Tissue patterning in the developing mouse limb.

The developing mouse fore- and hindlimbs begin as bumps on the flank of the embryo and grow out to form miniature models of the adult limb during a five day period from E9.5 to E14.5. In this paper I show a series of embryos taken at half-day intervals during limb development and outline the timetable of patterning for each of the component tissues of a limb: epidermis, connective tissues, muscle, nerves and blood vessels. Scanning electron micrographs, supplemented by histological sections, are presented to define a set of standard stages for the description of mouse limb development. I discuss my observations of the mouse limb in the light of current theories of vertebrate limb development, which are based on classic manipulation experiments in the chick as well as more recent molecular data from the mouse system. The limb skeletal pattern in a mouse is laid down in a proximodistal direction, as it is in a bird: the E11.5 forelimb reveals the first signs of a humerus and by E14.5 even the most distal phalanges of the hand are formed. At this late stage ossification sleeves are seen around the proximal limb elements as the cartilage template begins to be converted to a bony skeleton. Myogenic cells stainable with the MF20 antibody against early muscle myosin heavy chain are first seen in the mouse forelimb at E11.5, which is also when the first nerve fascicles begin to enter the limb. From E11.5 to E14.5 both muscle and nerve patterns mature to give distinct muscles at all proximodistal levels of the limb, each muscle with its own nerve branch, and a cutaneous nerve plexus that extends to the fingertips. The developing skin of the mouse limb matures from a bi-layered epidermis overlying an avascular, but otherwise nondescript, prospective dermal layer of mesenchyme at E9.5, to a 4- or 5-layered epidermis with early hair placodes and the first signs of a distinct dermal layer at E14.5. Notable differences between mouse and chick limb development lie in the relatively late formation of the apical ectodermal ridge in the mouse and its unexpectedly close relations with blood vessels, in the absence of anterior and posterior necrotic zones and, possibly, in a late migration of myogenic cells into the mouse limb bud.