Novel cell states arise in embryonic cells devoid of key reprogramming factors.

The capacity for embryonic cells to differentiate relies on a large-scale reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this reprogramming step is achieved by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Yet, it remains unclear whether cells lacking this reprogramming step are directed towards wild type states or towards novel developmental canals in the Waddington landscape of embryonic development. Here we investigate the developmental fate of embryonic cells mutant for NPS by analyzing their single-cell gene expression profiles. We find that cells lacking the first developmental reprogramming steps can acquire distinct cell states. These states are manifested by gene expression modules that result from a failure of nuclear reprogramming, the persistence of the maternal program, and the activation of somatic compensatory programs. As a result, most mutant cells follow new developmental canals and acquire new mixed cell states in development. In contrast, a group of mutant cells acquire primordial germ cell-like states, suggesting that NPS-dependent reprogramming is dispensable for these cell states. Together, these results demonstrate that developmental reprogramming after fertilization is required to differentiate most canonical developmental programs, and loss of the transient totipotent state canalizes embryonic cells into new developmental states in vivo.

Apollo-NADP+ reveals in vivo adaptation of NADPH/NADP+ metabolism in electrically activated pancreatic ß cells.

Several genetically encoded sensors have been developed to study live cell NADPH/NADP+ dynamics, but their use has been predominantly in vitro. Here, we developed an in vivo assay using the Apollo-NADP+ sensor and microfluidic devices to measure endogenous NADPH/NADP+ dynamics in the pancreatic ß cells of live zebrafish embryos. Flux through the pentose phosphate pathway, the main source of NADPH in many cell types, has been reported to be low in ß cells. Thus, it is unclear how these cells compensate to meet NADPH demands. Using our assay, we show that pyruvate cycling is the main source of NADP+ reduction in ß cells, with contributions from folate cycling after acute electrical activation. INS1E ß cells also showed a stress-induced increase in folate cycling and further suggested that this cycling requires both increased glycolytic intermediates and cytosolic NAD+. Overall, we show in vivo application of the Apollo-NADP+ sensor and reveal that ß cells are capable of adapting NADPH/NADP+ redox during stress.

Chromatin expansion microscopy reveals nanoscale organization of transcription and chromatin.

Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.

Live imaging and conditional disruption of native PCP activity using endogenously tagged zebrafish sfGFP-Vangl2.

Tissue-wide coordination of polarized cytoskeletal organization and cell behaviour, critical for normal development, is controlled by asymmetric membrane localization of non-canonical Wnt/planar cell polarity (PCP) signalling components. Understanding the dynamic regulation of PCP thus requires visualization of these polarity proteins in vivo. Here we utilize CRISPR/Cas9 genome editing to introduce a fluorescent reporter onto the core PCP component, Vangl2, in zebrafish. Through live imaging of endogenous sfGFP-Vangl2 expression, we report on the authentic regulation of vertebrate PCP during embryogenesis. Furthermore, we couple sfGFP-Vangl2 with conditional zGrad GFP-nanobody degradation methodologies to interrogate tissue-specific functions for PCP. Remarkably, loss of Vangl2 in foxj1a-positive cell lineages causes ependymal cell cilia and Reissner fiber formation defects as well as idiopathic-like scoliosis. Together, our studies provide crucial insights into the establishment and maintenance of vertebrate PCP and create a powerful experimental paradigm for investigating post-embryonic and tissue-specific functions for Vangl2 in development and disease.

Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development.

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

Understanding Idiopathic Scoliosis: A New Zebrafish School of Thought.

Idiopathic scoliosis (IS) refers to a 3D rotation of the spine that occurs in the absence of underlying vertebral anomalies or obvious physiological defects. Despite affecting approximately 4% of the population, the etiology and pathogenesis of IS remain poorly understood, largely due to genetic heterogeneity and historical lack of appropriate developmental models. Recently, zebrafish has emerged as a powerful system for studying IS, owing to well-developed genetic resources and a natural susceptibility to spinal curvatures. Here, we summarize the utility of zebrafish as a genetic and biological model of IS, examine current faithful mutant IS models, and focus on their recent advances towards understanding core mechanisms governing both normal spine morphogenesis and the pathogenesis of IS-like spinal deformities.