1-/- littermates displaying that phenotype (Bason et al., 2005). (B,C) Transverse sections through embryonic kidneys of the genotypes indicated, stained with hematoxylin and eosin. Note the characteristic lack of invasion of the MM by the UB (arrowhead) at E11.5 in an Itga8-/-;Spry1+/+ embryonic kidney. The UB has invaded the MM at E11.5 in an Itga8-/-;Spry1+/- embryonic kidney. (D-G) Transverse sections through embryonic kidneys of the genotypes indicated, showing expression at E11.5 of Gdnf and Eya1, as detected by in situ hybridization. (D,E) Note the substantial reduction in the level of Gdnf expression in the MM of the Itga8-/-;Spry1+/- mutant compared with that in the control (Itga8+/-;Spry1+/-) embryo, despite invasion of the UB into the MM (arrowhead). Arrows point to Gdnf expression in the limb buds, which is similar in both genotypes. (F,G) Eya1 expression is similar in Itga8-/-;Spry1+/- and control (Itga8+/-;Spry1+/-) embryos. Note invasion of the UB into the MM of the Itga8-/-;Spry1+/- and control (Itga8+/-;Spry1+/-) embryos (arrowheads). Scale bars: 50 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007