The Isthmus organizer (IsO) is a secondary organizer located where the expression of the more rostral Otx2 (light orange in the outer arch) and the more caudal Gbx2 (dark orange) transcription factors coincide, a region that later becomes the midbrain-hindbrain boundary. IsO cells secrete Wnt1, Fgf8 and Shh, which play a crucial role in the development of the meso-diencephalic progenitor cells, and of cell populations in the anterior rhombencehpalon. Floor plate-secreted Shh also reaches the meso-diencephalic progenitor birth area.
The anterior neural ridge (ANR) and the zona limitans intrathalamica (ZLI) are two additional secondary organizers critical to development of the early brain. The ANR is located in the most anterior part of the neural tube and secretes signaling molecules that regulate the anterior-posterior patterning of the forebrain. The ZLI is located between prosomers 2 and 3 of the diencephalon, where the expression of the transcription factors Six3 (grey in the inner arch) and Irx3 (dark green) coincide. It has a major role in patterning the diencephalon.
The border between the diencephalon and the mesencephalon is located where the expression of Pax6 (dark purple in the middle arch) and Pax2 (light purple in the middle arch) coincide.
Abbreviations: IsO, Isthmus Organizer; ANR, Anterior Neural Ridge; ZLI, Zona Limitans Intrathalamica.