Research Article: Investigation of Islet2a function in zebrafish embryos: Mutants and morphants differ in morphologic phenotypes and gene expression

Date Published: June 21, 2018

Publisher: Public Library of Science

Author(s): Rosa L. Moreno, Kristina Williams, Kenneth L. Jones, Angeles B. Ribera, Christoph Winkler.


Zebrafish primary motor neurons differ from each other with respect to morphology, muscle targets and electrophysiological properties. For example, CaP has 2-3-fold larger densities of both inward and outward currents than do other motor neurons. We tested whether the transcription factor Islet2a, uniquely expressed in CaP, but not other primary motor neurons, plays a role in specifying its stereotypic electrophysiological properties. We used both TALEN-based gene editing and antisense morpholino approaches to disrupt Islet2a function. Our electrophysiology results do not support a specific role for Islet2a in determining CaP’s unique electrical properties. However, we also found that the morphological phenotypes of CaP and a later-born motor neuron differed between islet2a mutants and morphants. Using microarrays, we tested whether the gene expression profiles of whole embryo morphants, mutants and controls also differed. Morphants had 174 and 201 genes that were differentially expressed compared to mutants and controls, respectively. Further, islet2a was identified as a differentially expressed gene. To examine how mutation of islet2a affected islet gene expression specifically in CaPs, we performed RNA in situ hybridization. We detected no obvious differences in expression of islet1, islet2a, or islet2b in CaPs of mutant versus sibling control embryos. However, immunolabeling studies revealed that an Islet protein persisted in CaPs of mutants, albeit at a reduced level compared to controls. While we cannot exclude requirement for some Islet protein, we conclude that differentiation of the CaP’s stereotypic large inward and outward currents does not have a specific requirement for Islet2a.

Partial Text

Mammalian spinal motor neurons comprise a heterogeneous population, as evidenced by their different morphological and functional properties [1] [2]. Several lines of evidence support the view that different combinations of LIM-homeodomain (LIM-HD) transcription factors direct specification of the diverse set of mammalian motor neuron subtypes [3][4].

Motor neuron subtypes differ not only with respect to peripheral axon trajectories and muscle targets but also electrical membrane properties [12, 20]. However, little is known about the mechanisms that direct differentiation of vertebrate motor neuron subtype-specific electrical membrane properties. In contrast, several studies implicate LIM-HD transcription factors in specification of motor neuron subtype-specific morphological properties (for review, [2, 3, 41, 42]. In Drosophila, islet (orthologous to zebrafish islet1 genes) is expressed in ventral, but not dorsal, motor neurons [19]. Further, loss of islet results in loss of dorsal motor neuron’s distinguishing axonal morphology and larger outward current densities [20, 43–45]. These studies suggest that the same transcription factor code might specify motor neuron subtype-specific differentiation of electrical membrane properties as well as morphological differentiation, a possibility not yet tested in vertebrates.




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