Highlights d A transcriptional single-cell atlas of the developing testes during human puberty d Distinctive phases of germ cell differentiation occur during puberty d Identification of a common progenitor for Leydig and myoid cells prior to puberty d Partial reversal of Sertoli and germ cell maturation upon testosterone suppression

Summary Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show SO synapses normally have significantly more mushroom spines and higher magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered these differences require the Type II classic cadherins, cadherins-6, 9, and 10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and 10 to regulate mushroom spine density and high magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, 9, and 10 are gatekeepers for high magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.

Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.

Genetic abnormalities in synaptic proteins are common in individuals with autism; however, our understanding of the cellular and molecular mechanisms disrupted by these abnormalities is limited. SHANK3 is a postsynaptic scaffolding protein of excitatory synapses that has been found mutated or deleted in most patients with 22q13 deletion syndrome and about 2% of individuals with idiopathic autism and intellectual disability. Here, we generated CRISPR/Cas9-engineered human pluripotent stem cells (PSC) with complete hemizygous SHANK3 deletion ( SHANK3 +/− ), which is the most common genetic abnormality in patients, and investigated the synaptic and morphological properties of SHANK3-deficient PSC-derived cortical neurons engrafted in the mouse prefrontal cortex. We show that human PSC-derived neurons integrate into the mouse cortex by acquiring appropriate cortical layer identities and by receiving and sending anatomical projections from/to multiple different brain regions. We also demonstrate that SHANK3-deficient human neurons have reduced AMPA-, but not NMDA- or GABA-mediated synaptic transmission and exhibit impaired dendritic arbors and spines, as compared to isogenic control neurons co-engrafted in the same brain region. Together, this study reveals specific synaptic and morphological deficits caused by SHANK3 hemizygosity in human cortical neurons at different developmental stages under physiological conditions and validates the use of co-engrafted control and mutant human neurons as a new platform for studying connectivity deficits in genetic neurodevelopmental disorders associated with autism.

Our understanding of the human brain is limited by the lack of experimental models to mechanistically probe the properties of brain cells at different developmental stages under normal and pathological conditions. We developed a new method for generating human cortico-striatal organoids from stem cell-derived single neural rosettes (SNRs) and used it to investigate cortico-striatal development and deficits caused by the deficiency of an autism- and intellectual disability-associated gene SHANK3. We show that SNR-derived organoids consist of different cortico-striatal cells, including pallial and subpallial progenitors, primary cortical and striatal neurons, interneurons, as well as macroglial and mural cells. We also demonstrate that neurons in SNR-derived organoids are predictably organized, functionally mature, and capable of establishing functional neural networks. Interestingly, we found that the cellular and electrophysiological deficits in SHANK3-deficient SNR-derived organoids are dependent on the level of SHANK3 expression and that organoids with complete hemizygous SHANK3 deletion have disrupted expression of several clustered protocadherins and multiple primate-specific zinc-finger genes. Together, this study describes a new method for using SNRs to generate organoids, provides new insights into the cell lineages associated with human cortico-striatal development, and identifies specific molecular pathways disrupted by hemizygous SHANK3 deletion, which is the most common genetic abnormality detected in patients with 22q13 deletion syndrome.