Identification of spinal neurons in the embryonic and larval zebrafish

Previous studies indicated that the developing fish spinal cord was a simple system containing a small number of distinguishable neuronal cell types (Eisen et al., Nature 320 :269–271, '86; Kuwada, Science, 233 :740–746, '86). To verify this we have characterized the cellular anatomy of the spinal cord of developing zebrafish in order to determine the number, identities, and organization of the spinal neurons. Spinal neurons were labeled by intracellular dye injections, application of an axonal tracer dye to all or subsets of the axonal tracts, and application of antibodies which recognize embryonic neurons. We found that nine classes of neurons could be identified based on soma size and position, pattern of dendrites, axonal trajectory, and time of axonogenesis. These are two classes of axial motor neurons, which have been previously characterized (Myers, J. Comp. Neurol. 236 :555–561, '85), one class of sensory neurons, and six classes of interneurons. One of the interneuron classes could be subclassified as primary and secondary based on criteria similar to those used to classify the axial motor neurons into primary and secondary classes. The early cord (18–20 hours) is an extremely simple system and contains approximately 18 lateral cell bodies per hemisegment, which presumably are post-mitotic cells. By this stage, five of the neuronal classes have begun axonogenesis including the primary motor neurons, sensory neurons, and three classes of interneurons. By concentrating on these early stages when the cord is at its simplest, pathfinding by growth cones of known identities can be described in detail. Then it should be possible to test many different mechanisms which may guide growth cones in the vertebrate central nervous system (CNS).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50048/1/903020315_ftp.pd

Self-organizing spots get under your skin.

Sixty-five years after Turing first revealed the potential of systems with local activation and long-range inhibition to generate pattern, we have only recently begun to identify the biological elements that operate at many scales to generate periodic patterns in nature. In this Primer, we first review the theoretical framework provided by Turing, Meinhardt, and others that suggests how periodic patterns could self-organize in developing animals. This Primer was developed to provide context for recent studies that reveal how diverse molecular, cellular, and physical mechanisms contribute to the establishment of the periodic pattern of hair or feather buds in the developing skin. From an initial emphasis on trying to disambiguate which specific mechanism plays a primary role in hair or feather bud development, we are beginning to discover that multiple mechanisms may, in at least some contexts, operate together. While the emergence of the diverse mechanisms underlying pattern formation in specific biological contexts probably reflects the contingencies of evolutionary history, an intriguing possibility is that these mechanisms interact and reinforce each other, producing emergent systems that are more robust

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD

<div><p>Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in <i>trans</i>) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in <i>cis</i>). As a result, forms of DeltaD that have the MID can activate Notch in <i>trans</i> and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in <i>trans</i> fails but ability to inhibit in <i>cis</i> is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.</p></div

Identification of the <i>Mib1</i>-interacting domain (MID) in the Notch ligand DeltaD.

<p>(A) DeltaD deletion constructs and point mutations. Asterisks represent relative positions of NN and KNxNKK motifs. (B) Mib1 does not interact effectively with DeltaD ∆B-D. Myc-Mib1 was co-immunoprecipitated with full-length DeltaD and truncation mutants (∆D, ∆C-D, ∆B-D) using zdd2 antibody (Ab) and detected with anti-Myc Ab. (C) ∆B-D is not effectively ubiquitylated by Mib1. Full-length and DeltaD truncation mutants, co-transfected with HA-ubiquitin (HA-Ub), with and without Myc-Mib1, were immunoprecipitated with zdd2 Ab and immunoblotted with anti-HA Ab to detect ubiquitylated DeltaD. (D) Delta ∆A (∆A) and Delta ∆B (∆B) interact poorly with Mib1. HA-tagged DeltaD and deletion constructs co-transfected with and without Myc-Mib1 are immunoprecipitated with anti-Myc Ab and detected with anti-HA Ab. Relative density of IP anti-Myc band normalized to lysate anti-HA band. (E) ∆B is not effectively ubiquitylated by Mib1. DeltaD-HA and deletion constructs are immunoprecipitated with anti-HA Ab to detect total ubiquitylated DeltaD with and without Myc-Mib1.</p

Endocytosis of DeltaD deletion mutants.

<p>(A,E,I,M,Q,U) Distribution of zdd2 (green) in COS7 cells transfected with DeltaD (A) or DeltaD ∆A, ∆B, ∆C, ∆D or ∆A-D deletion mutants (E, I, M, Q, U). Surface DeltaD was first labelled by incubation with zdd2 at 4°C for 30’ then, following washout of unbound zdd2, internalization was allowed for 30’ at 37°C. Nuclei were labelled with DAPI (blue). (B-D, F-H, J-L, N-P, R-T, V-X) Distribution of zdd2 (green) in COS7 cells co-transfected with DeltaD constructs and Mib1 (red) following internalization as described above. Each set of 3 panels, respectively, shows distribution of the DeltaD construct (green), Myc-Mib1 (red)/nuclei (blue), and the merged image. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127864#sec002" target="_blank">materials and methods</a> for details.</p

Identification of critical residues in the Mib1-Interacting Domain (MID).

<p>(A) Conserved amino acids (pink shading) in the putative <i>Mib1</i>-Interacting Domain in Delta ligands. (B) KK and NN/KK mutants do not significantly interact with mind bomb. DeltaD-HA, ∆B and point mutation (NN, KK and NN/KK) constructs are co-immunoprecipitated with Myc-Mib1 using anti-Myc Ab and detected with anti-HA Ab. (C) KK and NN/KK mutants are not significantly ubiquitylated by Mib1. DeltaD-HA, ∆B and point mutation constructs co-transfected with Flag-Ubiquitin (Flag-Ub) with and without Myc-Mib1 are immunoprecipitated with anti-HA Ab and detected with Anti-Flag Ab to assay ubiquitylation of full length and mutant forms of DeltaD.</p

Ectopic expression of deltaD deletion and point mutant recapitulates neurogenic phenotype of DeltaD (∆A-D).

<p>(A) The prospective distribution of neurons revealed by the distribution of <i>huC</i> as revealed by <i>in situ</i> hybridization probe (purple) in control embryos injected with only <i>ß-galactosidase</i> mRNA. (B-F) <i>huC</i> in embryos co-injected with <i>ß-galactosidase</i> and <i>deltaD</i> (B), <i>deltaD-∆C</i> (C), <i>deltaD ∆B</i> (D), <i>deltaD NN/KK</i> (E) or <i>DeltaD ∆A-D</i> (F) mRNA. Distribution of ectopic mRNA injected in one cell at the two-cell stage revealed by X-Gal distribution (blue). Dorsal view, rostral to the left. Embryos are at approximately the 3 somite stage. (G) Quantification of the effect of ectopic expression of mRNA encoding various forms of DeltaD on the distribution of early neurons. Red indicates fraction with a neurogenic phenotype (increased density of neurons), Green—fraction with no obvious effect on neuron density, Blue- fraction with suppression of neurogenesis (reduced neuron density). P-values for pairwise comparison based on Fisher’s Exact test of independence. P >. 05 does not meet the criteria for the Null hypothesis that pairs contain an equivalent distribution of phenotype classes.</p

Mutations in the zebrafish unmask shared regulatory pathways controlling the development of catecholaminergic neurons

AbstractThe mechanism by which pluripotent progenitors give rise to distinct classes of mature neurons in vertebrates is not well understood. To address this issue we undertook a genetic screen for mutations which affect the commitment and differentiation of catecholaminergic (CA) [dopaminergic (DA), noradrenergic (NA), and adrenergic] neurons in the zebrafish,Danio rerio.The identified mutations constitute five complementation groups.motionlessandfoggyaffect the number and differentiation state of hypothalamic DA, telencephalic DA, retinal DA, locus coeruleus (LC) NA, and sympathetic NA neurons. Thetoo fewmutation leads to a specific reduction in the number of hypothalamic DA neurons.no soullacks arch-associated NA cells and has defects in pharyngeal arches, andsoullesslacks both arch-associated and LC cell groups. Our analyses suggest that the genes defined by these mutations regulate different steps in the differentiation of multipotent CA progenitors. They further reveal an underlying universal mechanism for the control of CA cell fates, which involve combinatorial usage of regulatory genes