The vertebrate hindbrain is composed of a series of lineage-restricted segments termed rhombomeres. Segment-specific gene expression drives unique programs of neuronal differentiation. Two critical embryonic signaling pathways, Fibroblast Growth Factor (FGF) and Retinoic Acid (RA), regulate early embryonic rhombomere patterning. The earliest expressed hox genes, hoxb1b and hoxb1a in zebrafish, are logical candidates for establishing signaling networks that specify segmental identity. We sought to determine the mechanism by which hox genes regulate hindbrain patterning in zebrafish. We demonstrate that hoxb1a regulates r4-specific patterning, while hoxb1b regulates rhombomere segmentation and size. Hoxb1a and hoxb1b redundantly regulate vhnf1 expression. Loss of hoxb1b together with pbx4 reverts the hindbrain to a groundstate identity, demonstrating the importance of hox genes in patterning nearly the entire hindbrain, and a key requirement for Pbx in this process. Additionally, we provide evidence that while pbx genes regulate RA signaling, hoxb1b regulates hindbrain identity through complex regulation of FGF signaling.

BackgroundThe reiterated architecture of cranial motor neurons aligns with the segmented structure of the embryonic vertebrate hindbrain. Anterior-posterior identity of cranial motor neurons depends, in part, on retinoic acid signaling levels. The early vertebrate embryo maintains a balance between retinoic acid synthetic and degradative zones on the basis of reciprocal expression domains of the retinoic acid synthesis gene aldhehyde dehydrogenase 1a2 (aldh1a2) posteriorly and the oxidative gene cytochrome p450 type 26a1 (cyp26a1) in the forebrain, midbrain, and anterior hindbrain.ResultsThis manuscript investigates the role of zinc finger of the cerebellum (zic) transcription factors in regulating levels of retinoic acid and differentiation of cranial motor neurons. Depletion of zebrafish Zic2a and Zic2b results in a strong downregulation of aldh1a2 expression and a concomitant reduction in activity of a retinoid-dependent transgene. The vagal motor neuron phenotype caused by loss of Zic2a/2b mimics a depletion of Aldh1a2 and is rescued by exogenously supplied retinoic acid.ConclusionZic transcription factors function in patterning hindbrain motor neurons through their regulation of embryonic retinoic acid signaling.

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.

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