5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary

5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts

Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos

A common feature of development in most vertebrate models is the early segregation of the germ line from the soma. For example, in Xenopus and zebrafish embryos primordial germ cells (PGCs) are specified by germ plasm that is inherited from the egg; in mice, Blimp1 expression in the epiblast mediates the commitment of cells to the germ line. How these disparate mechanisms of PGC specification evolved is unknown. Here, in order to identify the ancestral mechanism of PGC specification in vertebrates, we studied PGC specification in embryos from the axolotl (Mexican salamander), a model for the tetrapod ancestor. In the axolotl, PGCs develop within mesoderm, and classic studies have reported their induction from primitive ectoderm (animal cap). We used an axolotl animal cap system to demonstrate that signalling through FGF and BMP4 induces PGCs. The role of FGF was then confirmed in vivo. We also showed PGC induction by Brachyury, in the presence of BMP4. These conditions induced pluripotent mesodermal precursors that give rise to a variety of somatic cell types, in addition to PGCs. Irreversible restriction of the germ line did not occur until the mid-tailbud stage, days after the somatic germ layers are established. Before this, germline potential was maintained by MAP kinase signalling. We propose that this stochastic mechanism of PGC specification, from mesodermal precursors, is conserved in vertebrates

NANOG is required to establish the competence for germ-layer differentiation in the basal tetrapod axolotl

Pluripotency defines the unlimited potential of individual cells of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programming of pluripotency evolved has been obscured in part by a lack of data from lower vertebrates; in model systems such as frogs and zebrafish, the function of the pluripotency genes NANOG and POU5F1 have diverged. Here, we investigated how the axolotl ortholog of NANOG programs pluripotency during development. Axolotl NANOG is absolutely required for gastrulation and germ-layer commitment. We show that in axolotl primitive ectoderm (animal caps; ACs) NANOG and NODAL activity, as well as the epigenetic modifying enzyme DPY30, are required for the mass deposition of H3K4me3 in pluripotent chromatin. We also demonstrate that all 3 protein activities are required for ACs to establish the competency to differentiate toward mesoderm. Our results suggest the ancient function of NANOG may be establishing the competence for lineage differentiation in early cells. These observations provide insights into embryonic development in the tetrapod ancestor from which terrestrial vertebrates evolved. [Abstract copyright: Copyright: © 2023 Simpson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Stepwise evolution of Elk-1 in early deuterostomes

Metazoans have multiple ETS paralogues with overlapping or indiscriminate biological functions. Elk- 1, one of three mammalian Ternary Complex Factors (TCFs), is a well-conserved, ETS domain-containing transcriptional regulator of mitogen-responsive genes that operates in concert with Serum Response Factor (SRF). Nonetheless, its genetic role remains unresolved because the elk-1 gene could be deleted from the mouse genome seemingly without adverse effect. Here we have explored the evolution of Elk-1 to gain insight into its conserved biological role. We identified antecedent Elk-1 proteins in extant early metazoans and used amino acid sequence alignments to chart the appearance of domains characteristic of human Elk-1. We then performed biochemical studies to determine whether putative domains apparent in the Elk-1 protein of a primitive hemichordate were functionally orthologous to those of human Elk-1. Our findings imply the existence of primordial Elk-1 proteins in primitive deuterostomes that could operate as mitogen-responsive ETS transcription factors but not as TCFs. The role of TCF was acquired later, but presumably prior to the whole genome duplications in the basal vertebrate lineage. Thus its evolutionary origins link Elk-1 to the appearance of mesoderm

The chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites

<p>Abstract</p> <p>Background</p> <p>Somitogenesis is the earliest sign of segmentation in the developing vertebrate embryo. This process starts very early, soon after gastrulation has initiated and proceeds in an anterior-to-posterior direction during body axis elongation. It is widely accepted that somitogenesis is controlled by a molecular oscillator with the same periodicity as somite formation. This periodic mechanism is repeated a specific number of times until the embryo acquires a defined specie-specific final number of somites at the end of the process of axis elongation. This final number of somites varies widely between vertebrate species. How termination of the process of somitogenesis is determined is still unknown.</p> <p>Results</p> <p>Here we show that during development there is an imbalance between the speed of somite formation and growth of the presomitic mesoderm (PSM)/tail bud. This decrease in the PSM size of the chick embryo is not due to an acceleration of the speed of somite formation because it remains constant until the last stages of somitogenesis, when it slows down. When the chick embryo reaches its final number of somites at stage HH 24-25 there is still some remaining unsegmented PSM in which expression of components of the somitogenesis oscillator is no longer dynamic. Finally, we identify a change in expression of retinoic acid regulating factors in the tail bud at late stages of somitogenesis, such that in the chick embryo there is a pronounced onset of <it>Raldh2 </it>expression while in the mouse embryo the expression of the RA inhibitor <it>Cyp26A1 </it>is downregulated.</p> <p>Conclusions</p> <p>Our results show that the chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites. In addition, endogenous retinoic acid is probably also involved in the termination of the process of segmentation, and in tail growth in general.</p

Notch activity is present but Notch-based cyclic gene expression is not dynamic in the PSM of <i>Hes7</i>−/− embryos.

<p>(A,B,B') Dorsal and (C–H) lateral views of E9.5–10.5 (A,C,D,G) wild type or (B,B',E,F,H) <i>Hes7</i>−/− embryos analysed by <i>in situ</i> hybridisation or immunocytochemistry using (A,B,B') an intronic <i>Lfng</i> probe, (C–E) an <i>Nrarp</i> probe, (F) an anti-NICD antibody and (G,H) a <i>Mesp2</i> probe. (B,B') <i>Lfng</i> and (F) NICD do not show different patterns of expression in the PSM of <i>Hes7</i>−/− embryos.</p

Cyclic gene expression and somite formation are lost after Notch-blocking drug treatment.

<p>(A,D,G,J,M,P) Half embryo explants from E9.5–10.5 wild type embryos cultured <i>in vitro</i> in the absence (left half) or presence (right half) of 100 µM of DAPT or 100 nM LY411575 for 3 hours and then analysed by <i>in situ</i> hybridisation for the expression of (A) <i>Lfng</i>, (D) <i>Hes7</i>, (G) <i>Axin2</i>, (J) <i>Snail1</i>, (M) <i>Dusp6</i> and (P) <i>Sprouty2</i>. (B,C,E,F,H,I,K,L,N,O,Q,R) Lateral views of E8.0–8.5 wild type embryos cultured in a roller culture system in the absence (B,E,H,K,N,Q) or presence (C,F,I,L,O,R) of 100 µM of DAPT or 100 nM LY411575 for 18–20 hours and then analysed by <i>in situ</i> hybridisation for the expression of (B,C) <i>Lfng</i>, (E,F) <i>Hes7</i>, (H,I) <i>Axin2</i>, (K,L) <i>Snail1</i>, (N,O) <i>Dusp6</i> and (Q,R) <i>Sprouty2</i>, showing that cyclic gene expression and somite formation are interrupted after drug treatment. Red bars demarcate the limit of the somites already formed at start of drug treatment and number indicates the somites formed during culture in the absence or presence of the drug.</p

Cyclic gene expression in the tail bud is lost or stops being dynamic after treatment with Notch-blocking drugs.

<p>(A–F) Half embryo explants from E9.5–10.5 wild type embryos cultured <i>in vitro</i> in the presence of 100 µM of DAPT or 100 nM LY411575 for 3 hours (left) or 4 hours (right) and then analysed by <i>in situ</i> hybridisation for the expression of (A) <i>Lfng</i>, (B) <i>Hes7</i>, (C) <i>Axin2</i>, (D) <i>Snail1</i>, (E) <i>Dusp6</i> and (F) <i>Sprouty2</i>, showing that after drug treatment the cyclic gene expression is lost in the medial and rostral PSM, and that remaining expression still present in the tail bud is not dynamic. (G) Half embryo explants from E9.5 wild type embryos cultured <i>in vitro</i> in the absence (left half) or presence (right half) of 100 µM of DAPT plus 50 µM SU5402, a FGF signalling inhibitor, and then analysed by <i>in situ</i> hybridisation for the expression of <i>Hes7</i>. (H) Schematic representation of the data from the <i>Psen1</i>−/−;<i>Psen2</i>−/− embryos and the pharmacological treatment with Notch-blocking drugs showing that the oscillations of all cyclic genes along the PSM detected in wild type or untreated embryos are lost in the absence of Notch signalling. The expression of cyclic genes is completely lost or is severely down regulated and restricted to the caudal region of the PSM where it is non dynamic. In addition, in <i>Psen1</i>−/−;<i>Psen2</i>−/− embryos or in wild type embryos treated with Notch-blocking drugs the formation of somites is lost.</p