Developmental Switch Research Articles

Transcriptomic and genetic analysis reveals a Zn2Cys6 transcription factor specifically required for conidiation in submerged cultures of Thermothelomyces thermophilus.

Filamentous fungi are important producers of enzymes and secondary metabolites. The industrial thermophilic species, Thermothelomyces thermophilus, is closely related to the model fungus, Neurospora crassa. A critical aspect of the filamentous fungal life cycle is the production of asexual spores (conidia), which are regulated by various stimuli, including nutrient availability. Several species of fungi, including T. thermophilus, produce conidia under submerged fermentation conditions, which can be detrimental to product yields. In this study, transcriptional profiling of T. thermophilus was used to map changes during asexual development in submerged cultures, which revealed commonalities of regulation between T. thermophilus and N. crassa. We further identified a transcription factor, res1, whose deletion resulted in a complete loss of conidia production under fermentation conditions, but which did not affect conidiation on plates. Under fermentation conditions, the ∆res1 deletion strain showed increased biomass production relative to the wild-type strain, indicating that the manipulation of res1 in T. thermophilus has the potential to increase productivity in industrial settings. Overexpression of res1 caused a severe growth defect and early conidia production on both plates and in submerged cultures, indicating res1 overexpression can bypass regulatory aspects associated with conidiation on plates. Using chromatin-immunoprecipitation sequencing, we identified 35 target genes of Res1, including known conidiation regulators identified in N. crassa, revealing common and divergent aspects of asexual reproduction in these two species.IMPORTANCEFilamentous fungi, such as Thermothelomyces thermophilus, are important industrial species and have been harnessed in the Biotechnology industry for the production of industrially relevant chemicals and proteins. However, under fermentation conditions, some filamentous fungi will undergo a switch from mycelial growth to asexual development. In this study, we use transcriptional profiling of asexual development in T. thermophilus and identify a transcription factor that specifically regulates the developmental switch to the production of unwanted asexual propagules under fermentation conditions, thus altering secreted protein production. Mutations in this transcription factor Res1 result in the loss of asexual development in submerged cultures but do not affect asexual sporulation when exposed to air. The identification of stage-specific developmental regulation of asexual spore production and comparative analyses of conidiation in filamentous ascomycete species have the potential to further manipulate these species for industrial advantage.

Activity-Dependent Internalization of Glun2B-Containing NMDARS Is Required For Synaptic Incorporation of Glun2A And Synaptic Plasticity.

NMDA-type glutamate receptors are heterotetrameric complexes composed of two GluN1 and two GluN2 subunits. The precise composition of the GluN2 subunits determines the channel's biophysical properties and influences its interaction with postsynaptic scaffolding proteins and signaling molecules involved in synaptic physiology and plasticity. The precise regulation of NMDAR subunit composition at synapses is crucial for proper synaptogenesis, neuronal circuit development, and synaptic plasticity, a cellular model of memory formation.In the forebrain during early development, NMDARs contain solely the GluN2B subunit, which is necessary for proper synaptogenesis and synaptic plasticity. In rodents, GluN2A subunit expression begins in the second postnatal week, replacing GluN2B-containing NMDARs at synapses in an activity- or sensory experience-dependent process. This switch in NMDAR subunit composition at synapses alters channel properties and reduces synaptic plasticity. The molecular mechanism regulating the switch remains unclear.We have investigated the role of activity-dependent internalization of GluN2B-containing receptors in shaping synaptic NMDAR subunit composition. Using molecular, pharmacological, and electrophysiological approaches in cultured organotypic hippocampal slices from rats of both sexes, we show that the process of incorporating GluN2A-containing NMDARs receptors requires activity-dependent internalization of GluN2B-containing NMDARs. Interestingly, blockade of GluN2A synaptic incorporation was associated with impaired potentiation of AMPA-mediated synaptic transmission, suggesting a potential coupling between the trafficking of AMPARs into synapses and that of GluN2A-containing NMDARs.These insights contribute to our understanding of the molecular mechanisms underlying synaptic trafficking of glutamate receptors and synaptic plasticity. They may also have implications for therapeutic strategies targeting NMDAR function in neurological disorders.Significance statement NMDARs play a critical role in synaptogenesis, synaptic stability, and activity-dependent regulation of synaptic strength. The developmental switch in their GluN2 subunits composition is part of normal synapse development and crucial for proper synaptic physiology, plasticity, and the formation of functional neuronal circuits, though the mechanisms governing it remain unclear. We show that internalization of GluN2B-containing NMDARs is required for synaptic incorporation of GluN2A-containing receptors. This process can be induced by long-term potentiation and requires Ca+2 Notably, GluN2A trafficking to synapses is linked to the incorporation of AMPA-type glutamate receptors, suggesting a shared pathway for synaptic incorporation. These findings provide greater insight into the molecular mechanisms behind glutamate receptor trafficking and synaptic plasticity, potentially informing therapeutic strategies for neurological disorders.

An unscheduled switch to endocycles induces a reversible senescent arrest that impairs growth of the Drosophila wing disc.

A programmed developmental switch to G / S endocycles results in tissue growth through an increase in cell size. Unscheduled, induced endocycling cells (iECs) promote wound healing but also contribute to cancer. Much remains unknown, however, about how these iECs affect tissue growth. Using the D. melanogaster wing disc as model, we find that populations of iECs initially increase in size but then subsequently undergo a heterogenous arrest that causes severe tissue undergrowth. iECs acquired DNA damage and activated a Jun N-terminal kinase (JNK) pathway, but, unlike other stressed cells, were apoptosis-resistant and not eliminated from the epithelium. Instead, iECs entered a JNK-dependent and reversible senescent-like arrest. Senescent iECs promoted division of diploid neighbors, but this compensatory proliferation did not rescue tissue growth. Our study has uncovered unique attributes of iECs and their effects on tissue growth that have important implications for understanding their roles in wound healing and cancer.

The transcriptional landscape of the developmental switch from regular pollen maturation towards microspore-derived plant regeneration in barley

Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors. Haploids/doubled haploids (DHs) derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering. However, the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined. Here, RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis (ME) in barley cultivars Golden Promise and Igri, which exhibit contrasting responsiveness to microspore-derived plant formation. Our experiments revealed fundamental regulatory networks, specific groups of genes, and transcription factor (TF) families potentially regulating the developmental switch. We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME. Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.

MicroRNA-mediated gene regulation and the resilience of multicellular animals.

MicroRNAs are small RNAs that enable parts of the genome to regulate the other parts of the genome by RNA::RNA complementarity. Genes that encode microRNAs function as trans-acting regulators of hundreds of other genes, primarily by inhibiting the production of protein from mRNAs to which the microRNAs can bind by base pairing. MicroRNAs and their Argonaute partner proteins constitute a regulatory complex (the miRISC) that exhibits astonishing regulatory versatility. microRNAs have been shown to perform diverse roles in genetic regulatory networks (GRNs) - to control developmental switches, to dampen gene expression noise, to coordinate multigene functional modules, and more broadly, to confer robustness and resilience to developmental and homeostatic processes. Genetic analysis reveals that the function of particular microRNAs can be conditional, such that the microRNA is required under particular environmental or physiological conditions, but relatively dispensable under other conditions. The diversity and versatility of microRNA function in animal systems reflects the many ways that miRISC can be regulated by cellular signaling pathways, and the structure-function interplay among microRNA, target, and Argonaute.

TSG-6-Mediated Extracellular Matrix Modifications Regulate Hypoxic-Ischemic Brain Injury.

Proteoglycans containing link domains modify the extracellular matrix (ECM) to regulate cellular homeostasis and can also sensitize tissues/organs to injury and stress. Hypoxic-ischemic (H-I) injury disrupts cellular homeostasis by activating inflammation and attenuating regeneration and repair pathways. In the brain, the main component of the ECM is the glycosaminoglycan hyaluronic acid (HA), but whether HA modifications of the ECM regulate cellular homeostasis and response to H-I injury is not known. In this report, employing both male and female mice, we demonstrate that link-domain-containing proteoglycan, TNFα-stimulated gene-6 (TSG-6), is active in the brain from birth onward and differentially modifies ECM HA during discrete neurodevelopmental windows. ECM HA modification by TSG-6 enables it to serve as a developmental switch to regulate the activity of the Hippo pathway effector protein, yes-associated protein 1 (YAP1), in the maturing brain and in response to H-I injury. Mice that lack TSG-6 expression display dysregulated expression of YAP1 targets, excitatory amino acid transporter 1 (EAAT1; glutamate-aspartate transporter) and 2 (EAAT2; glutamate transporter-1). Dysregulation of YAP1 activation in TSG-6-/- mice coincides with age- and sex-dependent sensitization of the brain to H-I injury such that 1-week-old neonates display an anti-inflammatory response in contrast to an enhanced proinflammatory injury reaction in 3-month-old adult males but not females. Our findings thus support that a key regulator of age- and sex-dependent H-I injury response in the mouse brain is modulation of the Hippo-YAP1 pathway by TSG-6-dependent ECM modifications.

MBNL1 Regulates Programmed Postnatal Switching Between Regenerative and Differentiated Cardiac States.

Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.

Selenium nanoparticles conferred drought tolerance in tomato plants by altering the transcription pattern of microRNA-172 (miR-172), bZIP, and CRTISO genes, upregulating the antioxidant system, and stimulating secondary metabolism.

Drought stress is one of the major limiting factors for the production of tomato in Iran. In this study, the efficiency of selenate and Se nanoparticle (SeNP) foliar application on tomato plants was assessed to vestigate mitigating the risk associated with water-deficit conditions. Tomato plants were treated with SeNPs at the concentrations of 0 and 4mg L-1; after the third sprays, the plants were exposed to water-deficit conditions. The foliar spraying with SeNPs not only improved growth, yield, and developmental switch to the flowering phase but also noticeably mitigated the detrimental risk associated with the water-deficit conditions. Gene expression experiments showed a slight increase in expression of microRNA-172 (miR-172) in the SeNP-treated plants in normal irrigation, whereas miR-172 displayed a downregulation trend in response to drought stress. The bZIP transcription factor and CRTISO genes were upregulated following the SeNP and drought treatments. Drought stress significantly increased the H2O2 accumulation that is mitigated with SeNPs. The foliar spraying with Se or SeNPs shared a similar trend to alleviate the negative effect of drought stress on the membrane integrity. The applied supplements also conferred drought tolerance through noticeable improvements in the non-enzymatic (ascorbate and glutathione) and enzymatic (catalase and peroxidase) antioxidants. The SeNP-mediated improvement in drought stress tolerance correlated significantly with increases in the activity of phenylalanine ammonia-lyase, proline, non-protein thiols, and flavonoid concentrations. SeNPs also improved the fruit quality regarding K, Mg, Fe, and Se concentrations. It was concluded that foliar spraying with SeNPs could mitigate the detrimental risk associated with the water-deficit conditions.

A coordinated switch in sucrose and callose metabolism enables enhanced symplastic unloading in potato tubers.

One of the early changes upon tuber induction is the switch from apoplastic to symplastic unloading. Whether and how this change in unloading mode contributes to sink strength has remained unclear. In addition, developing tubers also change from energy to storage-based sucrose metabolism. Here, we investigated the coordination between changes in unloading mode and sucrose metabolism and their relative role in tuber sink strength by looking into callose and sucrose metabolism gene expression combined with a model of apoplastic and symplastic unloading. Gene expression analysis suggests that callose deposition in tubers is decreased by lower callose synthase expression. Furthermore, changes in callose and sucrose metabolism are strongly correlated, indicating a well-coordinated developmental switch. Modelling indicates that symplastic unloading is not the most efficient unloading mode per se. Instead, it is the concurrent metabolic switch that provides the physiological conditions necessary to potentiate symplastic transport and thereby enhance tuber sink strength .

Sorghum bicolor INDETERMINATE1 is a conserved primary regulator of flowering.

A fundamental developmental switch for plants is transition from vegetative to floral growth, which integrates external and internal signals. INDETERMINATE1 (Id1) family proteins are zinc finger transcription factors that activate flowering in grasses regardless of photoperiod. Mutations in maize Id1 and rice Id1 (RID1) cause very late flowering. RID1 promotes expression of the flowering activator genes Early Heading Date1 (Ehd1) and Heading date 1 (Hd1), a rice homolog of CONSTANS (CO). Mapping of two recessive late flowering mutants from a pedigreed sorghum EMS mutant library identified two distinct mutations in the Sorghum bicolor Id1 (SbId1) homolog, mutant alleles named sbid1-1 and sbid1-2. The weaker sbid1-1 allele caused a 35 day delay in reaching boot stage in the field, but its effect was limited to 6 days under greenhouse conditions. The strong sbid1-2 allele delayed boot stage by more than 60 days in the field and under greenhouse conditions. When sbid1-1 and sbid1-2 were combined, the delayed flowering phenotype remained and resembled that of sbid1-2, confirming late flowering was due to loss of SbId1 function. Evaluation of major flowering time regulatory gene expression in sbid1-2 showed that SbId1 is needed for expression of floral activators, like SbCO and SbCN8, and repressors, like SbPRR37 and SbGhd7. These results demonstrate a conserved role for SbId1 in promotion of flowering in sorghum, where it appears to be critical to allow expression of most major flowering regulatory genes.

A Developmental Switch in Cholinergic Mechanisms of Modulation in the Medial Nucleus of the Trapezoid Body.

The medial nucleus of the trapezoid body (MNTB) has been intensively investigated as a primary source of inhibition in brainstem auditory circuitry. MNTB-derived inhibition plays a critical role in the computation of sound location, as temporal features of sounds are precisely conveyed through the calyx of Held/MNTB synapse. In adult gerbils, cholinergic signaling influences sound-evoked responses of MNTB neurons via nicotinic acetylcholine receptors (nAChRs; Zhang et al., 2021) establishing a modulatory role for cholinergic input to this nucleus. However, the cellular mechanisms through which acetylcholine (ACh) mediates this modulation in the MNTB remain obscure. To investigate these mechanisms, we used whole-cell current and voltage-clamp recordings to examine cholinergic physiology in MNTB neurons from Mongolian gerbils (Meriones unguiculatus) of both sexes. Membrane excitability was assessed in brain slices, in pre-hearing (postnatal days 9-13) and post-hearing onset (P18-20) MNTB neurons during bath application of agonists and antagonists of nicotinic (nAChRs) and muscarinic receptors (mAChRs). Muscarinic activation induced a potent increase in excitability most prominently prior to hearing onset with nAChR modulation emerging at later time points. Pharmacological manipulations further demonstrated that the voltage-gated K+ channel KCNQ (Kv7) is the downstream effector of mAChR activation that impacts excitability early in development. Cholinergic modulation of Kv7 reduces outward K+ conductance and depolarizes resting membrane potential. Immunolabeling revealed expression of Kv7 channels as well as mAChRs containing M1 and M3 subunits. Together, our results suggest that mAChR modulation is prominent but transient in the developing MNTB and that cholinergic modulation functions to shape auditory circuit development.

INDETERMINATE1-mediated expression of FT family genes is required for proper timing of flowering in Brachypodium distachyon.

The transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 (FT1) and FT-Like (FTL) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 (BdID1) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 (FT-LIKE9) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1. We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1, FT2, FTL12, and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses.

Abstract 11679: The Crucial Role of Estrogen-Related Receptor in Human Cardiomyocyte Maturation

During the developmental transition from fetal to adult, the heart undergoes dramatic mitochondrial and sarcomeric maturation. This process is partially reversed during development of heart failure. We have identified the nuclear receptors, estrogen-related receptor (ERR) α and γ, as critical transcriptional regulators of energy metabolic and sarcomeric genes during postnatal maturation of mouse heart. Here, we aimed to define the transcriptional mechanisms involved in ERR-mediated maturation in human cardiomyocytes. Genomic interrogation with published datasets revealed that approximately 50% of super-enhancer regions (SEs) overlapped with ERRγ peaks in human-induced pluripotent stem cell-derived cardiac myocytes. Intersection of the ERRγ cistrome with a published H3K27ac Hi-ChIP dataset from human hearts identified over 3,000 ERR-containing enhancer-promoter loops, which are associated with genes related to cardiac contractile function. These results highlighted the essential role of ERR in cardiomyocyte enhancer function. Among the SEs defined by mediator complex deposition, we found that both ERRα and γ occupied the enhancer region flanking the MYH6/MYH7 cluster, a well-characterized locus for fetal to adult developmental switching. Targeting ERR recruitment on the enhancer using catalytically-dead Cas9-KRAB or proteolysis targeting chimeric (PROTAC) strategies resulted in the reversal of MYH6/7 switching. RNA-sequencing analysis following PROTAC treatment revealed that deactivation of ERRα resulted in downregulation of a wide array of genes encoding cardiac structural proteins, particularly adult-type ventricular sarcomeric proteins. Additional cistrome analyses demonstrated that PROTAC treatment resulted in decreased ERRα peak signals on cardiac structural genes, including enhancer regions directing MYH6/7 transcription consistent with the observed gene expression changes. In summary, ERR signaling is essential for the transcriptional program of structural maturation in human cardiomyocytes. This process likely involves establishing proper cardiac chromatin architecture. These results support a novel therapeutic strategy of utilizing ERR activators to reverse fetal shifts in failing hearts.

Stomach encyclopedia: Combined single-cell and spatial transcriptomics reveal cell diversity and homeostatic regulation of human stomach

The stomach is an important digestive organ with various biological functions. However, because of the complexity of its cellular and glandular composition, its precise cellular biology has yet to be elucidated. In this study, we conducted single-cell RNA sequencing (scRNA-seq) and subcellular-level spatial transcriptomics analysis of the human stomach and constructed the largest dataset to date: a stomach encyclopedia. This dataset consists of approximately 380,000 cells from scRNA-seq and the spatial transcriptome, enablingintegrated analyses of transcriptional and spatial information of gastric and metaplastic cells. This analysis identified LEFTY1 as an uncharacterized stem cell marker, which was confirmed through lineage tracinganalysis. A wide variety of cell-cell interactions between epithelial and stromal cells, including PDGFRA+BMP4+WNT5A+ fibroblasts, was highlighted in the developmental switch of intestinal metaplasia. Our extensive dataset will function as a fundamental resource in investigations of the stomach, including studies of development, aging, and carcinogenesis.

Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.

MiR-425-5p suppression of Crebzf regulates oocyte aging via chromatin modification

Female infertility due to declining oocyte quality with age remains a significant challenge for patients and physicians, despite extensive research efforts. Recent studies suggest that microRNAs (miRNAs), which respond to various stressors in the aging process, may provide a promising solution. With the approval of small RNA drugs for clinical use, miRNA-based treatment of oocyte aging appears to be a viable option. Through high-throughput sequencing, miR-425-5p was identified as the only miRNA elevated under natural aging and oxidative stress. Microinjection of inhibitors to inhibit miR-425-5p effectively improved compromised phenotypes of old oocytes in vitro. Further investigation revealed that Crebzf acts as a mediator of miR-425-5p's age-related functions in old oocytes. In vivo treatment with miR-425-5p antagomirs significantly improved impaired oocyte development in reproductively old females by targeting Crebzf. Single-cell RNA sequencing revealed that Crebzf plays a vital role in regulating mRNAs targeting histone H3, trimethylated lysine 4 (H3K4me3), a crucial marker for transcriptional silencing. Overexpression of miR-425-5p could hinder oocyte maturation by downregulating Crebzf expression and disrupting transcriptional regulation. Our findings provide new insights into the potential of miR-425-5p antagomirs as a treatment for female infertility and highlight an elegant mechanism by which miR-425-5p inhibition of Crebzf inhibits a developmental switch in GV oocytes by regulating a group of histone methyltransferase mRNAs.

Endogenous epitope tagging of eEF1A2 in mice reveals early embryonic expression of eEF1A2 and subcellular compartmentalisation of neuronal eEF1A1 and eEF1A2

All vertebrate species express two independently-encoded forms of translation elongation factor eEF1A. In humans and mice eEF1A1 and eEF1A2 are 92 % identical at the amino acid level, but the well conserved developmental switch between the two variants in specific tissues suggests the existence of important functional differences. Heterozygous mutations in eEF1A2 result in neurodevelopmental disorders in humans; the mechanism of pathogenicity is unclear, but one hypothesis is that there is a dominant negative effect on eEF1A1 during development. The high degree of similarity between the eEF1A proteins has complicated expression analysis in the past; here we describe a gene edited mouse line in which we have introduced a V5 tag in the gene encoding eEF1A2. Expression analysis using anti-V5 and anti-eEF1A1 antibodies demonstrates that, in contrast to the prevailing view that eEF1A2 is only expressed postnatally, it is expressed from as early as E11.5 in the developing neural tube. Two colour immunofluorescence also reveals coordinated switching between eEF1A1 and eEF1A2 in different regions of postnatal brain. Completely reciprocal expression of the two variants is seen in post-weaning mouse brain with eEF1A1 expressed in oligodendrocytes and astrocytes and eEF1A2 in neuronal soma. Although eEF1A1 is absent from neuronal cell bodies after development, it is widely expressed in axons. This expression does not appear to coincide with myelin sheaths originating from oligodendrocytes but rather results from localised translation within the axon, suggesting that both variants are transcribed in neurons but show completely distinct subcellular localisation at the protein level. These findings will form an underlying framework for understanding how missense mutations in eEF1A2 result in neurodevelopmental disorders.

Contribution of the IGCR1 regulatory element and the 3′Igh CTCF-binding elements to regulation of Igh V(D)J recombination

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.

The TOPLESS corepressor regulates developmental switches in the bryophyte Physcomitrium patens that were critical for plant terrestrialisation.

The plant-specific TOPLESS (TPL) family of transcriptional corepressors is integral to multiple angiosperm developmental processes. Despite this, we know little about TPL function in other plants. To address this gap, we investigated the roles TPL plays in the bryophyte Physcomitrium patens, which diverged from angiosperms approximately 0.5 billion years ago. Although complete loss of PpTPL function is lethal, transgenic lines with reduced PpTPL activity revealed that PpTPLs are essential for two fundamental developmental switches in this plant: the transitions from basal photosynthetic filaments (chloronemata) to specialised foraging filaments (caulonemata) and from two-dimensional (2D) to three-dimensional (3D) growth. Using a transcriptomics approach, we integrated PpTPL into the regulatory network governing 3D growth and we propose that PpTPLs represent another important class of regulators that are essential for the 2D-to-3D developmental switch. Transcriptomics also revealed a previously unknown role for PpTPL in the regulation of flavonoids. Intriguingly, 3D growth and the formation of caulonemata were crucial innovations that facilitated the colonisation of land by plants, a major transformative event in the history of life on Earth. We conclude that TPL, which existed before the land plants, was co-opted into new developmental pathways, enabling phytoterrestrialisation and the evolution of land plants.

Calcium dynamics at the neural cell primary cilium regulate Hedgehog signaling–dependent neurogenesis in the embryonic neural tube

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.