Characterisation and expression of zebrafish frizzled-3a (zfzd3a) during embryonic development

Wolfgang Hofmeister (2007). Characterisation and expression of zebrafish frizzled-3a (zfzd3a) during embryonic development PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Wolfgang Hofmeister
Thesis Title Characterisation and expression of zebrafish frizzled-3a (zfzd3a) during embryonic development
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2007-11
Thesis type PhD Thesis
Supervisor Key, Brian
Rothnagel, Joseph A.
Smith, Ross
Subjects 270000 Biological Sciences
Formatted abstract The Frizzled (Fzd) family of seven-pass transmembrane receptors are known to bind members of the secreted Wnt ligand family via a highly conserved extracellular cysteine-rich domain. Frizzled receptors can activate at least three different signaling pathways depending on the Frizzled, Wnt ligand and co-receptors involved. These pathways result in the transcription of β-catenin target genes, changes in intracellular Ca2+ levels, or the polarisation of cells orthogonal to the apical-basal axis. Frizzled-3 provides an example of the multifaceted nature of Wnt/Frizzled signaling as it has been shown to be involved in all three pathways depending on the cellular context. Frizzled signaling is essential throughout various stages of neural development in metazoans. In the present study we have cloned the zebrafish orthologue of frizzled-3 named frizzled-3a (zfzd3a). The transcript is organised over seven exons with the main coding region beginning in exon 2. The predicted zebrafish protein shows ~74% similarity to its human, mouse and rat orthologues. zFzd3a is maternally provided and continues to be expressed zygotically during embryogenesis and in the adult fish. During embryonic development it was highly expressed throughout the developing central nervous system, consistent with a role in neural development. Knock down of zFzd3a protein levels with either of two non-overlapping anti-sense morpholino oligonucleotides resulted in gross-morphological defects of the brain with increased apoptosis, while the major transverse and longitudinal subdivisions of the embryonic brain were normally specified. Overexpression of zfzda RNA partially rescued the morpholino induced phenotype confirming the specificity of zfzd3a knock down. In addition, zFzd3a was required for the correct formation of the primary axon scaffold at 24-28hpf. Knock down of zFzd3a resulted in minor defasciculation of the supraoptic tract (SOT), defasciculation of the post-optic commissure (POC) and a complete or partial loss of the anterior commissure (AC). Development of neuronal clusters in the telencephalon and diencephalon was only minimally affected.
In bilaterally symmetrical organisms commissures traverse the developing neuroepithelium to connect apposing sides of the nervous system. The AC is pioneered by axons of the dorsal rostral cluster which projects axons across the rostral surface of the telencephalon to join the contralateral drc. The POC is formed by the ventral rostral cluster of the diencephalon projecting axons into the contralateral cluster. Formation of these commissures requires appropriate expression of the slit family of axon guidance molecules in the forebrain to help channel axons across the rostral midline. While slit1a is present in the neuroepithelium at the site of axon crossing, slit2 expression surrounds the site of commissure formation. This thesis shows that zFzd3a is required in the neuroectoderm for restricting the expression of slit2 at the rostral midline, while acting positively on slit1 expression at the commissures. Knock down of zFzd3a resulted in expansion of slit2 at the rostral midline and a reduction in slit1a expression at the commissures. Blocking Slit2 activity following knock down of zFzd3a rescued the anterior commissure defect. This suggests that slit2 is largely responsible for the loss of the AC. Hence, zFzd3a acts indirectly in the forebrain to facilitate formation of the AC through restricting the expression domain of slit2 in the neuroepithelium. This is the first in vivo data implicating zFzd3a in patterning of slit expression domains and presents a novel role for Frizzled signaling in the zebrafish forebrain.

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