Developmental Clock Research Articles

Oscillatory DeltaC Expression in Neural Progenitors Primes the Prototype of Forebrain Development.

Notch signaling plays a pivotal role in regulating various developmental processes, particularly in controlling the timing of neuronal production within the developing neocortex. Central to this regulatory mechanism is the oscillatory pattern of Delta, which functions as a developmental clock modulator. Its deficiency profoundly impairs mammalian brain formation, highlighting its fundamental role in brain development. However, zebrafish carrying a mutation in the functional ortholog DeltaC (dlc) within their functional ortholog exhibit an intact forebrain structure, implying evolutionary variations in Notch signaling within the forebrain. In this study, we unveil the distinct yet analogous expression profiles of Delta and Her genes in the developing vertebrate forebrain. Specifically, for the first time, we detected the oscillatory expression of the Delta gene dlc in the developing zebrafish forebrain. Although this oscillatory pattern appeared irregular and was not pervasive among the progenitor population, attenuation of the dlc-involved Notch pathway using a γ-secretase inhibitor impaired neuronal differentiation in the developing zebrafish forebrain, revealing the indispensable role of the dlc-involved Notch pathway in regulating early zebrafish neurogenesis. Taken together, our results demonstrate the foundational prototype of dlc-involved Notch signaling in the developing zebrafish forebrains, upon which the intricate patterns of the mammalian neocortex may have been sculpted.

Developmental trajectories of GABAergic cortical interneurons are sequentially modulated by dynamic FoxG1 expression levels

GABAergic inhibitory interneurons, originating from the embryonic ventral forebrain territories, traverse a convoluted migratory path to reach the neocortex. These interneuron precursors undergo sequential phases of tangential and radial migration before settling into specific laminae during differentiation. Here, we show that the developmental trajectory of FoxG1 expression is dynamically controlled in these interneuron precursors at critical junctures of migration. By utilizing mouse genetic strategies, we elucidate the pivotal role of precise changes in FoxG1 expression levels during interneuron specification and migration. Our findings underscore the gene dosage-dependent function of FoxG1, aligning with clinical observations of FOXG1 haploinsufficiency and duplication in syndromic forms of autism spectrum disorders. In conclusion, our results reveal the finely tuned developmental clock governing cortical interneuron development, driven by temporal dynamics and the dose-dependent actions of FoxG1.

Ror homolog nhr-23 is essential for both developmental clock and circadian clock in C. elegans

Animals have internal clocks that generate biological rhythms. In mammals, clock genes such as Period form the circadian clock to generate approximately 24-h biological rhythms. In C. elegans, the clock gene homologs constitute the “developmental clock”, which has an 8-h period during larval development to determine the timing of molting. Thus, the ancestral circadian clock has been believed to evolve into the oscillator with a shorter period in C. elegans. However, circadian rhythms have also been observed in adult C. elegans, albeit relatively weak. This prompts the question: if the clock gene homologs drive the developmental rhythm with 8-h period, which genes generate the circadian rhythms in C. elegans? In this study, we discovered that nhr-23, a homolog of the mammalian circadian clock gene Ror, is essential for circadian transcriptional rhythms in adult C. elegans. Interestingly, nhr-23 was also known to be essential for the molting clock. The bilaterian ancestral circadian clock genes might have evolved to function over multiple periods depending on developmental contexts rather than a single 8-h period in C. elegans.

Pharmacologic Therapies in Beta Hemoglobinopathies: Fetal Globin Gene Induction in the First Molecular Diseases

Beta hemoglobinopathies and thalassemias are caused by diverse molecular mutations of the βA globin chains and modulated by polymorphisms. These are important disorders in medical history, as they became prevalent globally in regions where P. falciparum malaria was endemic. Heterozygous or carrier states conferred a survival advantage; however, doubly heterozygous or homozygous states cause severe hemolytic anemia and multi-organ complications. These disorders are somewhat unique in that all humans have alternate genes for fetal globin chains, which are expressed in fetal life but are silenced on a developmental clock in infancy. An established approach to ameliorating conditions of abnormal adult globin genes is to reactivate, or increase expression, of the endogenous fetal globin genes to replace the missing protein chains in beta thalassemias or inhibit polymerization of hemoglobin S and reduce the red cell abnormalities. This review provides a high-level overview of cell and molecular mechanisms that mediate fetal to adult globin gene switching and pharmacologic therapies that can reactivate fetal globin through different actions. Intermittent or metronomic dosing regimens have overcome challenges of undesirable off-target effects by agents that cause erythroid cell growth arrest. Multiple pharmacologic candidates reactivate or increase the expression of fetal globin protein and proportions of red blood cells containing HbF (F-cells). Hydroxyurea maintains high levels of HbF if begun in infancy, with some decline in mid-childhood. It elicits lower responses in HbF in adult patients, but still has broad clinical benefit in reducing many complications. However, many adult patients do not tolerate optimal hydroxyurea dosing due to cytopenias. Parenterally administered therapies with differing molecular actions, such as demethylating agents and histone deacetylase inhibitors, have shown proof-of-principle in reactivating HbF. Orally bioavailable candidates with complementary molecular mechanisms are in trials. Combining fetal globin-inducing agents with other therapies with complementary mechanisms, such as recombinant erythropoietin that promotes the survival of red blood cells and therapeutics that promote erythroid cell metabolism, should have additive effects. These pharmaceutical candidates offer great clinical potential and global feasibility for ameliorating these serious diseases.

Creation of bioengineered lung using induced lung progenitor cells

In organ engineering, by combining stem cells, biomaterials and bioreactors, it may be possible for scientists to produce bioengineered organs for transplantation, as well as advancing understanding of organs and diseases. Researchers from the Department of Thoracic Surgery at Tohoku University in Japan and the University of Toronto in Canada are conducting multidisciplinary international research led by thoracic surgeon Assistant Professor Takaya Suzuki (Tohoku University) to create a bioengineering lung using induced lung progenitor cells. The research revolves around designing the respiratory system and involves utilising progenitor-generating cellular reprogramming using transient gene transfection. Ultimately, the researchers are seeking to identify a way to intentionally control the developmental clock of the human body and Suzuki believes that this will be possible if the team can unveil the key mechanisms that govern this clock. Suzuki is working closely with Professor Thomas K Waddell at the University of Toronto and Toronto General Hospital. It was in Waddellâ–™s lab that the concept of partial reprogramming was conceived, alongside global leading stem cell scientist Professor Andras Nagy. Alongside his collaborators, Suzuki is seeking to transform the landscape of transplantation medicine. As partial reprogramming can be employed in various organs and cells, any kind of organ bioengineering can benefit from the research.

Selective translation of epigenetic modifiers affects the temporal pattern and differentiation of neural stem cells

The cerebral cortex is formed by diverse neurons generated sequentially from neural stem cells (NSCs). A clock mechanism has been suggested to underlie the temporal progression of NSCs, which is mainly defined by the transcriptome and the epigenetic state. However, what drives such a developmental clock remains elusive. We show that translational control of histone H3 trimethylation in Lys27 (H3K27me3) modifiers is part of this clock. We find that depletion of Fbl, an rRNA methyltransferase, reduces translation of both Ezh2 methyltransferase and Kdm6b demethylase of H3K27me3 and delays the progression of the NSC state. These defects are partially phenocopied by simultaneous inhibition of H3K27me3 methyltransferase and demethylase, indicating the role of Fbl in the genome-wide H3K27me3 pattern. Therefore, we propose that Fbl drives the intrinsic clock through the translational enhancement of the H3K27me3 modifiers that predominantly define the NSC state.

Pathways of DNA Demethylation.

The regulation of the genome relies on the overlying epigenome to instruct, define, and restrict the activities of cellular differentiation and growth integral to embryonic development, as well as defining the key activities of terminally differentiated cell types. These instructions are positioned as readers, writers, and erasers in their functional roles. Among the sizeable repertoire of epigenetic instructions, DNA methylation is perhaps the best understood process. In mammals, multiple cycles of reprogramming, the addition and removal of DNA methylation coupled with modulation of chromatin post-translational modifications (PMTs), constitute critical phases when the developing embryo must negotiate lineage specification and commitment events which serve to canalise development. During these reprogramming events the DNA methylation instruction is often removed, thereby allowing a change in developmental restriction, resulting in a return to a more plastic and pluripotent state. Thus, in germline reprogramming, DNA demethylation is essential in order to give rise to fully functional gametes which are inherited across generations and poised to restore totipotency. A similar return to a less differentiated state can also be achieved experimentally. DNA methylation constitutes one of the significant barriers to erroneous induced pluripotency, and loss of DNA methylation is a prerequisite for the generation of induced pluripotent stem cells (iPSCs). Taking fully differentiated cells, such as skin fibroblast cells or peripheral blood cells, and turning back the developmental clock by generating iPSCs constituted a technological breakthrough in 2006, offering unprecedented promise in precision regenerative medicine. In this chapter, I will explore mechanistic possibilities for DNA demethylation in the context of natural and experimentally induced epigenetic reprogramming. The balance of the maintenance of DNA methylation as a heritable mark together with its potential for timely removal is essential for lifelong health and may be key in our understanding of aging and the potential to limit or reverse that process.

Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs.

Marked epigenetic reprogramming is essential to convert terminally differentiated gametes to totipotent embryos. It remains puzzling why postfertilization global DNA reprogramming occurs in mammals but not in nonmammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by extensive enhancer “dememorization” as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection, we eliminated DNA methylation in early embryos, which died around gastrulation with severe differentiation defects. Notably, methylation deficiency leads to derepression of adult tissue–specific genes and CG-rich enhancers, which acquire ectopic transcription factor binding and, unexpectedly, histone H3 lysine 4 trimethylation (H3K4me3). By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that “enhancer dememorization” underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and nonmammals.

Racing against the clock: How flies regenerate just in time.

In this issue of Developmental Cell, Cohen etal. show that the Drosophila hindgut is a genetically tractable model for studying tissue regeneration. This tissue exhibits different regeneration strategies at different developmental times, demonstrating that the hindgut developmental clock, not tissue type, dictates the mode and capacity for regeneration.

Developmental clock and mechanism of de novo polarization of the mouse embryo.

Embryo polarization is critical for mouse development; however, neither the regulatory clock nor the molecular trigger that it activates is known. Here, we show that the embryo polarization clock reflects the onset of zygotic genome activation, and we identify three factors required to trigger polarization. Advancing the timing of transcription factor AP-2 gamma (Tfap2c) and TEA domain transcription factor 4 (Tead4) expression in the presence of activated Ras homolog family member A (RhoA) induces precocious polarization as well as subsequent cell fate specification and morphogenesis. Tfap2c and Tead4 induce expression of actin regulators that control the recruitment of apical proteins on the membrane, whereas RhoA regulates their lateral mobility, allowing the emergence of the apical domain. Thus, Tfap2c, Tead4, and RhoA are regulators for the onset of polarization and cell fate segregation in the mouse.

Gene expression oscillations in C. elegans underlie a new developmental clock.

During C. elegans larval development, thousands of genes, accounting for >20% of the transcriptome, exhibit oscillatory expression with large amplitudes. The time of peaking varies for different genes, but expression generally peaks once per larval stage, with both the oscillation period and larval stage duration varying in concert with temperature. This and other evidence support the existence of a gene expression oscillator that functions as a developmental clock. In this article, we review what is known about the biology, architecture and possible mechanisms of this clock. We compare it to other oscillators, and highlight tools and approaches suited to its study. Finally, we point out implications of these wide-spread and dynamic changes of gene expression on any type of gene expression profiling experiment in C. elegans larvae and how such experiments need to be controlled.

SAT-448 Limited Adaptive Response by Deiodinase Enzymes in the Perinatal Hypothyroid Cochlea

Thyroid hormone (TH) is essential for cochlear development and normal auditory function. Considering the importance of TH in mediating cochlear development, understanding the degree to which developing tissues can adapt to perturbations in thyroid hormone signaling is extremely important. The deiodinases (Dio2 and Dio3) are enzymes that tightly control TH availability at the tissue level and have been proposed to function as adaptive mechanisms that maintain tissue TH homeostasis. Dio2 converts thyroxine (T4) to a biologically active ligand triiodothyronine (T3); locally amplifying a T3 signal. Conversely, Dio3 inactivates T3 and T4 by converting these iodothyronines to the inactive metabolites diiodothyronine (T2) and reverse T3 (rT3), respectively. During cochlear development, Dio3 expression is high prenatally while Dio2 expression is low. During the first postnatal week, the expression levels of these enzymes invert, resulting in high expression of Dio2 and low expression of Dio3. Together, the deiodinases control the timing of postnatal cochlear remodeling; suggesting a genetic developmental clock controls TH-mediated cochlear development. Considering the role deiodinases as adaptive mechanisms, it is important to understand whether such a developmental clock can be negated during times of developmental thyroid hormone insufficiency. We hypothesize that the perinatal change in deiodinase expression is controlled by a developmental clock rather than environmental clues and therefore have limited capacity function as compensatory mechanisms in response to low TH during development. To test this, timed-pregnant mice were treated with thyroid gland inhibitors to induced hypothyroidism from gestational day 12.5 until pup sacrifice. A parallel set of untreated timed-pregnant mice served as controls. Cochlea were harvest from control and hypothyroid mice at postnatal (P) ages P1, P5, P10 and P15 for qRT-PCR, and P7 for in situ hybridization. In situ hybridization for Dio2 mRNA demonstrated a striking reduction in Dio2 mRNA in hypothyroid animals compared to euthyroid animals. Additionally, we observed a significant effect of age (p<0.001) and an interaction between age and treatment (p=0.0035) on Dio2 mRNA levels analyzed by qRT-PCR. Specifically, Dio2 mRNA levels were similar or reduced in hypothyroid animals compared to controls at P1, P5, and P10. This finding indicates that Dio2 mRNA at these ages did not respond in manner consistent with compensation. However, at P15, Dio2 mRNA levels were increased in hypothyroid animals compared to controls; a finding that is consistent with a compensatory mechanism. Taken together, our results suggest that developmental programs in tissues may be dominant over potential compensatory mechanisms and that developing tissues may be more susceptible to perturbations in tissue TH levels due to a reduced capacity to compensate.

Automated minute scale RNA-seq of pluripotent stem cell differentiation reveals early divergence of human and mouse gene expression kinetics.

Pluripotent stem cells retain the developmental timing of their species of origin in vitro, an observation that suggests the existence of a cell-intrinsic developmental clock, yet the nature and machinery of the clock remain a mystery. We hypothesize that one possible component may lie in species-specific differences in the kinetics of transcriptional responses to differentiation signals. Using a liquid-handling robot, mouse and human pluripotent stem cells were exposed to identical neural differentiation conditions and sampled for RNA-sequencing at high frequency, every 4 or 10 minutes, for the first 10 hours of differentiation to test for differences in transcriptomic response rates. The majority of initial transcriptional responses occurred within a rapid window in the first minutes of differentiation for both human and mouse stem cells. Despite similarly early onsets of gene expression changes, we observed shortened and condensed gene expression patterns in mouse pluripotent stem cells compared to protracted trends in human pluripotent stem cells. Moreover, the speed at which individual genes were upregulated, as measured by the slopes of gene expression changes over time, was significantly faster in mouse compared to human cells. These results suggest that downstream transcriptomic response kinetics to signaling cues are faster in mouse versus human cells, and may offer a partial account for the vast differences in developmental rates across species.

Bilateral enucleation at birth modifies calcium spike amplitude, but not frequency, in neurons of the somatosensory thalamus and cortex: Implications for developmental cross-modal plasticity

Bilateral eye enucleation at birth (BE) leads to an expansion of the primary somatosensory cortex (S1) in rat pups. Although increased growth of the somatosensory thalamo-cortical afferents (STCAs) in part explains S1 expansion, timing mechanisms governing S1 formation are also involved. In this work, we begin the search of a developmental clock by intending to document the existence of putative clock neurons in the somatosensory thalamus (VPM) and S1 based upon changes of spontaneous spike amplitude; a biophysical property sensitive to circadian regulation; the latter known to be shifted by enucleation. In addition, we also evaluated whether STCAs growth rate and segregation timing were modified, as parameters the clock might time. We found that spontaneous spike amplitude transiently, but significantly, increased or decreased in VPM and S1 neurons of BE rat pups, respectively, as compared to their control counterparts. The growth rate and segregation timing of STCAs was, however, unaffected by BE. These results support the existence of a developmental clock that ticks differently in the VPM and S1 after BE. This observation, together with the fact that STCAs growth rate and segregation timing is unchanged, suggests that S1 expansion in BE rats may in part be controlled at the cortical level.

Species-specific developmental timing is maintained by pluripotent stem cells ex utero

How species-specific developmental timing is controlled is largely unknown. By following human embryonic stem (ES) cell and mouse epiblast stem (EpiS) cell differentiation through detailed RNA-sequencing time courses, here we show that pluripotent stem cells closely retain in vivo species-specific developmental timing in vitro. In identical neural differentiation conditions in vitro, gene expression profiles are accelerated in mouse EpiS cells compared to human ES cells with relative rates of differentiation closely reflecting the rates of progression through the Carnegie stages in utero. Dynamic Time Warping analysis identified 3389 genes that were regulated more quickly in mouse EpiS cells and identified none that were regulated more quickly in human ES cells. Interestingly, we also find that human ES cells differentiated in teratomas maintain the same rate of differentiation observed in vitro in spite of being grown in a mouse host. These results suggest the existence of a cell autonomous, species-specific developmental clock that pluripotent stem cells maintain even out of context of an intact embryo.

Inhibition of MLL1 histone methyltransferase brings the developmental clock back to naïve pluripotency.

Embryonic stem cells (ESCs) and the post-implantation epiblast stem cells (EpiSCs) portray two different states of pluripotency. They differ with respect to epigenetic signatures, dependency of growth factor signaling circuit and cell morphology. They are interconvertible, however, with poor reconversion efficiency. This is indicative of existence of other unknown regulatory pathways govern developmental stage transition. Zhang and colleagues have recently demonstrated that pharmacological inhibition of MLL1 histone methyltransferase is casually linked to efficient reprogramming of EpiSCs to developmentally competent ESCs. MLL1 controlled H3K4me1 serves as an epigenetic valve that ensures maintenance of EpiSCs. Removing this barrier leads to global redistribution of H3K4me1 at enhancers and target gene promoters, in turn represses EpiSC specific genes and reactivates ESC specific transcriptional network. This study underscores the critical role of MLL1 in establishing discrete chromatin states indispensible for early mammalian developmental events.

Plasticity Related Gene 3 (PRG3) overcomes myelin-associated growth inhibition and promotes functional recovery after spinal cord injury.

The Plasticity Related Gene family covers five, brain-specific, transmembrane proteins (PRG1-5, also termed LPPR1-5) that operate in neuronal plasticity during development, aging and brain trauma. Here we investigated the role of the PRG family on axonal and filopodia outgrowth. Comparative analysis revealed the strongest outgrowth induced by PRG3 (LPPR1). During development, PRG3 is ubiquitously located at the tip of neuronal processes and at the plasma membrane and declines with age. In utero electroporation of PRG3 induced dendritic protrusions and accelerated spine formations in cortical pyramidal neurons. The neurite growth promoting activity of PRG3 requires RasGRF1 (RasGEF1/Cdc25) mediated downstream signaling. Moreover, in axon collapse assays, PRG3-induced neurites resisted growth inhibitors such as myelin, Nogo-A (Reticulon/RTN-4), thrombin and LPA and impeded the RhoA-Rock-PIP5K induced neurite repulsion. Transgenic adult mice with constitutive PRG3 expression displayed strong axonal sprouting distal to a spinal cord lesion. Moreover, fostered PRG3 expression promoted complex motor-behavioral recovery compared to wild type controls as revealed in the Schnell swim test (SST). Thus, PRG3 emerges as a developmental RasGRF1-dependent conductor of filopodia formation and axonal growth enhancer. PRG3-induced neurites resist brain injury-associated outgrowth inhibitors and contribute to functional recovery after spinal cord lesions. Here, we provide evidence that PRG3 operates as an essential neuronal growth promoter in the nervous system. Maintaining PRG3 expression in aging brain may turn back the developmental clock for neuronal regeneration and plasticity.

Developmental synchrony in multivoltine insects: generation separation versus smearing

AbstractMany insect species undergo multiple generations each year. They are found across biomes that vary in their strength of seasonality and, depending on location and species, can display a wide range of population dynamics. Some species exhibit cycles with distinct generations (developmental synchrony/generation separation), some exhibit overlapping generations with multiple life stages present simultaneously (generation smearing), while others have intermediate dynamics with early season separation followed by late season smearing. There are two main hypotheses to explain these dynamics. The first is the ‘seasonal disturbance’ hypothesis where winter synchronizes the developmental clock among individuals, which causes transient generation separation early in the season that erodes through the summer. The second is the ‘temperature destabilization’ hypothesis where warm temperatures during the summer cause population dynamics to become unstable giving rise to single generation cycles. Both hypotheses are supported by detailed mathematical theory incorporating mechanisms that are likely to drive dynamics in nature. In this review, we synthesize the theory and propose a conceptual framework—where each mechanism may be seen as an independent axis shaping the developmental (a)synchrony—that allows us to predict dynamic patterns from insect life‐history characteristics. High fecundity, short adult life‐span and strong seasonality enhance synchrony, while developmental plasticity and environmental heterogeneity erode synchrony. We further review current mathematical and statistical tools to study multi‐generational dynamics and illustrate using case studies of multivoltine tortrix moths. By integrating two disparate bodies of theory, we articulate a deep connection among temperature, stability, developmental synchrony and inter‐generational dynamics of multivoltine insects that is missing in current literature.

Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation.

Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a ‘developmental clock’ using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model.

Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning.

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.