To the Editor:The article by Seto et al. ([1997] Teratology 56:262–

270) proposes that the basis of missing central digits inthe mouse mutant Dactylaplasia (Dac) is aberrant celldeath in the apical ectodermal ridge (AER). Becausethe aer is responsible for elongation of the limb inthe proximo/distal axis (Saunders [1948] J. Exp.Zool. 108:363–403), the proposal maintains biologicalplausibility.

This letter is meant to transmit concern about theconclusions of this study based on figures 4–6. Thesefigures contain mouse embryo limb buds stained withthe supravital dye Nile Blue sulfate to depict thepattern of cell death. A troubling aspect of these demon-strations is the absence of Nile Blue sulfate staining inthe ectoderm of non-mutant embryos (Figs. 4A, 5A).Dying cells are a prominent feature of the murine aerrepeatedly demonstrated by Milaire and his colleagues(e.g., Milaire and Rooze [1983] Arch Biol. 94:459–490).In fact, those limbs pictured as Dac heterozygotes havea Nile Blue sulfate staining pattern that one usuallysees in normal mouse embryo limb buds. Because theauthors did not genotype the embryos, their resultssuggest to me that the pictures shown represent theopposite of what was intended. That is, Figures 4A and5A are Dac/1 embryos, and the absence of Nile Bluesulfate staining indicates the lack of an AER, whereasFigures 4B and 5B are non-mutant embryos with anormal AER cell death pattern. Close inspection ofFigure 4A (also on the cover of the October 1997 issue)provides a hint of staining at the most proximal ante-rior and posterior extents of the putative AER. Such astaining pattern, indicative of an aer only at the extremesof hand or footplate, would concur with the phenotypeshown on the cover, i.e., absence of central digits.

This dilemma can be resolved quite easily. Mostsimple would be histological sectioning of the limbsdepicted in Figures 4 and 5 (or Nile Blue sulfate-stainedsubstitutes). If the authors are correct, then the mutantlimbs will show excessive cytotoxicity in the aer whencompared to non-mutant limbs. If the limb buds havebeen miscategorized, as I suspect, then histologicalsection will show a reduced or absent aer in the limbbuds labeled wild type (Figs. 4A,5B), whereas thoselabeled Dac/1 (Figs. 4B,5B) will possess a normal aercontaining the usual remnants of dead cells.

A second approach, mentioned in Materials andMethods, but not described in Results, would utilizeNile Blue sulfate staining of embryos from matings ofNZB/BINJ-Dac/Dac mice. All offspring are Dac homo-zygotes, so genotyping is unecessary; thus, the patternof cell death in the aer can be unequivocally associatedwith the Dac/Dac genotype. As a control NZB/BINJ-1/1 mice would provide wild-type embryos to docu-ment the normal Nile Blue sulfate staining pattern.

Alterations in episodes of programmed cell deathhave repeatedly been shown to have morphogeneticsignificance as evidenced by abnormal fetal outcomes.If Dactylaplasia works via this mechanism, then fur-ther study of this gene will provide important clues forthe tightly regulated spatiotemporal occurrence of theseepisodes. Thus, it is crucial that we be certain thatmutation of this gene leads to limb reduction by pre-mature death of AER cells as proposed by the authors.

William J. Scott, Jr., D.V.M., Ph.D.Department of PediatricsChildren’s Hospital Research FoundationCincinnati, Ohio 45229

TERATOLOGY 58:227 (1998)

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