Panagiotis Papasaikas

Multicopy transgene arrays remain widely used in Caenorhabditis elegans research. It is usually assumed that they behave neutrally, not impacting the phenotype under investigation. Here, we reveal that a previously reported heterochronic extra-molt phenotype associated with myrf-1(mg412) depends on the presence of the integrated molting reporter mgIs49. Nanopore long-read sequencing shows that mgIs49 is a massive 8.8-Mb insertion—around 50% of the size of its host chromosome—which disrupts the prmt-9 gene. Both mgIs49 and another array, maIs105, cause dysregulation of the transcriptome and accumulation of reads mapping in sense-direction to transgene promoter sequences. We identify additional arrays exceeding 4 Mb and show that variable molting defects occur across different transgenic lines when combined with myrf-1(mg412), implicating array size or composition in the synthetic phenotype. Our results underscore the necessity of replacing multicopy reporters in developmental studies with single-copy insertions or endogenous tagging whenever possible.

Regeneration can proceed through epimorphosis, involving regrowth of lost structures at the amputation site in a proximal-to-distal sequence, or through morphallaxis, which is characterized by extensive tissue remodeling with minimal cell proliferation. While the mechanisms underlying epimorphic regeneration are well studied, the spatiotemporal dynamics of morphallaxis remain less understood. Here we show, using time-course single-cell RNA sequencing (scRNA-seq) of regenerating Hydra tissue, that morphallaxis involves a temporal sequence in the re-emergence of terminal and subterminal cell identities, with transcriptional signatures associated with proximal structures appearing earlier than those of distal structures. We further find that Wnt signaling remains dynamically active at the amputation site throughout regeneration, with its composition changing over time, consistent with a transient regulatory state. Together, these findings provide a high-resolution view of dynamic cell state transitions and signaling activity during morphallactic regeneration and offer a framework for exploring how organizer function and axial patterning are re-established in Hydra.

Multicopy transgene arrays remain widely used in C. elegans research. It is usually assumed that they behave neutrally, not impacting the phenotype under investigation. Here, we reveal that a previously reported heterochronic extra-molt phenotype associated with myrf-1(mg412) depends on the presence of the integrated molting reporter mgIs49 . Nanopore long-read sequencing shows that mgIs49 is a massive 8.8-Mb insertion - around 50% of the size of its host chromosome - which disrupts the prmt-9 gene. Both mgIs49 and another array, maIs105 , cause dysregulation of the transcriptome and accumulation of reads mapping to the promoter sequences used as components of the array. We identify additional arrays exceeding 4 Mb and show that variable molting defects occur across different transgenic lines when combined with myrf-1(mg412) , implicating array size or composition in the synthetic phenotype. Our results underscore the necessity of replacing multicopy reporters in developmental studies with single-copy insertions or endogenous tagging whenever possible.

In Hydra, a simple cnidarian model, epithelio-muscular cells shape and maintain body architecture through continuous renewal. Undifferentiated cells from the mid-body region migrate passively toward the extremities, replacing shed cells and acquiring region-specific identities. This ongoing turnover, together with the Hydra stable axial organization, provides a powerful model to study how cell type specification is integrated with body patterning. Yet, the molecular mechanisms that govern epithelial identity remain largely unknown. Here, we identify a double-negative feedback loop between the transcription factors Zic4 and Gata3 that functions as a toggle switch to control terminal epidermal cell fates. Zic4, activated by Wnt signaling from the head organizer, induces battery cell specification in tentacles. In contrast, Gata3 promotes basal disk cell identity at the aboral end. These factors are mutually repressive: silencing one leads to expansion of the other's domain and ectopic cell fate induction. Notably, simultaneous knockdown of both restores normal patterning, suggesting that identity is dictated by their relative balance rather than absolute levels. Our study reveals how opposing transcriptional signals coordinate epithelial identity with axial patterning at Hydra poles.

In the germ line and during early embryogenesis, DNA methylation (DNAme) undergoes global erasure and re-establishment to support germ cell and embryonic development. While DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin are unknown. Through conditional gene deletion of the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b, we observe that DNMT3A primarily safeguards against DNA hypomethylation in undifferentiated spermatogonia, while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm. To assess the impact of altered sperm chromatin in formatting embryonic chromatin, we measure H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a transposon-based tagging approach. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.

SUMMARY: In mammalian somatic and male germline cells, genomes are extensively DNA methylated (DNAme). In oocytes, however, DNAme is largely limited to transcribed regions only. Regulatory CpG-island (CGI) sequences are also devoid of repressive DNAme in somatic and germ cells of both sexes. The mechanisms restricting de novo DNAme acquisition in developing oocytes, at CGIs and globally, and the relevance thereof for regulating zygotic gene expression and embryo development after fertilization are largely unknown. Here we show that the histone H3 lysine 36 dimethyl (H3K36me2) demethylases KDM2A and KDM2B prevent genome-wide accumulation of H3K36me2, thereby impeding global DNMT3A-catalyzed de novo DNAme, including at CGI gene promoters. By recruiting variant Polycomb Repressive Complex 1 (vPRC1), they further control H2A mono-ubiquitin deposition and vPRC1-dependent gene repression. Through genetic perturbations, we demonstrate that aberrant Dnmt3a -dependent DNAme established in Kdm2a/Kdm2b double mutant oocytes represses transcription from maternal loci in two-cell embryos. The lethality of Kdm2a/Kdm2b maternally deficient pre-implantation embryos is suppressed by Dnmt3a deficiency during oogenesis. Hence, KDM2A/KDM2B are essential for confining the oocyte DNA methylome, conferring competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient to erase aberrant DNAme from maternal chromatin, and that early development is vulnerable to gene dosage haplo-insufficiency effects. HIGHLIGHTS: Demethylation of H3K36me2 by KDM2A and KDM2B prevents aberrant de novo DNA methylation in mouse oocytes. Sequence composition and H3K4me3 modulate the probability for aberrant H3K36me2 and DNA methylation at CpG islands. Aberrant oocyte DNA methylation is not reprogrammed in early embryos and suppresses maternal gene transcription. Aberrant oocyte DNA methylation causes embryonic lethality during pre-implantation development. GRAPHICAL SUMMARY:

A central mechanism of mTOR complex 1 (mTORC1) signaling is the coordinated translation of ribosomal protein and translation factor mRNAs mediated by the 5′-terminal oligopyrimidine motif (5′TOP). Recently, La-related protein 1 (LARP1) was proposed to be the specific regulator of 5′TOP mRNA translation downstream of mTORC1, while eIF4E-binding proteins (4EBP1/2) were suggested to have a general role in translational repression of all transcripts. Here, we use single-molecule translation site imaging of 5′TOP and canonical mRNAs to study the translation of single mRNAs in living cells. Our data reveal that 4EBP1/2 has a dominant role in repression of translation of both 5′TOP and canonical mRNAs during pharmacological inhibition of mTOR. In contrast, we find that LARP1 selectively protects 5′TOP mRNAs from degradation in a transcriptome-wide analysis of mRNA half-lives. Our results clarify the roles of 4EBP1/2 and LARP1 in regulating 5′TOP mRNAs and provide a framework to further study how these factors control cell growth during development and disease.

The molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/β-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration.

The molecular mechanisms that maintain cell identities and prevent transdifferentiation remain mysterious. Interestingly, both dedifferentiation and transdifferentiation are transiently reshuffled during regeneration. Therefore, organisms that regenerate readily offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4 silencing is sufficient to induce transdifferentiation of tentacle into foot cells. We identified a Wnt-controlled Gene Regulatory Network that controls a transcriptional switch of cell identity. Furthermore, we show that this switch also controls the re-entry into the cell cycle. Our data indicate that maintenance of cell fate by a Wnt-controlled GRN is a key mechanism during both homeostasis and regeneration. One-Sentence Summary: A Wnt-controlled GRN controls fate maintenance in Hydra .