High-throughput mapping of cellular differentiation hierarchies from single-cell data promises to empower systematic interrogations of vertebrate development and disease. Here we applied single-cell RNA sequencing to >92,000 cells from zebrafish embryos during the first day of development. Using a graph-based approach, we mapped a cell-state landscape that describes axis patterning, germ layer formation, and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon-based barcoding approach (TracerSeq) for reconstructing single-cell lineage histories. Clonally related cells were often restricted by the state landscape, including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in embryos lacking the chordin gene. We provide web-based resources for further analysis of the single-cell data.

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Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo

Environmental Control of Astrocyte Pathogenic Activities in CNS Inflammation.

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain

The neuroendocrine hypothalamus is composed of the tuberal and anterodorsal hypothalamus, together with the median eminence/neurohypophysis It centrally governs wide-ranging physiological processes, including homeostasis of energy balance, circadian rhythms and stress responses, as well as growth and reproductive behaviours Homeostasis is maintained by integrating sensory inputs and effecting responses via autonomic, endocrine and behavioural outputs, over diverse time-scales and throughout the lifecourse of an individual Here, we summarize studies that begin to reveal how different territories and cell types within the neuroendocrine hypothalamus are assembled in an integrated manner to enable function, thus supporting the organism's ability to survive and thrive We discuss how signaling pathways and transcription factors dictate the appearance and regionalization of the hypothalamic primordium, the maintenance of progenitor cells, and their specification and differentiation into neurons We comment on recent studies that harness such programmes for the directed differentiation of human ES/iPS cells We summarize how developmental plasticity is maintained even into adulthood and how integration between the hypothalamus and peripheral body is established in the median eminence and neurohypophysis Analysis of model organisms, including mouse, chick and zebrafish, provides a picture of how complex, yet elegantly coordinated, developmental programmes build glial and neuronal cells around the third ventricle of the brain Such conserved processes enable the hypothalamus to mediate its function as a central integrating and response-control mediator for the homeostatic processes that are critical to life Early indications suggest that deregulation of these events may underlie multifaceted pathological conditions and dysfunctional physiology in humans, such as obesity

Development of the Neuroendocrine Hypothalamus

Critical Role for a Subset of Intestinal Macrophages in Shaping Gut Microbiota in Adult Zebrafish.

This article describes a procedure for the dissociation of zebrafish (Danio rerio) embryos to produce a suspension of single cells that is suitable for fluorescence-activated cell sorting (FACS). The method has been applied to embryos at stages from 14 h post fertilization (hpf) to larvae at 5 d post fertilization (dpf), and it has also been successfully used for isolating fluorescently tagged neurons from whole dissociated embryos and early larvae. The cell collection procedures described in this protocol may also be adapted for older embryos and juvenile zebrafish. RNA can be extracted from the sorted cells and used for subsequent quantitative real-time PCR (qRT-PCR), microarrays, or next-generation sequencing (NGS) experiments.

Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.

During ventral forebrain development, orthologs of the homeodomain transcription factor Nkx2.1 control patterning of hypothalamus, preoptic region, and ventral telencephalon. However, the relative contributions of Nkx2.1 and Nkx2.4 to prosencephalon development are poorly understood. Therefore we analysed functions of the previously uncharacterized nkx2.4-like zgc:171531 as well as of the presumed nkx2.1 orthologs nkx2.1a and nkx2.1b in zebrafish forebrain development. Our results show that zgc:171531 and nkx2.1a display overlapping expression patterns and a high sequence similarity. Together with a high degree of synteny conservation, these findings indicate that both these genes indeed are paralogs of nkx2.4. As a result, we name zgc:171531 now nkx2.4a, and changed the name of nkx2.1a to nkx2.4b, and of nkx2.1b to nkx2.1. In nkx2.1, nkx2.4a, and nkx2.4b triple morpholino knockdown (nkx2TKD) embryos we observed a loss of the rostral part of prosomere 3 and its derivative posterior tubercular and hypothalamic structures. Furthermore, there was a loss of rostral and intermediate hypothalamus, while a residual preoptic region still develops. The reduction of the ventral diencephalon was accompanied by a ventral expansion of the dorsally expressed pax6, revealing a dorsalization of the basal hypothalamus. Within the telencephalon we observed a loss of pallidal markers, while striatum and pallium are forming. At the neuronal level, nkx2TKD morphants lacked several neurosecretory neuron types, including avp, crh, and pomc expressing cells in the hypothalamus, but still form oxt neurons in the preoptic region. Our data reveals that, while nkx2.1, nkx2.4a and nkx2.4b genes act partially redundant in hypothalamic development, nkx2.1 is specifically involved in the development of rostral ventral forebrain including the pallidum and preoptic regions, whereas nkx2.4a and nkx2.4b control the intermediate and caudal hypothalamus.

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Martha Manoli

Papers

Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.

nkx2.1 and nkx2.4 genes function partially redundant during development of the zebrafish hypothalamus, preoptic region, and pallidum