During animal development, signaling centers (a.k.a., ‘organizers’) play fundamental roles in the formation of organs, such as the CNS. Organizers consist of groups of cells that produce secreted factors (morphogens) that act on adjacent cells, often in the form of gradients, to induce distinct cell fates at different positions in the gradient. Dysfunction of organizers causes developmental disorders (e.g., agenesis of neural structures and limb defects). Hence, a key goal of developmental biology is to understand how organizers form and function. Several organizers have been implicated in CNS development (e.g., the anterior neural ridge organizer (ANR) and the isthmic organizer; IsO), but we do not understand how a population of neural progenitors is imparted with the ability to secrete specific morphogens and act as an organizer. The IsO is the most-studied CNS organizer as initial observations over a century ago defined a constriction (isthmus) forming in the neurectoderm at the junction of the midbrain and hindbrain primordia. The isthmus was later shown to contain a signaling center with the ability to produce Fgf8 and to control differentiation and positioning of adjacent neural structures, but we do not understand the cellular and molecular components required for IsO function. While the isthmic constriction appears to be positioned at the interface of the midbrain and hindbrain primordia – the IsO itself has been variably predicted to be derived from the midbrain or the hindbrain primordium, and we do not understand how the IsO is formed and positioned in the embryonic CNS. One key barrier to filling these knowledge gaps has been a lack of comprehensive molecular data for organizers during their formation in the CNS. A simple gene regulatory network acting at the isthmus was delineated decades ago, but it contains few gene. As a result, there is no clear molecular signature for the IsO, and we therefore cannot study its formation and function. Using scMultiome analysis – that enables simultaneous characterization of gene expression and epigenetic profiles of individual cells – we overcame this barrier and molecularly identified two cell populations that express known isthmic genes at zebrafish segmentation stages. One population co-expresses midbrain genes and one hindbrain genes, suggesting that they correspond to isthmic cells in the midbrain (IsMB) and hindbrain (IsHB), respectively. The IsHB also expresses fgf8 – indicating that it contains the IsO – but not all IsHB cells are fgf8-positive. At earlier stages (end of gastrulation), our analysis detects the IsMB, but the IsHB is not fully formed, and it is unclear if the IsO is associated with the IsMB or the IsHB at this stage. Our analyses represent the first molecular resolution of the isthmic region and includes earlier stages than prior work. These advances allow us to address key questions: What isthmic cell populations are required for IsO function? Does the IsO arise from the...