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First published online 14 March 2007
doi: 10.1242/dev.000703

Development 134, 1583-1592 (2007)
Published by The Company of Biologists 2007
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Activities of N-Myc in the developing limb link control of skeletal size with digit separation

Sara Ota1,*, Zi-Qiang Zhou1,*, Doug R. Keene1, Paul Knoepfler2 and Peter J. Hurlin1,3,{dagger}

1 Shriners Hospitals for Children Portland, 3101 SW Sam Jackson Park Road, Portland, OR 97239, USA.
2 Department of Cell Biology and Human Anatomy, Davis School of Medicine, 3301 Tupper Hall, University of California, Davis, CA 95616, USA.
3 Department of Cell and Developmental Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.


Figure 1
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  		Fig. 1. Effect of N-Myc deficiency on mouse limb bud formation. (A) Comparison of PC control embryos with PC-NMycf/f and N-Myc null embryos at E10.5. Arrows indicate forelimb and hindlimb buds. Note that N-Myc null embryos die between E10.5 and 11.5. (B) Representative whole-mount in situ hybridization results showing forelimb expression of the indicated genes at E10.5. At least three sets of embryos were used for each probe. A, anterior; P, posterior.

 

Figure 2
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  		Fig. 2. Decreased proliferation in mouse limb bud mesenchyme caused by loss of N-Myc. (A-X) N-Myc expression is shown by in situ hybridization of transverse sectioned limb buds from PC, PC-NMycf/f at E9.5, 10.5 and 11.5 as indicated. N-Myc-/- embryos were analyzed only at E9.5 and 10.5 due to lethality by E11.5. Transverse limb bud sections were stained for BrdU incorporation (brown) and counterstained with Hematoxylin (blue). The boxed regions, representing areas in which N-Myc is expressed or predicted to be expressed in the mutants, are shown at higher magnification (40x of E9.5 sections and 20x of E10.5 and 11.5 sections) in panels on the right.

 

Figure 3
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  		Fig. 3. Effects of N-Myc deficiency on the size and pattern of the limb skeleton. (A) Alcian Blue (staining for cartilage) and Alizarin Red (staining for bone) staining of PC and PC-NMycf/f mouse embryos at E15.5. (B) E15.5 autopods stained with Alcian Blue. Note that the phalangeal elements of digit 1 (thumb, black arrows) are longer in PC-NMycf/f embryos and resemble those of the most posterior digit (digit 5 or little finger). (C) The distal skeleton of E18.5 PC and PC-NMycf/f embryos stained with Alcian Blue and Alizarin Red. Note the lack of well-defined joints (black arrows), or absence of joints, in the digits of PC-NMycf/f embryos. Note also the bony fusions that occur at the base of the anterior and posterior digits of the PC-NMycf/f autopod (red arrows). (D) Analysis of Sox9 expression, a marker of cartilage growth and development, by whole-mount and section in situ hybridization at the indicated embryonic stages. (E) Whole-mount in situ hybridization showing Noggin expression at E11.5. (F) Expression of Gdf5, a marker of joint development, at the indicated embryonic stages.

 

Figure 4
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  		Fig. 4. N-Myc deficiency causes syndactyly. (A) Dorsal view of left paws (autopods) of PC and PC-NMycf/f mouse limbs at E15.5. Note the syndactyly of the central three digits and protruding digits 1 and 5 of PC-NMycf/f limbs. (B) Absence of digit individualization in N-Myc-deficient autopods at postnatal day 1. Autopods were stained for E-cadherin (green, arrow) to mark the epithelium and counterstained with a pan-cytokeratin antibody (red). (C) Dorsal and ventral views of forelimbs and hindlimbs of 1-week-old mice.

 

Figure 5
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  		Fig. 5. The relationship between N-Myc expression and programmed cell death in the developing mouse forelimb autopod. (A-R) Whole-mount in situ hybridization of N-Myc is compared with whole-mount staining with LysoTracker, and sections stained for TUNEL at E12.5, 13.5 and 14.5 as indicated. For TUNEL staining at E13.5, higher magnification views of distal regions of digits 4 and 5 of Prx1-Cre (O) and PC-NMycf/f (R) are shown.

 

Figure 6
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  		Fig. 6. N-Myc deficiency does not affect expression of Fgf8 and Fgf4. Whole-mount in situ hybridization analysis of Fgf8 (A) and Fgf4 (B) expression in mouse forelimbs at the indicated embryonic stages. Note restriction of Fgf8 expression to epithelium at the distal tips of digits in both PC and PC-NMycf/f embryos.

 

Figure 7
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  		Fig. 7. Diminished or altered expression of interdigital and perichondrial markers in N-Myc-deficient mouse forelimbs. (A-E) Whole-mount in situ hybridization analysis for expression of the indicated genes was performed at the indicated embryonic stages.

 

Figure 8
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  		Fig. 8. Loss of interdigital tissue in N-Myc-deficient mouse limbs. (A) Staining for BrdU incorporation at E13.5. Note the interdigital channel of predominantly BrdU-negative cells of PC limbs is lost in interdigital regions of PC-NMycf/f limbs. The boxed regions are shown at higher magnification and the percentage of BrdU-positive cells in the specific regions marked by red boxes is indicated. (B) TEM of interdigital regions in PC and PC-NMycf/f paws. Scale bars: 10 µm. (C) Staining for the blood vessel marker Pecam (green) with propidium iodide. C, cartilage condensations; P, perichondrium; E, epithelium.

 

Figure 9
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  		Fig. 9. Model linking skeletal size determination with digit separation in the developing mouse limb. In this model [adapted from Mariani and Martin (Mariani and Martin, 2003Go)], N-Myc acts downstream of AER-derived FGFs in regulating the pool of undifferentiated mesenchyme (green) in the early limb bud. The undifferentiated mesenchyme gives rise to precartilaginous condensations that are pre-specified to generate proximal [stylopod: forelimb humerous (blue)], medial [zeugopod: forelimb radius and ulna (yellow)] and distal (autopod, red) skeletal elements. After contributing to the growth of the limb segments, residual undifferentiated cells are localized to the IDM, where they contribute to the final stages of autopod patterning and are then eliminated by programmed cell death. Their elimination terminates autopod patterning and initiates digit separation. In the absence of N-Myc, the pool of undifferentiated mesenchymal cells is reduced, leading to a proportional decrease in all skeletal segments. The reduced pool of undifferentiated mesenchymal cells is depleted during skeletal outgrowth, leading to loss of the undifferentiated IDM that is targeted for cell death and required for digit separation.

 

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© The Company of Biologists Ltd 2007