A 3D Bioprinted Cortical Organoid Platform for Modeling Human Brain Development.

The ability to promote three-dimensional (3D) self-organization of induced pluripotent stem cells into complex tissue structures called organoids presents new opportunities for the field of developmental biology. Brain organoids have been used to investigate principles of neurodevelopment and neuropsychiatric disorders and serve as a drug screening and discovery platform. However, brain organoid cultures are currently limited by a lacking ability to precisely control their extracellular environment. Here, this work employs 3D bioprinting to generate a high-throughput, tunable, and reproducible scaffold for controlling organoid development and patterning. Additionally, this approach supports the coculture of organoids and vascular cells in a custom architecture containing interconnected endothelialized channels. Printing fidelity and mechanical assessments confirm that fabricated scaffolds closely match intended design features and exhibit stiffness values reflective of the developing human brain. Using organoid growth, viability, cytoarchitecture, proliferation, and transcriptomic benchmarks, this work finds that organoids cultured within the bioprinted scaffold long-term are healthy and have expected neuroectodermal differentiation. Lastly, this work confirms that the endothelial cells (ECs) in printed channel structures can migrate toward and infiltrate into the embedded organoids. This work demonstrates a tunable 3D culturing platform that can be used to create more complex and accurate models of human brain development and underlying diseases.

Identification of ligand-receptor pairs that drive human astrocyte development.

Extrinsic signaling between diverse cell types is crucial for nervous system development. Ligand binding is a key driver of developmental processes. Nevertheless, it remains a significant challenge to disentangle which and how extrinsic signals act cooperatively to affect changes in recipient cells. In the developing human brain, cortical progenitors transition from neurogenesis to gliogenesis in a stereotyped sequence that is in part influenced by extrinsic ligands. Here we used published transcriptomic data to identify and functionally test five ligand-receptor pairs that synergistically drive human astrogenesis. We validate the synergistic contributions of TGFß2, NLGN1, TSLP, DKK1 and BMP4 ligands on astrocyte development in both hCOs and primary fetal tissue. We confirm that the cooperative capabilities of these five ligands are greater than their individual capacities. Additionally, we discovered that their combinatorial effects converge in part on the mTORC1 signaling pathway, resulting in transcriptomic and morphological features of astrocyte development. Our data-driven framework can leverage single-cell and bulk genomic data to generate and test functional hypotheses surrounding cell-cell communication regulating neurodevelopmental processes.

The γ-Protocadherins Regulate the Survival of GABAergic Interneurons during Developmental Cell Death.

Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins (Pcdhgs) from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of Pcdhgs from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in Pcdhg mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain.SIGNIFICANCE STATEMENT A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the Pcdhgs in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.

Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, Pcdh-α, Pcdh-ß, and Pcdh-γ (Pcdha, Pcdhb, and Pcdhg, respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between Pcdha and Pcdhg clusters. To circumvent neonatal lethality associated with deletion of Pcdhgs, we used Crispr-Cas9 genome editing in mice to combine a constitutive Pcdha mutant allele with a conditional Pcdhg allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both Pcdha and Pcdhg clusters led to far more dramatic defects in survival and self-avoidance than Pcdhg deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development.SIGNIFICANCE STATEMENT The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called Pcdh-α, Pcdh-ß, and Pcdh-γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between Pcdh-α and Pcdh-γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning.

The atypical cadherin fat directly regulates mitochondrial function and metabolic state.

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft(mito)) that is imported into mitochondria. Ft(mito) binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.

Mink1 regulates ß-catenin-independent Wnt signaling via Prickle phosphorylation.

ß-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos. We show that Mink1 phosphorylates Prickle on a conserved threonine residue and regulates its Rab5-dependent endosomal trafficking, a process required for the localized plasma membrane accumulation and function of Prickle. Mink1 also was found to be important for the clustering of Vangl within plasma membrane puncta. Our results provide a link between Mink and the Vangl-Prickle complex and highlight the importance of Prickle phosphorylation and endosomal trafficking for its function during Wnt-PCP signaling.

Initiation of actinorhodin export in Streptomyces coelicolor.

Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.