Retinoic acid production, regulation and containment through Zic1, Pitx2c and Cyp26c1 control cranial placode specification

Abstract

All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In Xenopus, Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here we identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain. Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that pitx2c is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a program of RA containment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain.

Repressive Interactions Between Transcription Factors Separate Different Embryonic Ectodermal Domains

“…While there are several examples of cell-cell signaling being important in placode and neural crest induction (Begbie et al, 1999;Brugmann et al, 2004;Ahrens and Schlosser, 2005;Litsiou et al, 2005;Monsoro-Burq et al, 2005;Watanabe et al, 2015;Hintze et al, 2017;Plouhinec et al, 2017;reviewed in;Milet and Monsoro-Burq, 2012;Stuhlmiller and Garcia-Castro, 2012;Saint-Jeannet and Moody, 2014;Pla and Monsoro-Burq, 2018;Streit, 2018;Schlosser, 2021), there also is evidence for indirect signaling. For example, Dlx5 indirectly induces epidermal and PPR genes (McLarren et al, 2003) and Zic1 affects PPR gene expression at a distance by regulating retinoic acid signaling (Jaurena et al, 2015;Dubey et al, 2021). Since an alternate explanation is that the nβgal mRNA was selectively diluted in part of the clone, it will be important to confirm our lineage tracing data by grafting TFexpressing cells into an ectopic domain and observing reduced expression in the adjacent host tissue, as has been elegantly shown for dlx5 and six1 in Xenopus (Woda et al, 2003;Ahrens and Schlosser, 2005).…”

Anterior patterning genes induced by Zic1 are sensitive to retinoic acid and its metabolite, 4‐oxo‐RA

“…We further found that the RA-catabolizing enzyme, Cyp26c1, and the RA-dependent transcription factor Pitx2c expressed in a domain between ANP and PPR to be critical for PPR formation. 5 Interestingly, we also discovered that one of the RA metabolites generated by Cyp26c1, 4-oxo-RA, to be present during development at stages critical for the induction and specification of PPR. Consistent with this observation, previous reports have shown that 4-oxo-RA is capable of inducing signaling via RA receptors (RARs) to modulate the development of the anterior-posterior (A-P) axis in the embryo.…”

“…We next sought to verify that Zic1 is required for the expression of these genes. Using a well-characterized Zic1 morpholino antisense oligonucleotides (Zic1MO [3][4][5]15 ), we knocked down Zic1 in one half of the embryo at the two-cell stage and assessed the consequences by ISH at NF stage 15 (Figure 2). Our analysis revealed that Zic1 knockdown indeed disrupted the expression of these genes, providing further evidence that they are Zic1 targets.…”

“…2.4 | Patterning genes respond to both RA and 4-oxo-RA Further assessment of the impact of RA signaling on the expression of Zic1 targets was performed through doseresponse experiments. Given our previous findings that Xenopus embryos contain a significant amount of the RA metabolite, 4-oxo-RA, 5 we wanted to determine if 4-oxo-RA is capable of regulating the expression of Zic1 targets. 4-oxo-RA is one of the polar metabolites generated by the action of Cyp26 enzymes on all-trans RA [18][19][20] ; however, the extent of its biological activity, if any, has been debated.…”

“…Figure 5 shows the summary findings of changes in gene expression of crx, fezf2, nkx3-1, pkdcc.2, and xanf1. We have previously shown that pitx2c and hnf1b show decreasing and increasing expression, respectively, upon treatment with increasing RA doses 5 and they serve as positive controls for the treatments. Our analysis reveals 4-oxo-RA as not only capable of regulating the expression of Zic1 targets, but that the effect of 4-oxo-RA is very similar to that of RA, albeit at a higher dose (Figure 4, compare 1 μM RA and 10 μM 4-oxo-RA).…”