digit 4 progenitors digit 5 progenitors Cells that make digit 4 and 5 and part

digit 4 progenitors digit 5 progenitors -Cells that make digit 4 and 5 (and part of digit 3) are direct descendants of Shh secreting cells. -Digit 5 cells spend more time exposed to Shh (autocrine) than cells in digit 4 -Digit 3 is specified by both time and Shh concentration (paracrine) -Digit 2 is specified by Shh concentration (paracrine) - Digit 1 is Shh-independent Shh effects are mediated by a gradient of BMP

Shh (ZPA) dual role in A/P limb patterning 1. Long range Controls digit number: define the regions in the bud that are competent to form digits. 2. Short range Establish and maintains a BMP2 morphogen gradient that specifies digit identity. Adding BMP inhibitors (Noggin) to limbs treated with Shh: - extra digits still form but they are all the same type

Regulation of digit identity by BMP in the interdigital space Noggin beads • Removal of interdigital region 2 transforms digit 2 into digit 1 • Removal of interdigital region 3 transforms digit 3 into digit 2 • Placing beads soaked with noggin (BMP inhibitor) into the interdigital spaces causes the same effect • BMP from Interdigital tissue specifies the identity of the digit anterior to it

Signaling in forelimb development Positive feedback loop between AER and ZPA Ectoderm AER ZPA AER

Dorso-Ventral limb axis • Determined by the ectodermal tissue that surrounds the limb bud ( non-AER ectoderm ). Not by mesoderm. • If ectoderm is rotated 180 o the dorsal-ventral polarity of the digits is reversed. • Signaling molecules are expressed asymmetrically in the limb ectoderm: – Wnt7a (dorsal) induces activation of Lmx1 (homeodomain) – Engrailed (ventral) • Lack of Wnt7a or LMx1 “ ventralizes ” limbs and digits (no nails, no knee caps). • The boundary where dorsal and ventral ectoderm meet is essential for AER placement: dorsalized limbs do not form AER.

Summary of signaling interactions in limb axes specification A. AER induction • Ffg10 from mesenchyme (lateral plate mesoderm) activates Wnt3 in ectoderm. • Wnt3 activates Fgf8 that in turns activates Fgf10 (positive feedback loop). • BMP from ventral ectoderm activates Engrailed (En1) which represses Wnt7 . B. AER maintenance As limb grows: • Shh from ZPA induces A-P polarity ( BMP gradient) • Shh induces gremlin ( Grem1 ) an inhibitor of BMP which allows continued Fgf8 synthesis by the AER.