Extracellular Movement of Signaling Molecules

Extracellular signaling molecules have crucial roles in development and homeostasis, and their incorrect deployment can lead to developmental defects and disease states. Signaling molecules are released from sending cells, travel to target cells, and act over length scales of several orders of magnitude, from morphogen-mediated patterning of small developmental fields to hormonal signaling throughout the organism. We discuss how signals are modified and assembled for transport, which routes they take to reach their targets, and how their range is affected by mobility and stability

Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development

SummaryError-prone repair of DNA double-strand breaks (DSBs) has been postulated to occur through classical non-homologous end joining (NHEJ) in systems ranging from nematode somatic tissues to zebrafish embryos. Contrary to this model, we show that zebrafish embryos mutant for DNA polymerase theta (Polq), a critical component of alternative end joining (alt-EJ), cannot repair DSBs induced by CRISPR/Cas9 or ionizing radiation. In the absence of DSBs, polq mutants are phenotypically normal, but they do not survive mutagenesis and display dramatic differences in the mutation profiles compared with the wild-type. These results show that alt-EJ repair is essential and dominant during the early development of a vertebrate

Single-cell internalization during zebrafish gastrulation

AbstractDuring gastrulation, germ layers are formed as prospective mesodermal and endodermal cells internalize and come to underlie the ectoderm [1–9]. Despite the pivotal role of gastrulation in animal development, the cellular interactions underlying this process are poorly understood. In zebrafish, mesoderm and endoderm formation requires the Nodal signals Cyclops and Squint and their cofactor One-eyed pinhead (Oep) [10–14]. We found that marginal cells in maternal-zygotic oep (MZoep) mutants do not internalize during gastrulation and acquire neural and tail fates at the expense of head and trunk mesendoderm. The lack of internalization in MZoep embryos and the cell-autonomous requirement for oep in Nodal signaling enabled us to test whether internalization can be achieved by individual cells or whether it depends on interactions within a group of cells. We found that individual MZoep mutant cells transplanted to the margin of wild-type blastula embryos initially internalize with their neighbors but are unable to contribute to the mesendoderm. In the reciprocal experiment, single wild-type cells transplanted to the margin of MZoep mutant embryos autonomously internalize and can express the mesendodermal markers axial/foxA2 and sox17. These results suggest that internalization and mesendoderm formation in zebrafish can be attained autonomously by single cells

Axis formation in annual killifish: Nodal coordinates morphogenesis in absence of Huluwa prepatterning

Axis formation in fish and amphibians is initiated by a prepattern of maternal gene products in the blastula. The embryogenesis of annual killifish challenges prepatterning models because blastomeres disperse and then re-aggregate to form the germ layers and body axes. This dispersion-aggregation process prompts the question how axis determinants such as Huluwa and germ layer inducers such as Nodal function in annual killifish. Here we show in Nothobranchius furzeri that huluwa, the factor thought to break symmetry by stabilizing β-catenin, is a non-functional pseudogene. Nuclear β-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the incipient aggregate. Inhibition of Nodal signaling blocks aggregation and disrupts coordinated cell migration, establishing a novel role for this signaling pathway. These results reveal a surprising departure from classic mechanisms of axis formation: canonical Huluwa-mediated prepatterning is dispensable and Nodal coordinates morphogenesis. One Sentence Summary Axis formation in annual killifish relies on Nodal to coordinate cell migration and is independent of Huluwa-mediated prepatterning

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system

The Structure and Timescales of Heat Perception in Larval Zebrafish

SummaryAvoiding temperatures outside the physiological range is critical for animal survival, but how temperature dynamics are transformed into behavioral output is largely not understood. Here, we used an infrared laser to challenge freely swimming larval zebrafish with “white noise” heat stimuli and built quantitative models relating external sensory information and internal state to behavioral output. These models revealed that larval zebrafish integrate temperature information over a time-window of 400 ms preceding a swim bout and that swimming is suppressed right after the end of a bout. Our results suggest that larval zebrafish compute both an integral and a derivative across heat in time to guide their next movement. Our models put important constraints on the type of computations that occur in the nervous system and reveal principles of how somatosensory temperature information is processed to guide behavioral decisions such as sensitivity to both absolute levels and changes in stimulation

Monitoring Neural Activity with Bioluminescence during Natural Behavior

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca$^{2+}$-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.Molecular and Cellular Biolog

Ancient pigs reveal a near-complete genomic turnover following their introduction to Europe

Archaeological evidence indicates that pig domestication had begun by ∼10,500 y before the present (BP) in the Near East, and mitochondrial DNA (mtDNA) suggests that pigs arrived in Europe alongside farmers ∼8,500 y BP. A few thousand years after the introduction of Near Eastern pigs into Europe, however, their characteristic mtDNA signature disappeared and was replaced by haplotypes associated with European wild boars. This turnover could be accounted for by substantial gene flow from local European wild boars, although it is also possible that European wild boars were domesticated independently without any genetic contribution from the Near East. To test these hypotheses, we obtained mtDNA sequences from 2,099 modern and ancient pig samples and 63 nuclear ancient genomes from Near Eastern and European pigs. Our analyses revealed that European domestic pigs dating from 7,100 to 6,000 y BP possessed both Near Eastern and European nuclear ancestry, while later pigs possessed no more than 4% Near Eastern ancestry, indicating that gene flow from European wild boars resulted in a near-complete disappearance of Near East ancestry. In addition, we demonstrate that a variant at a locus encoding black coat color likely originated in the Near East and persisted in European pigs. Altogether, our results indicate that while pigs were not independently domesticated in Europe, the vast majority of human-mediated selection over the past 5,000 y focused on the genomic fraction derived from the European wild boars, and not on the fraction that was selected by early Neolithic farmers over the first 2,500 y of the domestication process

Regulation of Vg1 biogenesis during mesendoderm induction

The TGF-beta signals Vg1 and Nodal form heterodimers to induce the vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal. Here we investigate the mechanisms underlying Vg1 retention, processing, secretion and signaling in zebrafish. First, using a newly devised Synthetic Processing (SynPro) system, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. Our results establish SynPro as a new in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active Vg1-Nodal heterodimers. These observations suggest two strategies for rapid mesendoderm induction: chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly