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First published online August 14, 2006
doi: 10.1242/10.1242/dev.02529

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Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial-mesenchymal signalling

Lia Panman^1,2,*,, Antonella Galli^1,*, Nadege Lagarde¹, Odysse Michos¹, Gwen Soete^2,, Aimee Zuniga^1, and Rolf Zeller^1,

¹ Developmental Genetics, DKBW Centre for Biomedicine, University of Basel Medical School, Mattenstrasse 28, CH-4058 Basel, Switzerland.
² Department of Developmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

View larger version (66K): [in a new window]   Fig. 1. Analysis of the spatial competence to activate and the temporal kinetics to upregulate the expression of SHH target signals in the limb bud mesenchyme. Left panels: contralateral control limb buds (not grafted). Right panels: contralateral, wild-type (A-D) or Shh-/- mutant (E-H) forelimb buds received posterior grafts of SHH-producing cell aggregates at E10.25 (31-33 somites). The red circles indicate the positions and approximate sizes of the grafts after culturing. White arrowheads indicate the endogenous gene expression domains. Blue arrowheads indicate the induced gene expression in response to a SHH graft. Limb buds are oriented with anterior towards the top. (A-D) Analysis of the temporal kinetics of the differential transcriptional response to SHH signalling in wild-type forelimb buds. (A) A ring of mesenchymal cells responds to SHH signalling by activating expression of the downstream target Gli1 within 6 hours of grafting. (B) Within 9 hours, Grem1 expression is expanded distoanteriorly in response to SHH signalling. (C,D) Jag1 expression is activated in the distal anterior mesenchyme after 9 hours (C), but upregulation of its expression in the distoanterior mesenchyme requires at least 15 hours (D). The small white arrowheads indicate localized endogenous Jag1 expression in the anterior limb bud mesenchyme. (E-H) Shh-deficient limb buds were cultured for 15 hours after receiving a graft of SHH-producing cells to detect strong expression of the gene of interest in all cases. Asterisks indicate the posteroproximal margins of the limb buds after culture. (E) SHH signalling induces transcriptional response in a large area of mesenchymal cells surrounding the graft as assessed by Gli1 expression. (F) Grem1 expression is upregulated in mesenchymal cells that are always located distally to the SHH graft. (G) Jag1 expression is activated in mesenchymal cells located also always distally to the SHH graft. The weak and speckled AER staining corresponds to crossreactivity that appears in a fraction of cultured limb buds. (H) A posterior SHH graft upregulates Bmp2 expression in mesenchymal cells located proximal to the graft (blue arrowhead). Upregulation of Bmp2 expression can be seen in the AER (striped arrowhead).

View larger version (99K): [in a new window]   Fig. 2. Restoration of GREM1/FGF mediated EM feedback signalling in Grem1In2/In2 limb buds rapidly rescues expression of Shh, Jag1 and Hoxd13 in the distal mesenchyme. Cell aggregates producing either gremlin 1 (GREM1, green circles) or FGF4 (FGF4, blue circles) were grafted into the posterior mesenchyme of Grem1In2/In2 forelimb buds (E10.25, 31-33 somites). In these studies, the grafts were placed more distally and further away from the posterior AER than in previous experiments in order to avoid disruption of the posterior mesenchyme competent to express Shh. Circles indicate the approximate size and positions of the grafts after culturing, coloured arrowheads indicate induced/upregulated gene expression. White arrowheads indicate endogenous gene expression in contralateral control limb buds. (A) GREM1 rescues Shh expression (red arrowhead) within 9 hours, indicative of restored EM feedback signalling. (B) GREM1 induces Fgf4 expression (arrowhead) in the posterior AER within 9 hours. (C) Grafts of FGF4-expressing cells in turn upregulate the expression of Shh within 9 hours (red arrowhead). (D,E) GREM1 upregulates the expression of the SHH targets Jag1 (D; blue arrowhead) and Hoxd13 (E; pink arrowhead) in the distal mesenchyme within 15 hours. (F,G) FGF4 also induces the upregulation of Jag1 (F; blue arrowhead) and Hoxd13 expression (G; pink arrowhead) in the distal mesenchyme of Grem1-deficient limb buds within 15 hours.

View larger version (48K): [in a new window]   Fig. 3. Blocking FGF signal transduction with SU5402 (10 µM final) in wild-type limb buds (E10.5, 34-36 somites) phenocopies aspects of the disruption of Jag1 and Hoxd13 expression in Grem1-deficient limb buds. (A-D) Asterisks indicate the anterior margins when necessary. (A) Jag1 expression in a wild-type forelimb bud cultured for 16 hours. Left panel: dorsal view with posterior towards the bottom and distal towards the left. Right panel: view onto the distal part of the same limb bud to reveal the anterior expansion of Jag1 expression at this stage. (B) Jag1 distribution in a wild-type forelimb bud cultured with SU5402 for 16 hours. Left panel: top view onto the distal limb to allow direct comparison with the control. Right panel: dorsal view. (C) Hoxd13 expression in a wild-type forelimb bud cultured for 16 hours. Left panel: dorsal view. Right panel: top view. (D) Hoxd13 distribution in a wild-type forelimb bud cultured with SU5402 for 16 hours. Left panel: top view of the distal limb to reveal the downregulation of Hoxd13 expression and lack of anterior expansion in the distal mesenchyme. Right panel: dorsal view. (E,F) Jag1 (E) and Hoxd13 expression (F) in wild-type and Grem1 deficient (Grem1In2/In2) forelimb buds at E11.0 (42 somites, not cultured), which corresponds to a stage similar to the limb buds shown in A-D.

View larger version (59K): [in a new window]   Fig. 4. GREM1 is required to time the mesenchymal response to SHH signalling. Forelimb buds of E10.25 (31-33 somites) embryos received anterior grafts of SHH cell aggregates (red circles indicate approximate positions and sizes of grafts after culture) and the temporal kinetics of inducing/expanding the distal expression domains of Jag1 and Hoxd13 were assessed. Anterior grafts were used to clearly distinguish endogenous (white arrowheads) from exogenous SHH induced gene expression (blue arrowheads). All whole-mount in situ results were developed for the same amount of time to clearly reveal the weak ectopic Jag1 and Hoxd13 expression in Grem1In2/In2 limb buds after 22 hours. (A) Wild-type limb buds. SHH induces ectopic Jag1 expression (blue arrowhead) within 22 hours. (B) Grem1-deficient limb buds. Left panel: only low levels of SHH induced ectopic Jag1 expression (blue arrowhead) are detected after 22 hours. Right panel: significant ectopic Jag1 expression (blue arrowhead) is detected only after 32 hours. (C) Wild-type limb buds. SHH induces significant anterior expansion of the Hoxd13 expression domain (blue arrowhead) within 22 hours. (D) Grem1-deficient limb buds. Left panel: only low levels of ectopic Hoxd13 expression (blue arrowhead) are detected after 22 hours. Right panel: significant levels of SHH induced anterior ectopic Hoxd13 expression (blue arrowhead) are detected after 32 hours.

View larger version (65K): [in a new window]   Fig. 5. The kinetics of blocking SHH signal transduction in cultured wild-type forelimb buds (E10.5, 34-36 somites) using 10 µM cyclopamine. (A) Loss of Gli1 transcription within 9 to 15 hours. T0: Gli1 expression in a non-cultured control limb bud. Wt: Gli1 expression in a wild-type limb bud cultured for 15 hours as a control. Cyc (9 hours): Gli1 expression in a wild-type limb bud cultured for 9 hours in the presence of cyclopamine. Cyc (15 hours): Gli1 expression in a wild-type limb bud cultured for 15 hours in the presence of cyclopamine. Complete loss of Gli1 expression occurs. Therefore, all subsequent experiments were carried out by culturing limb bud as in the presence of 10 µM cyclopamine for 15 hours. (B) Fgf4 expression in the AER is lost by culturing limb buds in the presence of cyclopamine for 15 hours (Cyc panel). (C) By contrast, Fgf8 remains expressed in the AER of cyclopamine treated limb buds (Cyc panel). All limb buds are oriented with anterior towards the top and posterior towards the bottom.

View larger version (47K): [in a new window]   Fig. 6. Anterior expansion of Grem1 expression in the distal mesenchyme and propagation of Jag1 expression do not require continuous SHH signal transduction. T0: non-cultured control forelimb bud at the stage indicated. Wt: wild-type control forelimb bud cultured for 15 hours. Cyc: wild-type forelimb bud cultured for 15 hours in the presence of 10 µM cyclopamine. E10.25, 31-33 somites; E10.5, 34-36 somites; E10.75, 37-39 somites. All limb buds are oriented with anterior towards the top and posterior towards the bottom. Blue broken lines indicate the approximate anterior limits of Grem1 expression when necessary in A-C. (A) Inhibition of SHH signal transduction at E10.25 does not significantly alter Grem1 expression. (B) Inhibition of SHH signal transduction at E10.5 reduces Grem1 levels in the distal mesenchyme (indicated by bracket), but does not significantly alter its anterior expression limit. (C) Grem1 expression in Shh-/- forelimb buds is rapidly downregulated, but is clearly detectable in the distoanterior mesenchyme at E9.75 (28 somites) and at E10.25 (32 somites). (D-F) Effects of cyclopamine treatment on Jag1 expression. (D) Inhibition of SHH signal transduction at E10.25 interferes to some extent with upregulation of Jag1 expression in the posterior limb bud mesenchyme. (E) Inhibition of SHH signal transduction at E10.5 does not significantly alter the onset of anterior expansion of Jag1 expression in the distal mesenchyme, albeit expression levels remain slightly lower. (F) Similarly, cyclopamine treatment at E10.75 does not interfere with continued anterior expansion of Jag1 expression in the distal limb bud mesenchyme.

View larger version (54K): [in a new window]   Fig. 7. Differential temporal requirement of SHH signal transduction for the establishment of the presumptive digit expression domains of 5'Hoxd genes. T0: non-cultured control forelimb bud at the stage indicated. Wt: wild-type control forelimb bud cultured for 15 hours. Cyc: wild-type forelimb bud cultured for 15 hours in the presence of 10 µM cyclopamine. E10.25, 31-33 somites; E10.5, 34-36 somites; E10.75, 37-39 somites. All limb buds are oriented with anterior towards the top and posterior towards the bottom. (A-C) Effects on Hoxd11 expression. (A) Cyclopamine treatment starting at E10.25 interferes with upregulation and distoanterior expansion of Hoxd11 expression. (B) By E10.5, the late Hoxd11 expression domain has been established and inhibition of SHH signal transduction no longer alters the spatial distribution, while Hoxd11 expression levels are still affected. (C) Inhibition of SHH signal transduction from E10.75 onwards no longer alters Hoxd11 expression. (D-F) Effects on Hoxd12 expression. (D) Cyclopamine treatment starting at E10.25 significantly interferes with upregulation and expansion of Hoxd12 expression. (E) Similarly, inhibition of SHH signal transduction at E10.5 blocks the ongoing distoanterior expansion of Hoxd12 expression (compare Cyc with Wt). (F) From E10.75 onwards, cyclopamine treatment no longer affects Hoxd12 expression significantly. (G,H) Effects on Hoxd13 expression. (G) At E10.5, the establishment of the distal Hoxd13 expression domain has been initiated (compare T0 with Wt). Cyclopamine treatment blocks the onset of anterior expansion of the Hoxd13 expression domain in the distal mesenchyme and upregulation of its expression (Cyc panel). (H) Cyclopamine treatment at E10.75 still efficiently blocks the ongoing anterior expansion of the Hoxd13 expression domain and upregulation of its expression levels.

View larger version (37K): [in a new window]   Fig. 8. The signalling interactions that control dynamic regulation of gene expression in the distal, digit-forming area of the limb bud. The scheme depicts three distinct phases of limb bud morphogenesis in a simplified manner (for details see text). Limb buds are drawn schematically with anterior towards the top and posterior towards the bottom. Only genes relevant to the present study are indicated. Phase I: setting up the signalling centres and differential mesenchymal responsiveness. The expression of various key regulator genes including Shh (dark blue), Grem1 (orange) and 5'Hoxd genes (green) in the mesenchyme and FGF genes in the AER (light blue) is activated locally and independent of SHH signalling. The interaction of GLI3R with HAND2 and other transcription factors pre-pattern the limb bud mesenchyme and regulate activation of their expression. Phase II: SHH signalling (dark blue) and GREM1/FGF-mediated feedback signalling (orange/light blue) are required to establish and propagate gene expression in the distal limb bud mesenchyme. Epithelial-mesenchymal (EM) feedback signalling regulates the temporally coordinated anterior expansion (yellow arrow) of gene expression in the distal mesenchyme (Jag1 and 5'Hoxd genes) and FGF gene expression in the AER (light blue). Jag1 becomes independent of SHH signalling early, while 5'Hoxd genes are progressively rendered SHH independent (with 3' to 5' polarity) as their presumptive digit expression domains are being established. Phase III: the expanding population of Shh descendants increasingly separates SHH signalling from GREM1 producing mesenchymal cells, which probably causes breakdown of SHH/GREM1/FGF feedback signalling and terminates limb bud patterning.