Hutlet B and Schlosser G (2025), Expression and function of new candidate regula...

XB-ART-61519

Dev Biol 2025 Dec 08;528:128-142. doi: 10.1016/j.ydbio.2025.08.026.

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Expression and function of new candidate regulators of placodal neurogenesis in Xenopus laevis.

Hutlet B , Schlosser G .

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The transcription factor Six1 and its co-activator Eya1 play central and varied roles during the development of sensory neurons derived from the cranial placodes in vertebrates. Previous studies suggested that these proteins promote both the maintenance of proliferative neuronal progenitors and neuronal differentiation. Context-specific interactions of Six1 and/or Eya1 with different cofactors are likely to contribute to the activation of distinct target genes during different stages of placodal neurogenesis. However, while some protein binding partners of Six1 have recently been identified, little is known about Eya1 interaction partners. To uncover additional Eya1 interaction partners with potential roles in placodal neurogenesis, we here conduct a yeast two-hybrid screen in Xenopus laevis. Apart from confirming Six1 (Six1.L) and Six4 (Six4.L and Six4.S) as known protein interaction partners of Eya1, this screen identified 25 additional candidate binding partners including several transcription factors, chromatin modifiers and enzymes involved in posttranslational modifications. Analysis of the expression of genes encoding five of these candidates (garre1, msh6, zmym3, pias4, smarce1) during X. laevis embryogenesis by in situ hybridization, revealed extensive co-expression of each of these with eya1 during placodal development. This suggests potential roles of these candidates in the regulation of placodal neurogenesis possibly in conjunction with Eya1. In gain and loss of function studies of the E3 SUMO-ligase Pias4 and the chromatin modifier Smarce1, we confirm essential roles of both of these proteins for placodal neurogenesis but their mode of interaction with Eya1 remains to be further clarified.

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Species referenced: Xenopus laevis
Genes referenced: ankfy1 ccdc15 cfap184 exosc10 eya1 garre1 iars1 klhl41 kmt2a LOC105946211 msh6 myg1 pax3 pi4ka pias4 ppard six1 six4 smarce1 smg1 sox11 sox7 tmtc3 XB5896327 zmym3 znf319 znf630
GO keywords: neurogenesis [+]
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	Graphical Abstract
	Fig. 1. Expression of eya1 and its candidate interaction partners at neural plate stage. Xenopus laevis embryos after whole-mount in situ hybridization at stage 14–18 in lateral views (A, C, E, G, I, K- anterior is to the left) and dorsal views (B, D, F, H, J, L - anterior is up). Red arrows indicate expression in the preplacodal ectoderm; blue and black arrowheads indicate expression in the neural crest and neural plate, respectively.
	Fig. 2. Expression patterns of eya1 and its candidate interaction partners at early tailbud stage. Xenopus laevis embryos after whole-mount in situ hybridization at stage 22, in lateral views (anterior is to the left). The schematic drawing (A) shows the distribution of placodes in an early tailbud stage embryo. Arrows indicate expression in placodes and display the colour code used in the drawing (yellow: profundal placode; dark green: trigeminal placode; light green: olfactory placode, pink: otic placode; brown: lateral line placode). Arrowheads and asterisks indicate other areas of expression. Pink arrowhead: somites; black arrowhead: anteroventral noradrenergic cells: white asterisks: pharyngeal pouches. Blue asterisks (neural crest streams) and arrowheads indicate expression in areas where eya1 is not expressed. Panels D, F, H, J, L are magnifications of panels C, E, G, I, K, respectively. Abbreviations: cg, cement gland; E, eye, including overlying lens placode; F: forebrain; H: hindbrain; IM: intermediary mesoderm; LL/Ot/EB: posterior placodal area, from which lateral line (LL), otic (Ot), and epibranchial (EB) placodes develop; Ol, olfactory placode; Pr, profundal placode; V, trigeminal placode. Schematic drawing from Schlosser and Ahrens (2004). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. Expression patterns of eya1 and its candidate interaction partners at mid-tailbud stage. Xenopus laevis embryos after whole-mount in situ hybridization at stage 27, in lateral views (anterior is to the left). The schematic drawing (A) shows the distribution of placodes in a mid-tailbud stage embryo. Arrows indicate expression in placodes and display the colour code used in the drawing (yellow: profundal placode; dark green: trigeminal placode; light green: olfactory placode, pink: otic placode; brown: lateral line placode; orange: epibranchial placodes). For placodes derived from the posterior placodal area, only the otic placode and one lateral line placode are indicated in panels C–L. Arrowheads and asterisks indicate other areas of expression. Pink arrowhead: somites; white asterisks: pharyngeal pouches. Blue asterisks (neural crest streams) and arrowheads indicate expression in areas where eya1 is not expressed. Panels D, F, H, J, L are magnifications of panels C, E, G, I, K, respectively. Abbreviations: B, blood islands; E, prospective eye, including lens (L) placode; EB, epibranchial placodes; F: forebrain; H: hindbrain; LL, lateral line placodes; LPM, lateral plate mesoderm; M: midbrain; Ol, olfactory placode; Ot, otic placode; P, pronephros; Pr, profundal placode; V, trigeminal placode. Schematic drawing from Schlosser and Ahrens (2004).
	Fig. 4. Expression patterns of eya1 and its candidate interaction partners at late tailbud stage. Xenopus laevis embryos after whole-mount in situ hybridization at stage 34, in lateral views (anterior is to the left). The schematic drawing (A) shows the distribution of placodes in a late tailbud stage embryo. Arrows indicate expression in placodes and display the colour code used in the drawing (dark green: trigeminal placode; light green: olfactory placode, pink: otic placode; brown: lateral line placode; orange: epibranchial placodes). For placodes derived from the posterior placodal area, only the otic placode, one lateral line placode and one epibranchial placode are indicated in panels C–L. Arrowheads and asterisks indicate other areas of expression. Pink arrowhead: somites (with hypaxial muscle precursors indicated separately at ventral edge of somites in B and F); white asterisks: pharyngeal pouches. Blue asterisks (neural crest streams) and arrowheads indicate expression in areas where eya1 is not expressed. Panels D, F, H, J, L are magnifications of panels C, E, G, I, K, respectively. Abbreviations: B, blood islands; E, prospective eye, including lens (L) placode; EB, epibranchial placodes; F: forebrain; H: hindbrain; LL, lateral line placodes; LPM: lateral plate mesoderm; M, midbrain; Ol, olfactory placode; Ot, otic placode; P, pronephros; V, trigeminal placode. Schematic drawing from Schlosser and Ahrens (2004).
	Fig. 5. Effects of pias4 knockdown. Neural plate stage embryos after unilateral injection of pias4.L MO. Embryos were injected at 16- to 64-cell stage in a blastomere contributing to cranial placodes and/or ganglia. LacZ mRNA was co-injected as a lineage tracer and X-gal staining (blue) marks the injected side (lower half). In all panels, asterisks indicate decreased expression and arrows increased expression in non-neural (red) or neural (blue) ectoderm. A-D: Placodal and neural crest markers. Alternative phenotypes shown for foxd3 (C) in main panel and insert. E-H: Neurogenesis markers. Alternative phenotypes shown for neurog2 (F) in main panel and insert. For n numbers and frequency of phenotypes see Table 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 6. Effects of pias4 overexpression. Neural plate stage embryos after unilateral injection of pias4.S mRNA. Embryos were injected at 16- to 64-cell stage in a blastomere contributing to cranial placodes and/or ganglia. LacZ mRNA was co-injected as a lineage tracer and X-gal staining (blue) marks the injected side (lower half). In all panels, asterisks indicate decreased expression and arrows increased expression in non-neural (red) or neural (blue) ectoderm. A-D: Placodal and neural crest markers. E-H: Neurogenesis markers. Insert in D shows an embryo with reduction of Sox3 in the neural plate. For n numbers and frequency of phenotypes see Table 2.
	Fig. 7. Effects of smarce1 knockdown. Neural plate stage embryos after unilateral injection of smarce1.LS MO1. Embryos were injected at 16- to 64-cell stage in a blastomere contributing to cranial placodes and/or ganglia. LacZ mRNA was co-injected as a lineage tracer and X-gal staining (blue) marks the injected side (lower half). In all panels, asterisks indicate decreased expression and arrows increased expression in non-neural (red) or neural (blue) ectoderm. A-D: Placodal and neural crest markers. E-H: Neurogenesis markers. For n numbers and frequency of phenotypes see Table 3.
	Suppl. Fig. 1. In situ hybridization with sense probes. Xenopus laevis embryos after whole-mount in situ hybridization at stage 27, in lateral views (anterior is to the left), using sense probes for five candidate interaction partners of eya1. No staining was present using these probes, except weak, diffuse staining often observed in the pharyngeal pouches (A to E), the optic and otic vesicles (A, D, E) and/or at the level of the notochord (B, C, E).
	Suppl. Fig. 2. Expression patterns of eya1 candidate interaction partners in the eye at late tailbud stage. Cross-sections through the eye in stage 32 embryos. All genes are expressed in the retina (ret) and all genes except msh6 are also expressed in the lens. The diameter of the lens differs between panels because cross-sections were not always exactly through the center of the eye.
	Suppl. Fig. 3. Expression of eya1 and genes encoding candidate interacting proteins in single cell RNA-Seq. Single cell expression data from the Harvard Xenopus Embryo Cell Atlas (Petrova et al., 2024), for eya1 and its candidate interactants, in the placodal area of stage 14 Xenopus tropicalis embryos. (A) The anterior and posterior placodal cell subpopulations were selected. (B) Eya1 is expressed in many placodal cells. (C-G) Genes coding for the five selected candidate Eya1 interactants are all expressed as well in placodal cells, including in at least some of the individual cells expressing eya1.
	Suppl. Fig. 4. One-by-one yeast two-hybrid solid growth tests confirming Eya1-Smarce1 and Eya1-Pias4 interactions. Baits were expressed in a LexA DNA-Binding-Domain vector (pB27), preys were expressed in a Gal4 Activation Domain vector (pP6 or pP7). Yeast cells that were co- transformed with vectors coding for fragments of Eya1 + Smarce1, and with vectors coding for fragments of Eya1 + Pias4, were able to grow on a medium lacking histidine, indicating a physical interaction between these two protein fragments. DO-2: selective medium without tryptophane and leucine; DO-3: selective medium without tryptophane, leucine and histidine; pB27ø: empty pB27 vector; pP7ø: empty pP7 vector.
	Suppl. Fig.5. Smarce1.LS MO2 efficacy test. Western blot analysis on total protein extracts from embryos injected with smarce1-eGFP mRNA (lane 1) or co-injected with smarce1-eGFP + smarce1.LS MO2 (lane 2). The extract from co-injected embryos displays a lower amount of Smarce1-eGFP protein. Coomassie staining performed on the same blot (lanes 3-4) shows that the total protein input is equivalent between both lanes.