Illusionary Self-Motion Perception in Zebrafish

Zebrafish mutant belladonna (bel) carries a mutation in the lhx2 gene (encoding a Lim domain homeobox transcription factor) that results in a defect in retinotectal axon pathfinding, which can lead to uncrossed optic nerves failing to form an optic chiasm. Here, we report on a novel swimming behavior of the bel mutants, best described as looping. Together with two previously reported oculomotor instabilities that have been related to achiasmatic bel mutants, reversed optokinetic response (OKR) and congenital nystagmus (CN, involuntary conjugate oscillations of both eyes), looping opens a door to study the influence of visual input and eye movements on postural balance. Our result shows that looping correlates perfectly with reversed OKR and CN and is vision-dependent and contrast sensitive. CN precedes looping and the direction of the CN slow phase is predictive of the looping direction, but is absent during looping. Therefore, looping may be triggered by CN in bel. Moreover, looping in wild-type fish can also be evoked by whole-field motion, suggesting that looping in a bel mutant larvae is a result of self-motion perception. In contrary to previous hypotheses, our findings indicate that postural control in vertebrates relies on both direct visual input (afference signal) and eye-movement-related signals (efference copy or reafference signal)

Attenuation of Notch and Hedgehog Signaling Is Required for Fate Specification in the Spinal Cord

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling

Visualization of Gli Activity in Craniofacial Tissues of Hedgehog-Pathway Reporter Transgenic Zebrafish

The Hedgehog (Hh)-signaling pathway plays a crucial role in the development and maintenance of multiple vertebrate and invertebrate organ systems. Gli transcription factors are regulated by Hh-signaling and act as downstream effectors of the pathway to activate Hh-target genes. Understanding the requirements for Hh-signaling in organisms can be gained by assessing Gli activity in a spatial and temporal fashion.We have generated a Gli-dependent (Gli-d) transgenic line, Tg(Gli-d:mCherry), that allows for rapid and simple detection of Hh-responding cell populations in both live and fixed zebrafish. This transgenic line expresses a mCherry reporter under the control of a Gli responsive promoter, which can be followed by using fluorescent microscopy and in situ hybridization. Expression of the mCherry transgene reporter during embryogenesis and early larval development faithfully replicated known expression domains of Hh-signaling in zebrafish, and abrogating Hh-signaling in transgenic fish resulted in the suppression of reporter expression. Moreover, ectopic shh expression in Tg(Glid:mCherry) fish led to increased transgene production. Using this transgenic line we investigated the nature of Hh-pathway response during early craniofacial development and determined that the neural crest skeletal precursors do not directly respond to Hh-signaling prior to 48 hours post fertilization, suggesting that earlier requirements for pathway activation in this population of facial skeleton precursors are indirect.We have determined that early Hh-signaling requirements in craniofacial development are indirect. We further demonstrate the Tg(Gli-d:mCherry) fish are a highly useful tool for studying Hh-signaling dependent processes during embryogenesis and larval stages

Linking Human Diseases to Animal Models Using Ontology-Based Phenotype Annotation

A novel method for quantifying the similarity between phenotypes by the use of ontologies can be used to search for candidate genes, pathway members, and human disease models on the basis of phenotypes alone

Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles