Pax3/7 regulates neural tube closure and patterning in a non-vertebrate chordate

Abstract

Pax3/7 factors play numerous roles in the development of the dorsal nervous system of vertebrates. From specifying neural crest at the neural plate borders, to regulating neural tube closure and patterning of the resulting neural tube. However, it is unclear which of these roles are conserved in non-vertebrate chordates. Here we investigate the expression and function of Pax3/7 in the model tunicate Ciona. Pax3/7 is expressed in neural plate border cells during neurulation, and in central nervous system progenitors shortly after neural tube closure. We find that separate cis-regulatory elements control the expression in these two distinct lineages. Using CRISPR/Cas9-mediated mutagenesis, we knocked out Pax3/7 in F0 embryos specifically in these two separate territories. Pax3/7 knockout in the neural plate borders resulted in neural tube closure defects, suggesting an ancient role for Pax3/7 in this chordate-specific process. Furthermore, knocking out Pax3/7 in the neural impaired Motor Ganglion neuron specification, confirming a conserved role for this gene in patterning the neural tube as well. Taken together, these results suggests that key functions of Pax3/7 in neural tube development are evolutionarily ancient, dating back at least to the last common ancestor of vertebrates and tunicates.

A gene regulatory network for specification and morphogenesis of a Mauthner Cell homolog in non-vertebrate chordates

“…To verify whether the expression of the selected reporter plasmids in the ddNs depends on Pax3/7, we performed tissue-specific CRISPR/Cas9-mediated mutagenesis in the MG in F0 larvae ( Figure 3A ), as routinely carried out in Ciona (Gandhi et al, 2018). We used the Fgf8/17/18 promoter to drive expression of Cas9 ( Fgf8/17/18>Cas9 ) in the A9.30 lineage, which gives rise to most of the neurons in the MG including the ddNs (Cole and Meinertzhagen, 2004; Imai et al, 2009; Stolfi and Levine, 2011), and previously published single-chain guide RNA (sgRNA) expression plasmids targeting Pax3/7 (Kim et al, 2022). Previously, this MG-specific CRISPR mutagenesis of Pax3/7 resulted in significant loss of the Dmbx/Defcab reporter expression in the ddNs (Kim et al, 2022).…”

“…We used the Fgf8/17/18 promoter to drive expression of Cas9 ( Fgf8/17/18>Cas9 ) in the A9.30 lineage, which gives rise to most of the neurons in the MG including the ddNs (Cole and Meinertzhagen, 2004; Imai et al, 2009; Stolfi and Levine, 2011), and previously published single-chain guide RNA (sgRNA) expression plasmids targeting Pax3/7 (Kim et al, 2022). Previously, this MG-specific CRISPR mutagenesis of Pax3/7 resulted in significant loss of the Dmbx/Defcab reporter expression in the ddNs (Kim et al, 2022). Here we assayed the expression of the three remaining ddN reporters ( Saxo, Ddpep, and Seldom ), comparing between Pax3/7 CRISPR and “negative control” CRISPR larvae electroporated instead with an sgRNA that does not target any known Ciona sequences (Stolfi et al, 2014).…”

“…Because Pax3/7, but not Pou4 or Lhx1/5, was sufficient and necessary for Ddpep reporter expression in the ddNs, we hypothesized that this regulatory logic might also operate in the ACINs. Immunostaining with a pan-bilaterian Pax3/7 antibody (Davis et al, 2005) and Pax3/7 reporter plasmids (Horie et al, 2018; Kim et al, 2022) suggested that ACINs also express Pax3/7 ( Supplemental Figure 5A,B ). Because ACINs derive from the A9.29 cell lineages (Nishitsuji et al, 2012), we used the Ephrin A.b promoter ( Supplemental Figure 5C ) to drive expression of Cas9 in these cells to disrupt Pax3/7 in ACIN progenitors.…”

“…Embryos, larvae, and juveniles were fixed in MEM-FA solution (3.7% formaldehyde, 0.5M NaCl, 0.1M MOPS pH 7.4, 1 mM EGTA, 2 mM MgSO4, 0.1% Triton-X100) and washed first in 1X PBS/0.4% Triton-X100/50mM NH4Cl, then in 1X PBS/0.1% Triton-X100 before mounting in 1X PBS/2% DABCO/50% glycerol. Pax3/7 and mCherry immunostains were performed as previously described (Kim et al, 2022) using mouse anti-Pax3/7 antibody graciously provided by Nipam Patel (Davis et al, 2005), rabbit anti-mCherry (BioVision antibody 5993), and Alexa Fluor 555 anti-mouse or rabbit secondary antibodies (Thermo Fisher). Slides were imaged on a Leica DMI8 or DM IL LED inverted epifluorescence microscope with a K3M or DFC3000 G monochrome digital camera.…”

“…Previous work on the ddNs revealed that the transcription factor Pax3/7 is necessary and sufficient for the specification of ddN fate and its unique morphology. Tissue-specific CRISPR/Cas9 mutagenesis of Pax3/7 abolished ddN fate (Kim et al, 2022), while overexpression of Pax3/7 throughout the MG resulted in ectopic ddN-like neurons that also projected axons across the midline (Stolfi et al, 2011). Furthermore, microarray-based transcriptome of isolated ddNs revealed several novel candidate effectors of ddN development and function (Gibboney et al, 2020).…”