Axial elongation of caudalized human organoids mimics aspects of neural tube development

ABSTRACT Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development. Elongating organoids consist largely of neuroepithelial compartments and contain TBXT+SOX2+ neuro-mesodermal progenitors in addition to PAX6+NES+ neural progenitors. A critical threshold of Wnt agonism stimulated singular axial extensions while maintaining multiple cell lineages, such that organoids displayed regionalized anterior-to-posterior HOX gene expression with hindbrain (HOXB1) regions spatially distinct from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR interference-mediated silencing of TBXT, a Wnt pathway target, increased neuroepithelial compartmentalization, abrogated HOX expression and disrupted uniaxial elongation. Together, these results demonstrate the potent capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early human nervous system development.

Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.

The developmental hourglass model is applicable to the spinal cord

SummaryThe developmental hourglass model predict that embryonic morphology is most conserved at mid-embryonic stage and diverge at early and late stage. This model is generally considered by whole embryonic level. Here, we demonstrate that the hourglass model is also applicable to the more reduced element, the spinal cord. In the middle of the spinal cord development, dorsoventrally arrayed neuronal progenitor domains are established, which is conserved among vertebrates. We found that, by comparing the single-cell transcriptomes between mice and zebrafish, V3 interneurons, a subpopulation of the post-mitotic spinal neurons, display the divergent molecular profiles. We also found non-conservation of cis-regulatory elements located around the progenitor fate determinants, indicating the rewiring of the upstream gene regulatory network. These results demonstrate that, despite the conservation of the progenitor domains, processes before and after the progenitor domain specification has diverged. This study may help understand the molecular basis of the developmental hourglass model.

Human spinal cord organoids exhibiting neural tube morphogenesis for a quantifiable drug screening system of neural tube defects

The human spinal cord forms well-organized neural circuits for environment sensing and motor behavior. The three-dimensional (3D) induction of the spinal cord-like tissue from human pluripotent stem cells has been reported, but they often do not mimic morphological features of neurulation and their maturity is limited. Here, we report an advanced 3D culture system for the production of human spinal cord-like organoids (hSCOs) suitable for the scale-up and quantitative studies. The hSCOs exhibited many aspects of spinal cord development, including neurulation-like tube-forming morphogenesis, differentiation of the major spinal cord neurons and glial cells, and mature synaptic functional activities. We further demonstrated that hSCOs platform allowed quantitative and systematic high-throughput examination of the potential risk of neural tube defects induced by antiepileptic drugs. Thus, hSCOs can be used for understanding human spinal cord development, disease modeling, and toxicology screening.

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development

Transcription factors that contain a homeodomain DNA-binding domain have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a subfamily of NK homeodomain-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here, we show that zebrafish (Danio rerio) hmx2 and hmx3a are coexpressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1, and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a. In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a. More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development.

Alterations in gene expression in the spinal cord of mice lacking Nfix

Objective Nuclear Factor One X (NFIX) is a transcription factor expressed by neural stem cells within the developing mouse brain and spinal cord. In order to characterise the pathways by which NFIX may regulate neural stem cell biology within the developing mouse spinal cord, we performed an microarray-based transcriptomic analysis of the spinal cord of embryonic day (E)14.5 Nfix−/− mice in comparison to wild-type controls. Data description Using microarray and differential gene expression analyses, we were able to identify differentially expressed genes in the spinal cords of E14.5 Nfix−/− mice compared to wild-type controls. We performed microarray-based sequencing on spinal cords from n = 3 E14.5 Nfix−/− mice and n = 3 E14.5 Nfix+/+ mice. Differential gene expression analysis, using a false discovery rate (FDR) p-value of p < 0.05, and a fold change cut-off for differential expression of > ± 1.5, revealed 1351 differentially regulated genes in the spinal cord of Nfix−/− mice. Of these, 828 were upregulated, and 523 were downregulated. This resource provides a tool to interrogate the role of this transcription factor in spinal cord development.

Spinal motoneuron firing properties mature from rostral to caudal during post-natal development of the mouse

Altricial mammals are born with immature nervous systems comprised of circuits that do not yet have the neuronal properties and connectivity required to produce future behaviours. During the critical period of post-natal development, neuronal properties are tuned to participate in functional circuits. In rodents, cervical motoneurons are born prior to lumbar motoneurons, and spinal cord development follows a sequential rostro-caudal sequence. Here we asked whether birth order is reflected in the post-natal development of electrophysiological properties. We show that motoneurons of both segments have similar properties at birth and follow the same developmental profile, with maximal firing increasing and excitability decreasing into the 3rd post-natal week. However, these maturative processes occur in cervical prior to lumbar motoneurons, correlating to the timing of arrival of descending systems. These results suggest that motoneuron properties do not mature by cell autonomous mechanisms alone, but rather depend on developing descending and spinal circuits.