Fig. 7. The mutant ENCCs form aggregates on a 2D substrate. Rings of E13.5 control and mutant distal midgut were plated on a mixture of ECM gel and fibronectin. After 2 days in culture, neuron cell bodies were detected with HuD (red), neuronal processes with NF160 (red), glial cells with B-FABP (green) and nuclei with DAPI. (A-G) Control culture, at low (A) and high (B-G) magnification. Neurons and glial cells form scattered networks on SMC (B,C) or directly on ECM (D-G). (H-N) Mutant culture, at low (H) and high (I-N) magnification. Green arrows indicate aggregates of neurons and glial cells that adhere directly to ECM. White arrows show spheroid aggregates that do not adhere to SMC or ECM. (I,J) Aggregates on the SMC layer. (K,L) An aggregate adhering to ECM. (M,N) High magnifications of spheroid aggregates stained for HuD and NF160 (red) and B-FABP (green) (M) and for X-Gal (N). Red arrows indicate neuronal processes linking aggregates to the explants.