Early inflammation dysregulates neuronal circuit formation in vivo via upregulation of IL-1ß.

Neuro-immune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well understood. We performed in vivo imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyper-dynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish we showed that these morphological changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1ß (IL-1ß) is upregulated by the inflammatory insult, and that down-regulation of IL-1ß abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the RGC arbors in IL-1ß morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1ß in mediating the pathological effects of inflammation on neuronal circuit development.SIGNIFICANCE STATEMENTMaternal immune activation (MIA) can increase the risk of neurodevelopmental disorders in offspring, however the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphological changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1ß plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders.

Maternal immune activation in neurodevelopmental disorders.

Converging lines of evidence from basic science and clinical studies suggest a relationship between maternal immune activation (MIA) and neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. The mechanisms through which MIA increases the risk of neurodevelopmental disorders have become a subject of intensive research. This review aims to describe how dysregulation of microglial function and immune mechanisms may link MIA and neurodevelopmental pathologies. We also summarize the current evidence in animal models of MIA. Developmental Dynamics 247:588-619, 2018. © 2017 Wiley Periodicals, Inc.

Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.

This corrects the article DOI: 10.1038/icb.2016.51.

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain.

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons.

A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells.

E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 (ITF-2/ME2/SEF2/TCF4), E2A (TCF3), and HEB (ME1/REB/TCF12). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog (SpE-protein). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site (SpE-Alt). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form (SpE-Can), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt(+) cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E-protein biology.

Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.

The purple sea urchin (Strongylocentrotus purpuratus) genome sequence contains a complex repertoire of genes encoding innate immune recognition proteins and homologs of important vertebrate immune regulatory factors. To characterize how this immune system is deployed within an experimentally tractable, intact animal, we investigate the immune capability of the larval stage. Sea urchin embryos and larvae are morphologically simple and transparent, providing an organism-wide model to view immune response at cellular resolution. Here we present evidence for immune function in five mesenchymal cell types based on morphology, behavior and gene expression. Two cell types are phagocytic; the others interact at sites of microbial detection or injury. We characterize immune-associated gene markers for three cell types, including a perforin-like molecule, a scavenger receptor, a complement-like thioester-containing protein and the echinoderm-specific immune response factor 185/333. We elicit larval immune responses by (1) bacterial injection into the blastocoel and (2) seawater exposure to the marine bacterium Vibrio diazotrophicus to perturb immune state in the gut. Exposure at the epithelium induces a strong response in which pigment cells (one type of immune cell) migrate from the ectoderm to interact with the gut epithelium. Bacteria that accumulate in the gut later invade the blastocoel, where they are cleared by phagocytic and granular immune cells. The complexity of this coordinated, dynamic inflammatory program within the simple larval morphology provides a system in which to characterize processes that direct both aspects of the echinoderm-specific immune response as well as those that are shared with other deuterostomes, including vertebrates.

An ancient role for Gata-1/2/3 and Scl transcription factor homologs in the development of immunocytes.

Although vertebrate hematopoiesis is the focus of intense study, immunocyte development is well-characterized in only a few invertebrate groups. The sea urchin embryo provides a morphologically simple model for immune cell development in an organism that is phylogenetically allied to vertebrates. Larval immunocytes, including pigment cells and several blastocoelar cell subtypes, emerge from a population of non-skeletal mesodermal (NSM) precursors that is specified at the blastula stage. This ring of cells is first partitioned into oral and aboral fields with distinct blastocoelar and pigment cell gene regulatory programs. The oral field is subsequently specified into several distinct immune and non-immune cell types during gastrulation. Here we characterize the oral NSM expression and downstream function of two homologs of key vertebrate hematopoietic transcription factors: SpGatac, an ortholog of vertebrate Gata-1/2/3 and SpScl, an ortholog of Scl/Tal-2/Lyl-1. Perturbation of SpGatac affects blastocoelar cell migration at gastrulation and later expression of immune effector genes, whereas interference with SpScl function disrupts segregation of pigment and blastocoelar cell precursors. Homologs of several transcription regulators that interact with Gata-1/2/3 and Scl factors in vertebrate hematopoiesis are also co-expressed in the oral NSM, including SpE-protein, the sea urchin homolog of vertebrate E2A/HEB/E2-2 and SpLmo2, an ortholog of a dedicated cofactor of the Scl-GATA transcription complex. Regulatory analysis of SpGatac indicates that oral NSM identity is directly suppressed in presumptive pigment cells by the transcription factor SpGcm. These findings provide part of a comparative basis to understand the evolutionary origins and regulatory biology of deuterostome immune cell differentiation in the context of a tractable gene regulatory network model.

Cell lineage tracing techniques for the study of brain development and regeneration.

Characterization of the means by which cells are generated and organized to make an organ as complex as the brain is a formidable task. Understanding how adult stem cells give rise to progeny that integrate into the existing structures during regeneration or in response to injury is equally challenging. Lineage tracing techniques are essential to studying cell behaviors such as proliferation, migration and differentiation, since they allow stem or precursor cells to be marked and their descendants followed and characterized over time. Here, we describe some of the key lineage tracing techniques available to date, highlighting advantages and drawbacks and focusing on their application in neural fate mapping. The more traditional methods are now joined by exciting new approaches to provide a vast array of tools at the disposal of neurobiologists.