We next analyzed the molecular signatures that distinguished each NGN2-iN cluster. We compared the signatures with in situ hybridization (Ravasi et al., 2010) data from the Allen Developing Mouse Brain Atlas, and with single-cell transcriptome atlases containing primary neural cells (Figure 3A). We also assessed the expression of neurotransmitters and other markers of neuron specialization. Based on whole-embryo mouse ISH data (Thompson et al., 2014), we find that Ngn2 is expressed in progenitor zones in the developing telencephalon and many other brain structures (Figures 3B and S4). Prph is expressed in the neural retina, trigeminal nerve, and nuclei within the gray horn of the spinal cord. Phox2b is expressed in rhombencephalon/brain stem neurons as well as neurons in the peripheral nervous system (PNS). Pou4f1 is expressed in the retina, mesencephalon derivatives, trigeminal nerve, and gray horn nuclei. We next compared each NGN2-iN cluster with PNS and CNS neurons from primary reference cell atlases (Clark et al., 2019; La Manno et al., 2020; Zeisel et al., 2018) (Figure 3C). Unlike neurons in the iPSC-derived cerebral and retinal organoids, the NGN2-iN clusters
