Zebrafish spinal cord oligodendrocyte formation requiresbocfunction

Abstract

The axis of the vertebrate neural tube is patterned, in part, by a ventral to dorsal gradient of Shh signaling. In the ventral spinal cord, Shh induces concentration-dependent expression of transcription factors, subdividing neural progenitors into distinct domains that subsequently produce distinct neuronal and glial subtypes. In particular, progenitors of the pMN domain express the bHLH transcription factor Olig2 and produce motor neurons followed by oligodendrocytes, the myelinating glial cell type of the central nervous system. In addition to its role in patterning ventral progenitors, Shh signaling must be maintained through development to specify pMN progenitors for oligodendrocyte fate. Using a forward genetic screen in zebrafish for mutations that disrupt development of oligodendrocytes, we identified a new mutant allele of boc, which encodes a type I transmembrane protein that functions as a coreceptor for Shh. Embryos homozygous for the bocco25 allele, which creates a missense mutation in a Fibronectin type III domain that binds Shh, have normally patterned spinal cords but fail to maintain pMN progenitors, resulting in a deficit of oligodendrocytes. Using a sensitive fluorescent detection method for in situ RNA hybridization, we found that spinal cord cells express boc in a graded fashion that is inverse to the gradient of Shh signaling activity and that boc function is necessary to maintain pMN progenitors by shaping the Shh signaling gradient.

cdonandbocaffect trunk neural crest cell migration non-cell autonomously through a reduction of hedgehog signaling in zebrafish slow-twitch muscle

“…We found cdon and boc transcripts to be abundant in the dorsal central nervous system (CNS) of 24hpf-48hpf zebrafish embryos (Figure 2A-D), consistent with similar data from prior studies (Bergeron et al, 2011;Cardozo et al, 2014;Kearns et al, 2021). This pattern is established early in development.…”

“…The boc/uml allele (Bergeron et al, 2011) results in a predicted early stop (hereafter boc uml ). The boc co25 allele (Kearns et al, 2021) results in an non-synonymous isoleucine to serine substitution in the third FNIII domain (Figure 1C). As boc uml and boc co25 alleles do not complement each other (Kearns et al, 2021), we focus our analyses on boc co25 and note that we see similar results with boc uml .…”

“…We next used multiplexed hybrid chain reaction in situ hybridization (HCR) in order to ask where cdon and boc transcripts are expressed, and whether transcripts are detectable in tNCCs and slow-twitch muscle progenitors. Unlike traditional enzymatic methods for in situ hybridization, HCR allows single molecule measurement of gene expression at cellular resolution, which enables identifying cells and cell types that express transcripts at relatively low levels (Kearns et al, 2021).…”

“…At 2 somites, cdon is expressed in the presumptive notochord, while both cdon and boc are both expressed along the border of the neural plate in cells that we suspect contribute to the dorsal neural tube at 24hpf (Figure 2A). Importantly, while boc transcripts in the neural tube form a broad dorsal-ventral gradient (Kearns et al, 2021), cdon transcripts are restricted to the dorsal-most roofplate cells and the ventral most floorplate cells of the neural tube by 24hpf. In this way, cdon creates a dorsal sub-domain of boc expression in the neural tube (Figure 2D).…”

Fmrp regulates oligodendrocyte lineage cell specification and differentiation

“…Moreover, OL formation appears to require slightly higher levels of Shh signaling than OPCs, as pharmacological inhibition of Shh leads to more OPCs and fewer OLs and overexpression of Shh shifts the balance toward OL fate (Ravanelli et al, 2018). To test whether altered populations of OLCs and motor neurons in fmr1 mutants are associated with changes in Shh signaling, we used fluorescent RNA in situ hybridization at early embryonic stages to examine the expression of genes marking ventral progenitor domains and Shh signaling: olig2 , a marker of the pMN progenitor domain; nkx2.2 , a marker of the p3 progenitor domain; ptch2 , a positive transcriptional target of Shh (Concordet et al, 1996); and boc , a Shh co‐receptor and negative transcriptional target of Shh (Kearns et al, 2021; Tenzen et al, 2006). fmr1 mutant embryos had no obvious changes relative to wild‐type in the expression of olig2 at 24 or 30 hpf (Figure 7(a), (b), (e), (f)) or nkx2.2 at 30 hpf (Figure 7(e′), (f′)), which again suggests a general maintenance of spinal cord progenitor boundaries in fmr1 mutants.…”