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

Knockdown of Bardet-Biedl Syndrome Gene BBS9/PTHB1 Leads to Cilia Defects

Bardet-Biedl Syndrome (BBS, MIM#209900) is a genetically heterogeneous disorder with pleiotropic phenotypes that include retinopathy, mental retardation, obesity and renal abnormalities. Of the 15 genes identified so far, seven encode core proteins that form a stable complex called BBSome, which is implicated in trafficking of proteins to cilia. Though BBS9 (also known as PTHB1) is reportedly a component of BBSome, its direct function has not yet been elucidated. Using zebrafish as a model, we show that knockdown of bbs9 with specific antisense morpholinos leads to developmental abnormalities in retina and brain including hydrocephaly that are consistent with the core phenotypes observed in syndromic ciliopathies. Knockdown of bbs9 also causes reduced number and length of cilia in Kupffer's vesicle. We also demonstrate that an orthologous human BBS9 mRNA, but not one carrying a missense mutation identified in BBS patients, can rescue the bbs9 morphant phenotype. Consistent with these findings, knockdown of Bbs9 in mouse IMCD3 cells results in the absence of cilia. Our studies suggest a key conserved role of BBS9 in biogenesis and/or function of cilia in zebrafish and mammals

Pathfinding by identified growth cones in the embryonic zebrafish brain.

The embryonic zebrafish (Brachydanio rerio) brain was studied to explore its potential as a system to investigate mechanisms of growth cone guidance in the early vertebrate brain. The early brain tracts were identified and their development was traced with (1) antibodies that label neurons soon after axonogenesis, (2) fluorescent dyes that retrogradely or orthogradely label neurons, and (3) electron microscopy. The embryonic zebrafish brain was found to contain a few longitudinal tracts connected by commissures, which together form an early axon scaffold. Small clusters of neurons were identified that either establish the axon scaffold or later extend growth cones along specific parts of the axon scaffold. Neurons within an identifiable cluster were found to extend growth cones along well defined stereotyped pathways suggesting that their growth cones follow cell specific trajectories by responding to specific cues in their environment. Neurons in the nucleus of the posterior commissure (nucPC), one such identifiable cluster, project growth cones ventrally along the posterior commissure (PC) to an intersection in the anterior tegmentum where the commissure crosses two longitudinal tracts, the TPOC (tract of the postoptic commissure) and the MLF (medial longitudinal fasciculus). Once at the intersection nucPC growth cones turn posteriorly onto the TPOC in the dorsal tegmentum and follow it to the hindbrain. Elimination of the TPOC caused nucPC growth cones to make more errors and extend along aberrant paths in the anterior tegmentum and at the midbrain/hindbrain boundary. This suggests that fasciculation with axons in the TPOC helps to guide the growth cones along their normal pathway in these two regions. However, many nucPC growth cones select an appropriate pathway in the absence of the TPOC. This suggests that other cues associated with the anterior tegmentum and the midbrain/hindbrain boundary, independent of the TPOC, also help to guide the nucPC growth cones. These studies show that the embryonic zebrafish is an attractive system to investigate mechanisms of growth cone guidance in the vertebrate brain. They suggest that error-free navigation in the CNS may normally be brought about by the simultaneous operation of multiple guidance mechanisms.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105506/1/9135571.pdfDescription of 9135571.pdf : Restricted to UM users only

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