Abstract
F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.
Highlights
The extracellular matrix (ECM) is an essential component of many tissues, providing for structural support and guidance, and affecting signaling and homeostasis [1]
The onset of spon1b mRNA expression occurred at 9–10 hours post fertilization, increasing 6.8 fold by the end of the first day of development, and reaching 16.3 fold by 72 hpf (Fig. 1A)
Proceeded to characterize the expression patterns for spon1b in developing and adult zebrafish using three complementary approaches. These included the imaging of a fluorescent transgene signal, immunohistochemical (IHC) staining for GFP, and in situ hybridization (ISH) to localize spon1b mRNA expression
Summary
The extracellular matrix (ECM) is an essential component of many tissues, providing for structural support and guidance, and affecting signaling and homeostasis [1]. The F-spondin molecule consists of about 800 amino acids, and contains an N-terminal domain homologous to the amino terminus of reelin, a spondin domain and six C-terminal thrombospondin repeats [4] This structure allows F-spondin to affect different processes through binding to the ECM or membrane receptors. Whereas the C-terminal fragment binds to the ECM, promoting neuronal outgrowth along the basement membrane beneath the floor plate, the N-terminal fragment binds to several members of the low density lipoprotein receptor family (ApoER2, LRP2/megalin, and LRP4), inhibiting neuronal outgrowth and preventing these neurons from growing through the floor plate [9] These effects of F-spondin are likely to be conserved, since it is present in the embryonic floor plate in the frog, mouse, chick, and zebrafish [4,10,11,12]. Studies in vitro found that F-spondin accumulates in the ECM that ensheaths the developing peripheral nerves [14], promotes neurite outgrowth from embryonic hippocampal and commissural neurons [2,13], and potentiates nerve precursor differentiation [5]
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