Fig. 5.

Generation of characteristic dorsal and ventral hypothalamic neurons. (A) Otp and neurophysin II (NP II) expression in the mouse hypothalamus at E15.5. At this stage, although Otp expression is found predominantly in the dorsal domain, a small number of neurons in ventrally located nuclei such as the lateroanterior hypothalamic nucleus and arcuate nucleus (data not shown) also express Otp. (B–K) Long-term neuronal culture following FACS sorting (day 7) of Rax–GFP⁺ progenitors from SFEBq/gfCDM-cultured ES cells. (B) Schematic of the culture procedure for rostral hypothalamic differentiation. (C–H) Immunostaining of a Transwell-cultured aggregate cultured without Shh. A number of NP II⁺ neurons were seen (C and F). High magnification views of NP II⁺ neurons showed predominantly large somas with a well developed NP II⁺ Golgi area (D) and characteristic varicosities in their neurites (E). (G) Immunostaining for NP II and synaptophysin in dissociation culture of Rax–GFP⁺ cells on day 35. (H) AVP-release analysis upon high K⁺ stimulation on day 25. AVP concentrations in conditioned media of Rax–GFP⁺ aggregates were measured by RIA. (I–K) Immunostaining of dissociation culture of Rax–GFP⁺ cells cultured with Shh (day 25). (I) SF1 (green), VGluT2 (located mainly in the growth cones; red), and TuJ1 (white). (J) Immunostaining for TH and Nkx2.1. (K) Immunostaining for AgRP and NPY. (Scale bars, 20 μm.)