Figure 4

Whole mount columella extracted from embryo. Alcian Blue/Alizarin Red S staining of the columella (col), E7-E16. (A) The columella at E7.5 with chondrogenic centers in the footplate/shaft and two more in the infracolumella (inf) arm (black arrowheads). The supracolumella (sup) and extracolumella (exc) condensations (white arrowheads) have no chondrocytes. (B) By E8 five condensation centers can be identified: columella shaft, supracolumella, extracolumella, and two in the infracolumella. (C, D) During E9/10 continued chondrogenesis and growth occurs. (E) At E12, the extent of the delicately sculpted infracolumella arm is visible. (F) At E13, pre-hypertrophic chondrocytes appear in the shaft of the columella (area between two black lines). (G) E13.5, Alizarin Red S staining is detected in the periosteal bony collar of the shaft. Platner's ligament is inserted onto the supracolumella/extracolumella base (arrowhead). The adjacent section shows chondrocytes, pre-hypertrophic, and hypertrophic chondrocyte progression. (H) A second periosteum becomes apparent at E14 in the base of the footplate (arrowed). (I) A section with periosteum labeled red (arrowed). The extracolumella arm inserts into the tympanic membrane (tm). (J) By E15 the periosteum regions merge, with the annular ligament negative (arrowed). (K) E16, note Alizarin Red S staining compared to earlier stages in the shaft (compare H-K). Abbreviations: al-annular ligament, ch-chondrocytes, fp-footplate, h-hypertrophic chondrocytes, i-insertion point, p-perichondrium/periosteum, ph-pre-hypertrophic chondrocytes, pl-Platner's ligament.