Significantly, live cell time-lapse imaging of radial migration of MECP2-deficient neurons from the VZ to CP inside intact live 3D organoids has not been performed so far. However, the use of fixed tissues did not allow cell tracking and analysis of parameters such as speed and trajectory of neuronal displacement thus the dynamics and cellular mechanisms of the presumptive migration deficit remain to be characterized.
Using fixed tissue slices from 3D cerebral organoids, we have recently demonstrated impaired proliferation of the progenitor pool and delayed maturation and presumed migration of neurons ( Mellios et al., 2011), consistent with human postmortem deficits. 2D human stem cell models of RTT, generated by reprogramming of patient cells or genome editing, have revealed deficits in human RTT neurons including aberrant transcription ( Lyst and Bird, 2015 Li et al., 2013 Chen et al., 2013 Gomes et al., 2020 Trujillo et al., 2021), impaired neuronal maturation and electrophysiological function ( Li et al., 2013 Tang et al., 2016 Kim et al., 2011 Farra et al., 2012), and up- or down-regulation of key signaling pathways and activity-related genes ( Li et al., 2013). These deficits are paralleled by reductions in dendritic arborization, soma size and spine density described in RTT mouse models ( Fukuda et al., 2005 Kishi and Macklis, 2004 Shahbazian et al., 2002 Smrt et al., 2007). Structural deficits described in postmortem RTT human brains include reduced cortical thickness, cell size and dendritic arborization ( Armstrong et al., 1995 Bauman et al., 1995) and reduced cerebral volume in MR imaging of RTT patients ( Carter et al., 2008). MeCP2 is a pleiotropic regulator of gene expression and impacts multiple components of brain development and function ( Ip et al., 2018). Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene encoding Methyl CpG binding protein 2 (MeCP2). Thus, they have been used to model neurogenesis-relevant human pathologies such as microcephaly ( Lancaster et al., 2013 Zhang et al., 2019), licencephaly ( Bershteyn et al., 2017), heterotopia ( Klaus et al., 2019), Zika virus infection ( Qian et al., 2016 Garcez et al., 2016 Dang et al., 2016), and idiopathic autism ( Mariani et al., 2015). Human cerebral organoids also recapitulate gene expression programs of the fetal cortex ( Qian et al., 2016 Velasco et al., 2019 Quadrato et al., 2017 Pollen et al., 2019 Luo et al., 2016 Camp et al., 2015) as well as the fetal brain epigenome ( Luo et al., 2016). These spheroid structures contain progenitor-rich zones around ventricle-like cavities akin to ventricular zones (VZ) from which neuronal progenitors migrate radially to generate the cortical plate (CP) ( Lancaster et al., 2013 Qian et al., 2016 Paşca et al., 2015 Mariani et al., 2012 Kadoshima et al., 2013). Human cerebral organoids derived from embryonic or induced pluripotent stem cells are unique in their ability to recapitulate early events of embryonic brain development ( Lancaster et al., 2013). Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially.
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