An E box comprises a positional sensor for regional differences in skeletal muscle gene expression and methylation

AbstractTo dissect the molecular mechanisms conferring positional information in skeletal muscles, we characterized the control elements responsible for the positionally restricted expression patterns of a muscle-specific transgene reporter, driven by regulatory sequences from the MLC1/3 locus. These sequences have previously been shown to generate graded transgene expression in the segmented axial muscles and their myotomal precursors, fortuitously marking their positional address. An evolutionarily conserved E box in the MLC enhancer core, not recognized by MyoD, is a target for a nuclear protein complex, present in a variety of tissues, which includes Hox proteins and Zbu1, a DNA-binding member of the SW12/SNF2 gene family. Mutation of this E box in the MLC enhancer has only a modest positive effect on linked CAT gene expression in transfected muscle cells, but when introduced into transgenic mice the same mutation elevates CAT transgene expression in skeletal muscles, specifically releasing the rostral restriction on MLC-CAT transgene expression in the segmented axial musculature. Increased transgene activity resulting from the E box mutation in the MLC enhancer correlates with reduced DNA methylation of the distal transgenic MLC1 promoter as well as in the enhancer itself. These results identify an E box and the proteins that bind to it as a positional sensor responsible for regional differences in axial skeletal muscle gene expression and accessibility

Kidney Development in the Absence of Gdnf and Spry1 Requires Fgf10

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf−/−;Spry1−/− or Ret−/−;Spry1−/− double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf−/−;Spry1−/− mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals

Kidney development in the absence of Gdnf and Spry1 requires Fgf10.

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf-/-;Spry1-/- or Ret-/-;Spry1-/- double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf-/-;Spry1-/- mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals

Model: GDNF and FGF10 cooperate to promote ureteric bud branching morphogenesis, via <i>Etv4</i> and <i>Etv5</i>, while Sprouty1 regulates signaling downstream of both RET and FGFR2.

<p>(A) In wild-type, GDNF/RET signaling plays a major role and FGF10/FGFR2 a minor role in promoting UB outgrowth and branching morphogenesis. The response to these signals is modulated by SPRY1, leading to a normal kidney at birth (right panel). The transcription factors ETV4 and ETV5 are downstream effectors of GDNF and FGF10 signaling. (B) In the absence of GDNF, there is presumably less SPRY1 produced <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000809#pgen.1000809-Basson1" target="_blank">[17]</a> (indicated by smaller text), but FGF10 is insufficient to overcome negative regulation by SPRY1, causing reduced downstream signaling to induce UB budding and branching (indicated by thinner arrows), one manifestation of which is a severe reduction in <i>Etv4/Etv5</i> expression <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000809#pgen.1000809-Lu1" target="_blank">[8]</a>. Consequently, renal agenesis or severe hypodysplasia is observed. (C) When GDNF and SPRY1 are both absent, the lack of negative regulation of signaling by FGFR2 allows for <i>Etv4/Etv5</i> expression, UB branching, and kidney development; however, the pattern of UB branching is altered, suggesting a unique role of GDNF in this process. (D) When FGF10 and GDNF are both absent, there is too little RTK signaling, even in the absence of negative regulation by SPRY1, to allow UB outgrowth from the Wolffian duct, resulting in renal agenesis (whether <i>Etv4/Etv5</i> would be expressed is not known, as there is no ureter or kidney to analyze). (E) Renal agenesis in <i>Etv4−/−</i>;<i>Etv5−/−</i> mice is not rescued by loss of <i>Spry1</i>, showing that increased RTK signaling is insufficient for kidney development in the absence of <i>Etv4</i> and <i>Etv5</i> (dashed arrow). The observation that ureters develop in <i>Etv4;Etv5;Spry1</i> triple mutants suggests that UB outgrowth, but not later branching, can occur independently of <i>Etv4/Etv5</i>. Insets in <b>a</b> and <b>c</b> show the pattern of branching UB tips in stage P0 wild-type and double mutant kidneys.</p

<i>Fgf10</i> is required for ureter and kidney development in the absence of <i>Gdnf</i> and <i>Spry1</i>.

<p>(A) Frequency of absence of the UB at E12.5 and renal agenesis at P0, in <i>Gdnf−/−</i>;<i>Spry1−/−</i> mice with 0, 1, or 2 <i>Fgf10</i> null alleles. (B) Example of normally branched wild-type UB at E12.5. (C) <i>Gdnf−/−</i>;<i>Spry1−/−</i> UB with moderately reduced branching at E12.5. (D) Absence of the UB in a <i>Gdnf−/−</i>;<i>Spry1−/−</i>;<i>Fgf10+/−</i> embryo at E12.5. n = number of (potential) kidneys.</p

Numerous nephron and normal nephron–UB connections are observed in double mutant kidneys.

<p>(A,B) Podocalyxin staining of nascent glomeruli in wild-type (A) and <i>Ret−/−;Spry1−/−</i> (B) kidneys at P0, showing numerous, cortically located nephrons in the double mutant, as in the wild-type. (C–H) Six optical sections at different Z-levels of a <i>Gdnf−/−</i>;<i>Spry1−/−</i> E15.5 kidney carrying <i>Hoxb7/myrVenus</i>. The sites where the UB connects to nephrons are visible as “holes” in the myrVenus-labeled UB, as the connecting tubule expresses little or no myrVenus. The connections of three nephrons (1, 2, 3) can be followed at different levels of the image stack. (I) Volume rendering of a wild-type kidney, with nephron connection sites indicated by the pink dots. (J) Volume rendering of double mutant kidneys shown in (C–H), showing normal number and positions of nephron connections per UB tip.</p

Extensive but irregular UB branching in <i>Gdnf−/−; Spry1−/−</i> and <i>Ret−/−; Spry1−/−</i> double mutant kidneys.

<p>(A–D) Newborn stage kidneys, all carrying the <i>Hoxb7/myrVenus</i> transgene to label the UB branches. Each panel shows a high magnification view of the kidney surface, revealing the shape and organization of branching UB tips; insets show the entire kidney in whole mount. Wild-type kidneys (A) have evenly spaced UB tips with a regular branching pattern, whereas <i>Gdnf−/−</i>;<i>Spry1−/−</i> (B) and <i>Ret−/−</i>;<i>Spry1−/−</i> (C) double mutant kidneys have highly irregular branching. <i>Spry1−/−</i> kidneys (D) have regularly branched, but swollen UB tips. (E–P) 3D volume rendering of E15.5 kidneys. (E–H) Whole kidneys from embryos of the indicated genotypes, carrying <i>Hoxb7/myrVenus</i>. (I–P) Higher power views of two representative surface regions of each genotype. The 3D images were generated from confocal Z-stacks, using Volocity (E–H) or ImageJ (I–P). The yellow dashed lines indicate an interpretation of the branching patterns. While most UB branches in the wild-type (I,M) and <i>Spry1−/−</i> (L,P) kidneys show a reiterative pattern of terminal bifurcation, with branches forming at right angles to their predecessors, most UB branches in the double mutants (J,K,N,O) fail to conform to this pattern, and instead display a variety of abnormal shapes and branching patterns.</p

Differential gene expression in tip and trunk domains is retained in <i>Gdnf−/−;Spry1−/−</i> and <i>Ret−/−;Spry1−/−</i> double mutant kidneys.

<p>Whole mount <i>in situ</i> hybridization for the UB tip markers <i>Ret</i>, <i>Wnt11</i> and <i>Etv4</i> and the trunk marker <i>Wnt7b</i>, in wild-type (A,C,E,G) and double mutant E12.5 kidneys (B,F and H, <i>Gdnf−/−</i>;<i>Spry1−/−</i>, D, <i>Ret−/−</i>;<i>Spry1−/−</i>). Solid arrows indicate UB tips and open arrows indicate trunks. Scale bars 100 µm.</p

<i>Fgf10</i> expression and function in early ureter and kidney development.

<p>(A,B) <i>In situ</i> hybridization in transverse sections of E10.5 wild type embryos reveals that <i>Fgf10</i> and <i>Gdnf</i> are expressed in metanephric mesenchyme (arrows). (C,D) Whole-mount <i>in situ</i> hybridization at E11.0 (dorsal view) shows that <i>Fgf10</i> and <i>Gdnf</i> are expressed in metanephric mesenchyme (MM) surrounding the UB epithelium. The schematic diagram illustrates <i>Fgf10</i> expression, with purple indicating where the hybridization signal was detected. (E–G) Visualization of <i>Hoxb7/myrVenus</i> shows (E) normal UB branching in an <i>Fgf10+/−</i> kidney, (F) reduced branching in an <i>Fgf10−/−</i> kidney, and (G) rescue of UB branching in an <i>Fgf10−/−</i> kidney when <i>Spry1</i> dosage is reduced (<i>Spry1+/−</i>). Scale bars, 100 µm. (H–J) Induction of ectopic budding from the Wolffian duct by FGF10. Dissected E10.5 urogenital regions were cultured with control PBS-soaked beads (H) or beads soaked in FGF10 (I,J) placed between the two Wolffian ducts (dotted yellow circles). FGF10 induces multiple ectopic UB outgrowths (marked by asterisks) in both control <i>Gdnf+/−</i> (I) and <i>Gdnf−/−</i> (J) samples. Open arrowhead in H, Wolffian duct; arrows in H-I, normal ureteric buds.</p