Tingler M et al. (2022), dmrt2 and myf5 Link Early Somitogenesis to Left...

XB-ART-59198

Front Cell Dev Biol 2022 Jan 01;10:858272. doi: 10.3389/fcell.2022.858272.

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dmrt2 and myf5 Link Early Somitogenesis to Left-Right Axis Determination in Xenopus laevis.

Tingler M , Brugger A , Feistel K , Schweickert A .

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The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-β growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: chrd dand5 dmrt2 foxj1 foxj1.2 gsc myf5 myod1 nodal nodal1 odc1 pitx2 slc35b1 sub1 tbx6 tubal3
GO keywords: somitogenesis
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	FIGURE 1. Dmrt2 is required for left-right development. Expression of the LR marker pitx2 in the left LPM was lost in 60% of specimens after left-sided injection of dmrt2 MO (A–C). Co-injection of full-length dmrt2 mRNA statistically significantly restored pitx2 asymmetry (C). Leftward fluid flow in controls and bilaterally injected dmrt2 morphants which was perturbed (D,E) in velocity (F) and directionality (G). LROs of controls and dmrt2 knockdown embryos were analyzed by immunofluorescence, detecting F-actin (green) and Tuba4a [red; (H,I)]. Compared to controls, cilia were statistically significantly shorter in morphants (G). Note enhanced F-actin signals in lateral sensory LRO cells [(H), sLRO] compared to flow-generating central LRO cells [(H), cLRO]. Enhanced F-actin signals were lost on the dmrt2 MO-injected side (I). SM expression of foxj1 in wildtype embryos (K) was diminished by dmrt2 loss of function (L,M). Numbers (n) in (C,F,G,J,M) represent analyzed specimens. N represents the number of independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test (C,J,M) or Wilcoxon-Match-Pair test (F,G); * significant p < 0.05; ***, very highly significant p < 0.001. Asterisks mark injected sides; a = anterior; co = control; cLRO = central left-right organizer; l = left; p = posterior; r = right; sLRO = sensory left-right organizer.
	FIGURE 2. Loss of nodal1 in sLRO cells by dmrt2 knockdown correlates with impaired expression of the somitic marker genes tbx6 and myf5 at gastrula stages. nodal1 mRNA was analyzed at stage (st.) 19 in untreated controls [co, (A)], unilaterally injected dmrt2 MO morphants (B) or specimens that received a mix of dmrt2 MO and rescue dmrt2 mRNA (C). nodal1 expression was lost or reduced in dmrt2 morphants (B,D). Statistical analysis shows that nodal1 was very highly significantly rescued (C,D). Early gastrula embryos were stained for tbx6 and myf5 (E–L). Controls showed horseshoe-like tbx6 expression, omitting the Spemann organizer (E). tbx6 signals were reduced by dmrt2 MO on the injected side (F,H), which by statistics was significantly rescued upon co-injection of dmrt2 mRNA (G,H). The angel wing-like expression pattern of myf5 (I) was lost (J,L) or reduced in dmrt2 morphants as well (J,L). The asterisk mark the injected side. Numbers in (D,H,L) represent analyzed specimens. N represents the number of independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test (D,H,L); very highly significant, p < 0.001. a = anterior; co = control; l = left; p = posterior; r = right; d = dorsal; v = ventral.
	FIGURE 3. The myogenic transcription factor Myf5 is required for LR asymmetry. In order to connect paraxial patterning to LR development, a myf5 knockdown was conducted in the left sLRO lineage and assayed for pitx2 asymmetry at tadpole (A–D) or nodal1 at neurula stages (E–H). In contrast to controls [co;(A)], myf5 morphants lost left pitx2 expression (B), which was regained by the introduction of myf5 rescue mRNA (C). Statistical analysis demonstrates the specificity of results (D). At late neurula, nodal1 expression was lost or reduced by myf5 knockdown (F) which was partially rescued by co-injecting a myf5 rescue mRNA (G). Statistical analysis is depicted in (H). Asterisks mark injected sides. Numbers in (D,H) represent analyzed specimens. N represents the number of independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test. * significant p < 0.05; highly significant p < 0.01; * very highly significant p < 0.001.
	FIGURE 4. Myf5 specifies sLRO cells downstream of Dmrt2. Using suboptimal dmrt2 MO and myf5 MO dosages, the cooperation of both transcription factors was demonstrated at the level of pitx2 asymmetry (A–D) and nodal1 expression in sLRO cells (D–G). The combination of both MOs resulted in an efficient loss of pitx2 and nodal1 expression. Note that individual MO injections had a weak impact on nodal1 in sLRO cells. Co-injecting myf5 mRNA rescued nodal1 expression in dmrt2 morphants (H–K). Asterisks mark injected sides. Numbers in (C,G,K) represent analyzed specimens. N represents the number of independent experiments. Statistical analyses were done with one-sided Pearson’s chi-square test. highly significant p < 0.01; * very highly significant p < 0.001. a = anterior; co = control; l = left; p = posterior; r = right.
	FIGURE 5. Dmrt2 regulates symmetry breakage and early somitogenesis in Xenopus laevis. Dmrt2 intervenes with two processes during symmetry breakage. It specifies the LRO by inducing foxj1 in the SM that gives rise to the ciliated epithelium, which generates a leftward flow of extracellular fluid (left panel). Simultaneously, Dmrt2 activates tbx6 and myf5 expression in the early gastrula embryo, leading to the specification of the paraxial mesoderm (right panel), which later differentiates into muscles and vertebrae. In addition, the somatic functions of Dmrt2 and Myf5 are required for sensory LRO morphogenesis and thus for Nodal-cascade induction in the left LPM. N = notochord; S = somites.
	Supplemental Figure 1: dmrt2 expression in cLRO cells / dissected SM/DM tissues and organizer gene expression in dmrt2 morphants. WMISH and subsequent histological sections of neurula stage (st.) embryos revealed dmrt2 transcription in cLRO cells and the notochord (N). Note that lateral sLRO cells and somites (S) lack dmrt2 expression (A, white arrowheads in A'). Detection of dmrt2 transcripts by RT-PCR (B) and RT-qPCR (C) in dissected superficial mesoderm (SM), underlying deep mesoderm (DM) and whole embryo (WE) at stage (st.) 10.5. odc served as loading control in A. Spemann Organizer marker genes chordin (D, E) and goosecoid (F, G ) were not perturbed in dmrt2 morphants (. a=anterior; d=dorsal; l=left; r=right; co=control; cLRO=central left-right organizer; sLRO= sensory left-right organizer.
	Supplemental Figure 2: sLRO cells are lost in dmrt2 and myf5 morphant neurulae. Histological sections showed myod1 expression in control (A), unilaterally dmrt2 MO (B) or myf5 MO (C) injected embryos at neurula stage (st. 18). In controls (co) and in the non-injected side of morphants, myod1 positive cells border to the extra cellular space of the archenteron sLRO. Following dmrt2 or myf5 knockdown, myod1 expression is restricted to deep cells, which are covered by the endodermal cell layer (dashed line). White arrow heads point to the position of sLRO cells. Asterisks mark injected sides; l=left; r=right; co=control; sLRO= sensory left-right organizer.
	Supplemental Figure 3: myf5 and tbx6 are expressed in sLRO cells. Both, myf5 (A) and tbx6 (B), are expressed in the posterior presomitic mesoderm in neurula stages (st. 18). Transversal sectioning demonstrated that myf5 (A’) and tbx6 (B’) positive signals were present in the presomitic mesoderm, including sLRO cells. White arrow heads point to the position of sLRO cells. Plain of section is indicated in (A, B) by a dashed line. cbc, circular blastoporal collar; sLRO, sensory left-right organizer; cLRO, central left-right organizer.
	Supplemental Figure 4: myf5 expression in early gastrula embryos is restricted to deep cells. myf5 staining of a stage (st.) 10.5 gastrula, shown in a dorso-vegetal view (A). Transversal section revealed that myf5 mRNA is present only in deep mesodermal cell populations (DM) but not in the superficial layer (A'). Dashed line in (A) indicated the plain of section.

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