Enhancement of neural crest formation by mechanical force in Xenopus development.

In vertebrate development, ectoderm is specified into neural plate (NP), neural plate border (NPB), and epidermis. Although such patterning is thought to be achieved by molecular concentration gradients, it has been revealed, mainly by in vitro analysis, that mechanical force can regulate cell specification. During in vivo patterning, cells deform and migrate, and this applies force to surrounding tissues, shaping the embryo. However, the role of mechanical force for cell specification in vivo is largely unknown. In this study, with an aspiration assay and atomic force microscopy, we have demonstrated that tension on ectodermal cells decreases laterally from the midline in Xenopus early neurula. Ectopically applied force laterally expanded the neural crest (NC) region, a derivative of the NPB, whereas force relaxation suppressed it. Furthermore, force application activated both the FGF and Wnt pathways, which are required for NC formation during neuroectodermal patterning. Taken together, mechanical force is necessary for NC formation in order to regulate signaling pathways. Furthermore, molecular signals specify the NP and generate force on neighboring tissue, the NPB, with its closure. This force activates signals, possibly determining the appropriate width of a narrow tissue, the NC.

Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Maternal factor Trim75 contributes to zygotic genome activation program in mouse early embryos.

BACKGROUND: Zygotic genome activation (ZGA) is an important event in the early embryo development, and human embryo developmental arrest has been highly correlated with ZGA failure in clinical studies. Although a few studies have linked maternal factors to mammalian ZGA, more studies are needed to fully elucidate the maternal factors that are involved in ZGA. METHODS AND RESULTS: In this study, we utilized published single-cell RNA sequencing data from a Dux-mediated mouse embryonic stem cell to induce a 2-cell-like transition state and selected potential drivers for the transition according to an RNA velocity analysis. CONCLUSIONS: An overlap of potential candidate markers of 2-cell-like-cells identified in this research with markers generated by various data sets suggests that Trim75 is a potential driver of minor ZGA and may recruit EP300 and establish H3K27ac in the gene body of minor ZGA genes, thereby contributing to mammalian preimplantation embryo development.

Laboratory Course Using Zebrafish to Uncover Changing Roles of Wnt Signaling in Early Vertebrate Development.

Coordinated signaling pathway activity directs early patterning to set up the vertebrate body plan. Perturbations in the timing or location of signal molecule expression impacts embryo morphology and organ formation. In this study, we present a laboratory course to use zebrafish for studying the role of Wnt signaling in specifying the early embryonic axes. Students are exposed to basic techniques in molecular and developmental biology, including embryo manipulation, fluorescence microscopy, image processing, and data analysis. Furthermore, this course incorporates student-designed experiments to stimulate independent inquiry and improve scientific learning, providing an experience resembling graduate-level laboratory research. Students appreciated following vertebrate development in real-time, and principles of embryogenesis were reinforced by observing the morphological changes that arise due to signaling alterations. Scientific and research skills were enhanced through practice in experimental design, interpretation, and presentation.

RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

Cell-type-specific mRNA transcription and degradation kinetics in zebrafish embryogenesis from metabolically labeled single-cell RNA-seq.

During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.

Dynamic Hippo pathway activity underlies mesenchymal differentiation during lung alveolar morphogenesis.

Alveologenesis, the final stage in lung development, substantially remodels the distal lung, expanding the alveolar surface area for efficient gas exchange. Secondary crest myofibroblasts (SCMF) exist transiently in the neonatal distal lung and are crucial for alveologenesis. However, the pathways that regulate SCMF function, proliferation and temporal identity remain poorly understood. To address this, we purified SCMFs from reporter mice, performed bulk RNA-seq and found dynamic changes in Hippo-signaling components during alveologenesis. We deleted the Hippo effectors Yap/Taz from Acta2-expressing cells at the onset of alveologenesis, causing a significant arrest in alveolar development. Using single cell RNA-seq, we identified a distinct cluster of cells in mutant lungs with altered expression of marker genes associated with proximal mesenchymal cell types, airway smooth muscle and alveolar duct myofibroblasts. In vitro studies confirmed that Yap/Taz regulates myofibroblast-associated gene signature and contractility. Together, our findings show that Yap/Taz is essential for maintaining functional myofibroblast identity during postnatal alveologenesis.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Karyopherin α2 is a maternal effect gene required for early embryonic development and female fertility in mice.

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene.

SRRM2 splicing factor modulates cell fate in early development.

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development.

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

A geometrical model of cell fate specification in the mouse blastocyst.

The lineage decision that generates the epiblast and primitive endoderm from the inner cell mass (ICM) is a paradigm for cell fate specification. Recent mathematics has formalized Waddington's landscape metaphor and proven that lineage decisions in detailed gene network models must conform to a small list of low-dimensional stereotypic changes called bifurcations. The most plausible bifurcation for the ICM is the so-called heteroclinic flip that we define and elaborate here. Our re-analysis of recent data suggests that there is sufficient cell movement in the ICM so the FGF signal, which drives the lineage decision, can be treated as spatially uniform. We thus extend the bifurcation model for a single cell to the entire ICM by means of a self-consistently defined time-dependent FGF signal. This model is consistent with available data and we propose additional dynamic experiments to test it further. This demonstrates that simplified, quantitative and intuitively transparent descriptions are possible when attention is shifted from specific genes to lineages. The flip bifurcation is a very plausible model for any situation where the embryo needs control over the relative proportions of two fates by a morphogen feedback.

Multiple transcriptome analyses reveal mouse testis developmental dynamics.

The testes are the organs of gamete production and testosterone synthesis. Up to date, no model system is available for mammalian testicular development, and only few studies have characterized the mouse testis transcriptome from no more than three postnatal ages. To describe the transcriptome landscape of the developing mouse testis and identify the potential molecular mechanisms underlying testis maturation, we examined multiple RNA-seq data of mouse testes from 3-week-old (puberty) to 11-week-old (adult). Sperm cells appeared as expected in 5-week-old mouse testis, suggesting the proper sample collection. The principal components analysis revealed the genes from 3w to 4w clustered away from other timepoints, indicating they may be the important nodes for testicular development. The pairwise comparisons at two adjacent timepoints identified 7,612 differentially expressed genes (DEGs), resulting in 58 unique mRNA expression patterns. Enrichment analysis identified functions in tissue morphogenesis (3-4w), regulation of peptidase activity (4-5w), spermatogenesis (7-8w), and antigen processing (10-11w), suggesting distinct functions in different developmental periods. 50 hub genes and 10 gene cluster modules were identified in the testis maturation process by protein-protein interaction (PPI) network analysis, and the miRNA-lncRNA-mRNA, miRNA-circRNA-mRNA and miRNA-circRNA-lncRNA-mRNA competing endogenous RNA (ceRNA) networks were constructed. The results suggest that testis maturation is a complex developmental process modulated by various molecules, and that some potential RNA-RNA interactions may be involved in specific developmental stages. In summary, this study provides an update on the molecular basis of testis development, which may help to understand the molecular mechanisms of mouse testis development and provide guidance for mouse reproduction.

SUMO-dependent transcriptional repression by Sox2 inhibits the proliferation of neural stem cells.

Sox2 is known for its roles in maintaining the stem cell state of embryonic stem cells and neural stem cells. In particular, it has been shown to slow the proliferation of these cell types. It is also known for its effects as an activating transcription factor. Despite this, analysis of published studies shows that it represses as many genes as it activates. Here, we identify a new set of target genes that Sox2 represses in neural stem cells. These genes are associated with centrosomes, centromeres and other aspects of cell cycle control. In addition, we show that SUMOylation of Sox2 is necessary for the repression of these genes and for its repressive effects on cell proliferation. Together, these data suggest that SUMO-dependent repression of this group of target genes is responsible for the role of Sox2 in regulating the proliferation of neural stem cells.

Temporospatial hierarchy and allele-specific expression of zygotic genome activation revealed by distant interspecific urochordate hybrids.

Zygotic genome activation (ZGA) is a universal process in early embryogenesis of metazoan, when the quiescent zygotic nucleus initiates global transcription. However, the mechanisms related to massive genome activation and allele-specific expression (ASE) remain not well understood. Here, we develop hybrids from two deeply diverged (120 Mya) ascidian species to symmetrically document the dynamics of ZGA. We identify two coordinated ZGA waves represent early developmental and housekeeping gene reactivation, respectively. Single-cell RNA sequencing reveals that the major expression wave exhibits spatial heterogeneity and significantly correlates with cell fate. Moreover, allele-specific expression occurs in a species- rather than parent-related manner, demonstrating the divergence of cis-regulatory elements between the two species. These findings provide insights into ZGA in chordates.

Comparison of genes involved in brain development: insights into the organization and evolution of the telencephalic pallium.

The mechanisms underlying the organization and evolution of the telencephalic pallium are not yet clear.. To address this issue, we first performed comparative analysis of genes critical for the development of the pallium (Emx1/2 and Pax6) and subpallium (Dlx2 and Nkx1/2) among 500 vertebrate species. We found that these genes have no obvious variations in chromosomal duplication/loss, gene locus synteny or Darwinian selection. However, there is an additional fragment of approximately 20 amino acids in mammalian Emx1 and a poly-(Ala)6-7 in Emx2. Lentiviruses expressing mouse or chick Emx2 (m-Emx2 or c-Emx2 Lv) were injected into the ventricle of the chick telencephalon at embryonic Day 3 (E3), and the embryos were allowed to develop to E12-14 or to posthatchling. After transfection with m-Emx2 Lv, the cells expressing Reelin, Vimentin or GABA increased, and neurogenesis of calbindin cells changed towards the mammalian inside-out pattern in the dorsal pallium and mesopallium. In addition, a behavior test for posthatched chicks indicated that the passive avoidance ratio increased significantly. The study suggests that the acquisition of an additional fragment in mammalian Emx2 is associated with the organization and evolution of the mammalian pallium.

The organizer: What it meant, and still means, to developmental biology.

This article is about how the famous organizer experiment has been perceived since it was first published in 1924. The experiment involves the production of a secondary embryo under the influence of a graft of a dorsal lip from an amphibian gastrula to a host embryo. The early experiments of Spemann and his school gave rise to a view that the whole early amphibian embryo was "indifferent" in terms of determination, except for a special region called "the organizer". This was viewed mainly as an agent of neural induction, also having the ability to generate an anteroposterior body pattern. Early biochemical efforts to isolate a factor emitted by the organizer were not successful but culminated in the definition of "neuralizing (N)" and "mesodermalizing (M)" factors present in a wide variety of animal tissues. By the 1950s this view became crystallized as a "two gradient" model involving the N and M factors, which explained the anteroposterior patterning effect. In the 1970s, the phenomenon of mesoderm induction was characterized as a process occurring before the commencement of gastrulation. Reinvestigation of the organizer effect using lineage labels gave rise to a more precise definition of the sequence of events. Since the 1980s, modern research using the tools of molecular biology, combined with microsurgery, has explained most of the processes involved. The organizer graft should now be seen as an experiment which involves multiple interactions: dorsoventral polarization following fertilization, mesoderm induction, the dorsalizing signal responsible for neuralization and dorsoventral patterning of the mesoderm, and additional factors responsible for anteroposterior patterning.

Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Segregation of morphogenetic regulatory function of Shox2 from its cell fate guardian role in sinoatrial node development.

Shox2 plays a vital role in the morphogenesis and physiological function of the sinoatrial node (SAN), the primary cardiac pacemaker, manifested by the formation of a hypoplastic SAN and failed differentiation of pacemaker cells in Shox2 mutants. Shox2 and Nkx2-5 are co-expressed in the developing SAN and regulate the fate of the pacemaker cells through a Shox2-Nkx2-5 antagonistic mechanism. Here we show that simultaneous inactivation of Nkx2-5 in the SAN of Shox2 mutants (dKO) rescued the pacemaking cell fate but not the hypoplastic defects, indicating uncoupling of SAN cell fate determination and morphogenesis. Single-cell RNA-seq revealed that the presumptive SAN cells of Shox2-/- mutants failed to activate pacemaking program but remained in a progenitor state preceding working myocardium, while both wildtype and dKO SAN cells displayed normal pacemaking cell fate with similar cellular state. Shox2 thus acts as a safeguard but not a determinant to ensure the pacemaking cell fate through the Shox2-Nkx2-5 antagonistic mechanism, which is segregated from its morphogenetic regulatory function in SAN development.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis.

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.