Biology subjects

Hou, Y.

Publications and source records attributed to Hou, Y..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

The discovery of phages in the Substantia Nigra and its implication for Parkinson's Disease

BackgroundA century ago, a mystery between virus and Parkinsons disease (PD) was described. Owing to the limitation of human brain biopsy and the challenge of electron microscopy in observing virions in human brain tissue, it has been difficult to study the viral etiology of PD. Recent discovery of virobiota reveals that viruses coexist with humans as symbionts. Newly-developed transcriptomic sequencing and novel bioinformatic approaches for mining the encrypted virome in human transcriptome make it possible to study the relationship between symbiotic viruses and PD. Nevertheless, whether viruses exist in the human substantial nigra (SN), and whether symbiotic viruses underlie PD pathogenesis remain unknown. MethodsWe collected current worldwide human SN transcriptomic datasets from the United States, the United Kingdom, the Netherlands and Switzerland. We used bioinformatic approaches including viruSITE and the Virus-Track to identify the existence of viruses in the SN of patients. The comprehensive RNA sequencing-based virome analysis pipeline was used to characterize the virobiota in the SN. The Pearsons correlation analysis was used to examine the association between the viral RNA fragment counts (VRFC) and PD-related human gene sequencing reads in the SN. The differentially expressed genes (DEGs) in the SN between PD patients and non-PD individuals were used to examine the molecular signatures of PD and also evaluate the impact of symbiotic viruses on the SN. FindingsWe observed the existence of viruses in the human SN. A dysbiosis of virobiota was found in the SN of PD patients. A significant correlation between VRFC and PD-related human gene expression was detected in the SN of PD patients. These PD-related human genes correlated to VRFC were named as the virus-correlated PD-related genes (VPGs). We identified three bacteriophages (phages), including the Proteus phage VB_PmiS-Isfahan, the Escherichia phage phiX174 and the Lactobacillus phage Sha1, that might impair the gene expression of neural cells in the SN of PD patients. The Proteus phage VB_PmiS-Isfahan was a common virus in the SN of patients from the UK, the Netherlands, and Switzerland. VPGs and DEGs together highlighted that the phages might dampen dopamine biosynthesis and weaken cGAS-STING function. InterpretationThis is the first study to discover the involvement of phages in PD pathogenesis. A life-long low symbiotic viral load in the SN may be a contributor to PD pathogenesis. Our findings unlocked the black box between brain virobiota and PD, providing a novel insight into PD etiology from the perspective of phages-human symbiosis.

bioinformatics↗

bioRxiv2024

Pre-Assembly NGS Correction of ONT Reads Achieves HiFi-Level Assembly Quality

BackgroundRecently developed hybrid assemblies can achieve Telomere-to-Telomere (T2T) completeness of some chromosomes. However, such approaches involve sequencing a large volume of both Pacific Biosciences high-fidelity (HiFi) and Oxford Nanopore Technologies (ONT) sequencing reads. Along with this, third-generation sequencing techniques are rapidly advancing, increasing the available length and accuracy. To reduce the final cost of genome assembly, here we investigated the possibility of assembly from low-coverage samples and with only ONT corrected by Next-Generation Sequencing (NGS) sequencing reads. We demonstrated that ONT-based assembly approaches corrected by NGS can achieve performance metrics comparable to more expensive hybrid approaches based on HiFi sequencing. ResultsWe investigated the assembly of different chromosomes of HG002 and the low-coverage performance of state-of-the-art hybrid assembly tools, including Verkko and Hifiasm, and ONT-based assemblers Shasta and Flye. Our study shows that even with the best NG50, Hifiasm has many misassemblies in the centromere region, raising doubts about its T2T capabilities. Additionally, ONT-based assemblies perform well at low coverage, offering a cost-effective solution. To achieve better assembly quality, we evaluated the performance of different NGS technologies across various aspects of hybrid genome assembly, including pre-assembly correction, haplotype phasing, and polishing, and found them to be similarly effective. For the NGS technology stLFR, we proposed two-round assembly methods that utilize stLFR linked-read data to achieve assembly phasing performance comparable to that obtained with trio data. ConclusionWe found that assemblies based on ONT corrected by NGS can match or exceed HiFi-based assemblies in accuracy. It appears that no assembly tool investigated here can achieve T2T completeness, even with 50X coverage. Therefore, we recommend using a combination of regular R9 or R10 simplex ONT reads and accurate NGS reads for assembly without aiming for T2T completeness. These ONT-based assemblies perform well even at low coverage, offering a cost-effective solution.

genomics↗

bioRxiv2024

A unique mechanism of snRNP core assembly

The assembly of spliceosomal snRNP cores involves seven Sm proteins (D1/D2/F/E/G/D3/B) forming a ring around each snRNA and typically requires several essential assembly chaperones, particularly the SMN-Gemins complex implicated in spinal muscular atrophy (SMA). Strikingly, in budding yeast, snRNP core assembly only involves Brr1, a nonessential homolog of Gemin2. To unravel this enigma, we systematically investigated snRNP core assembly in budding yeast using biochemical and genetic approaches. We discovered two distinct pathways in budding yeast: chaperone-mediated and direct. The chaperone-mediated pathway involves two proteins, Brr1 and a novel protein, Lot5, but is inefficient. Lot5 binds D1/D2/F/E/G to form a heterohexameric ring (6S). Brr1 binds D1/D2/F/E/G and 6S but cannot displace Lot5 to facilitate assembly. Disruption of both BRR1 and LOT5 genes caused mild growth retardation, but LOT5 overexpression substantially impeded growth. The direct pathway uniquely involves F/E/G as a trimer and a stable D1/D2/F/E/G intermediate complex, explaining the non-essentiality of assembly chaperones. These findings unveil a unique assembly mechanism of snRNP cores, illuminate the evolution of assembly chaperones, and suggest avenues for studying SMA pathophysiology.

molecular biology↗

bioRxiv2024

The RAE1-STOP1 Module Reduces Sensitivity to Exogenous ABA Treatment by Regulating ABI5 in Arabidopsis

The SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor plays a pivotal role in maintaining cellular ion balance and governing aluminum tolerance in plants. Abscisic acid (ABA) participates in aluminum tolerance by inducing the expression of several genes that are STOP1 targets. However, the mechanisms underlying ABA signaling and STOP1-mediated gene expression remain poorly understood. The F-box protein RAE1, an SCF-type E3 ligase component, recognizes STOP1 and controls its ubiquitination and degradation. This study revealed that exogenous ABA supplementation reduced STOP1 levels by promoting the expression of RAE1. Notably, both RAE1 loss-of-function mutants and STOP1 overexpressing lines showed enhanced sensitivity to exogenous ABA treatment, which correlated with early stage post-transcriptional upregulation of ABSCISIC ACID INSENSITIVE5 (ABI5). Our observations strongly suggest that RAE1 operates as an ABA-responsive factor, exerting control over STOP1 homeostasis, and thus establishing a negative feedback loop that controls ABA responses in Arabidopsis. Thus, our study revealed a novel function of the RAE1-STOP1 module in ABA signaling, highlighting its role in reducing ABA sensitivity by preventing ABI5 increase. Single Sentence SummaryF-box protein RAE1 functions as an exogenous ABA responsive mediator to reduce STOP1-mediated ABA sensitivity.

plant biology↗

bioRxiv2024

CRISPR screens reveal convergent targeting strategies against evolutionarily distinct chemoresistance in cancer

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.

cancer biology↗

bioRxiv2024

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu

The estrogen receptor- (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations --receptor conformational heterodimers--mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. SignificanceThe estrogen receptor- (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

biophysics↗

bioRxiv2024

Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. Here, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-day time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds the molecular foundation and a comparative single-cell genomic framework to identify neutrophil markers of tissue damage using model organisms.

immunology↗

bioRxiv2024

Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy

A fundamental challenge in fluorescence microscopy is the defocused background caused by scattering light, optical aberration, or limited axial resolution. Severe defocus backgrounds will submerge the in-focus information and cause artifacts in the following processing. Here, we leverage a priori knowledge about dark channels of biological structures and dual frequency separation to develop a single-frame defocus removal algorithm. It stably improves the signal-to-background ratio and structural similarity index measure of images by approximately 10-fold, and recovers in-focus signal with 85% accuracy, even when the defocus background is 50 times larger than in-focus information. Our Dark-based optical sectioning approach (Dark sectioning) is fully compatible with various microscopy techniques, such as wide-filed microscopy, polarized microscopy, laser-scanning / spinning-disk confocal microscopy, stimulated emission depletion microscopy, lightsheet microscopy, and light-field microscopy. It also complements reconstruction or processing algorithms such as deconvolution, structure illumination microscopy, and super-resolution optical fluctuation imaging.

bioinformatics↗

bioRxiv2024

Platinum drug reprogramming of protein phosphorylation

Cisplatin is a DNA-targeting chemotherapeutic. Here we investigate how the cisplatin damaged gene loci are linked to specific protein-driven signalling pathways. Using forward chemical genetics methods, cisplatin damage to specific genes has been mapped in human lung cancer cells, a total of 16216 cisplatin-damaged genes (CDGs) with fold-enrichment > 1.5. Surprisingly, bioinformatics analysis demonstrates that cisplatin targets the majority of human protein kinase and phosphatase genes and involved in 300 core signalling pathways (-log p >4). The most associated key signalling pathways are sperm motility and protein kinase A. The highest related disease is cancer, and tissue toxicities related to CDGs are about hepato- and nephro- toxicities. Notably, cisplatin damaged 85% (440) of human protein kinase genes and 81% (110) of human protein phosphatase genes. This suggests that cisplatin acts as a multi-targeting protein-phosphorylation regulator, confirmed by a significant decrease in expression of a series of key protein kinase genes. These results reveal that cisplatin disrupts protein phosphorylation signalling genome-wide. Key points summarising the key messages of your articleA total of 16216 genes damaged by the clinical drug cisplatin in human lung cancer cells were identified using a forward chemical genetics method. Bioinformatics analysis revealed that cisplatin targets 85% of protein kinase and 81% of phosphatase genes. Cisplatin acts as a multi-targeting protein-phosphorylation regulator, disrupting protein phosphorylation signalling genome-wide. The disturbance of protein phosphorylation by cisplatin can be related to anticancer activity and tissue toxicities of the drug. These findings suggests novel strategies for rational design of next-generation anticancer metallodrugs involving specific targeting of protein phosphorylation.

cancer biology↗

bioRxiv2024

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+ hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+ HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

molecular biology↗

bioRxiv2024

Urolithin A improves Alzheimers disease cognition and restores mitophagy and lysosomal functions

BackgroundCompromised autophagy, including impaired mitophagy and lysosomal function, is thought to play a pivotal role in Alzheimers disease (AD). Urolithin A (UA) is a gut microbial metabolite of ellagic acid that stimulates mitophagy. The effects of early and/or long-term treatment, as well as more detailed mechanisms of action, are not known. MethodsWe addressed these questions in three mouse models of AD, and behavioral, electrophysiological and biochemistry assays were performed. ResultsLong-term UA treatment significantly improved learning, memory and olfactory function in different AD transgenic mice. UA also reduced A{beta} and Tau pathologies, and improved long-term potentiation. We found that UA activated autophagy/mitophagy via increasing lysosomal functions. At the cellular level, UA improved lysosomal function and normalized lysosomal cathepsins, especially targeting cathepsin Z, to restore lysosomal function in AD, indicating the important role of cathepsins in UA-induced therapeutic effects of AD. ConclusionsCollectively, our study highlights the importance of lysosomal dysfunction in AD etiology, and points to the high translational potential of UA.

neuroscience↗

bioRxiv2024

Increased regional P2X7R expression detected by GSK1482160 PET in a tauopathy mouse model

Neuroinflammation plays an important role in Alzheimers disease and primary tauopathies. The aim of the current study was to map [18F]GSK1482160 for imaging of purinergic P2X7R in Alzheimers disease and primary tauopathy mouse models. MicroPET was performed using [18F]GSK1482160 in widely used mouse models of Alzheimers disease (APP/PS1, 5xFAD and 3xTg), 4-repeat tauopathy (rTg4510) mice and age-matched wild-type mice. Increased uptake of [18F]GSK1482160 was observed in the cortex and basal forebrain of 7-month-old rTg4510 mice compared to age-matched wild-type mice and compared to 3-month-old rTg4510 mice. Nonparametric Spearmans rank analysis revealed a positive correlation between tau [18F]APN-1607 uptake and [18F]GSK1482160 in the hippocampus of rTg4510 mice. No significant differences in the uptake of [18F]GSK1482160 were observed between wild-type mice and APP/PS1 mice (5, 10 months), 5xFAD mice (3, 7 months) or 3xTg mice (10 months). Immunofluorescence staining further indicated the distribution of P2X7Rs in the brains of 7-month-old rTg4510 mice with accumulation of tau inclusion compared to wild-type mice. These findings provide in vivo imaging evidence for increased P2X7R in the brains of tauopathy model mice.

neuroscience↗

bioRxiv2024

Blocker-SELEX: A Structure-guided Strategy for Developing Inhibitory Aptamers Disrupting Undruggable Transcription Factor Interactions

Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges associated with modulating their protein-protein and protein-DNA interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs do not favor the use of small-molecule inhibitors, or neutral antibodies, in blocking TF interactions. Aptamers are short oligonucleotides exhibiting high affinity and specificity for a diverse range of targets. Large molecular weights, expansive blocking surfaces and efficient cellular internalization make aptamers a compelling molecular tool for use as traditional TF interaction modulators. Here, we report a structure-guided design strategy called Blocker-SELEX to develop inhibitory aptamers (iAptamer) that selectively block TF interactions. Our approach led to the discovery of iAptamers that cooperatively disrupts SCAF4/SCAF8-RNA Polymerase II (RNAP2) interactions, thereby dysregulating RNAP2-dependent gene expression and splicing and, in turn, leading to the impairment of cell proliferation. This approach was further applied to develop iAptamers to efficiently block WDR5-MYC interaction with a nexus in cancer. Taken together, our study highlights the potential of Blocker-SELEX in developing iAptamers that effectively disrupt potentially pathogenic TF interactions with attendant implications for iAptamers as chemical tools for use in the study of biological functions of TF interactions, but also for potential use in nucleic acids drug discovery.

biochemistry↗

bioRxiv2023

AVM: a manually curated database of aerosol-transmitted virus mutations, human diseases, and drugs

Aerosol-transmitted viruses, with aerosol particles floating in the air to long distances, have the characteristics of strong infectivity and wide spread that is difficult to control. They cause various human diseases, posing a huge threat to human health. Some mutations can increase the transmissibility and virulence of the strain, which can reduce the protection of vaccines and weaken efficacy of antiviral drugs. Here, we established a manually curated database, AVM, to store this information. The current version of the AVM contains a total of 42,041 virus mutations, including 2613 immune escape mutations, 45 clinical information datasets, and 407 drugs, antibodies, or vaccines. In addition, we recorded 88 human diseases associated with viruses, and we found that the same virus can attack multiple target organs in the body and lead to diversified diseases. Further, the AVM database offers a straightforward user interface to expediently browse, retrieve, and download details. The AVM database is a comprehensive resource that provides timely and valuable assistance regarding the transmission, treatment, and related diseases of aerosol-transmitted viruses (http://bio-bigdata.hrbmu.edu.cn/AVM or http://www.bio-bigdata.center/AVM).

bioinformatics↗

bioRxiv2023

HELZ promotes R loop resolution to facilitate DNA double-strand break repair by homologous recombination

R loop homeostasis is critical for DNA double-strand break (DSB) repair; however, how R loops are resolved in this context is poorly understood. Here, we define HELZ as a unique RNA-DNA helicase that resolves R loops to facilitate homologous recombination (HR) repair. From a synthetic lethal etoposide resistance siRNA screen, we found that HELZ depletion causes R loop-mediated hypersensitivity to DSB-inducing agents, and HELZ localizes and binds to DSBs. HELZ preferentially binds to and unwinds RNA-DNA hybrids with 5ssRNA overhangs to promote R loop resolution genome-wide and at DSBs. Interestingly, HELZ facilitates BRCA1 recruitment to DSBs by preventing R loop accumulation, thereby promoting DNA end resection and HR to prevent R loop mediated genomic instability. Our findings define a role for HELZ in resolving R loops critical for HR that promotes genome stability and governs DSB-inducing agent resistance.

molecular biology↗

bioRxiv2023

High-speed Auto-Polarization Synchronization Modulation Three-dimensional Structured Illumination Microscopy

In recent years, notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy (SIM). Nevertheless, the advancement of 3DSIM has been impeded by challenges arising from the speed and intricacy of polarization modulation. In this study, we introduce a high-speed modulation 3DSIM system, leveraging the polarization maintaining and modulation capabilities of a digital micro-mirror device (DMD) in conjunction with an electro-optic modulator. The DMD-3DSIM system yields a 2-fold enhancement in both lateral (133 nm) and axial (300 nm) resolution compared to wide-field imaging, and can acquire a data set comprising 29 sections of 1024x1024 pixels, with 15 ms exposure time and 6.75 s per volume. The versatility of the DMD-3DSIM approach was exemplified through the imaging of various specimens, including fluorescent beads, nuclear pores, microtubules, actin filaments, and mitochondria within cells, as well as plant and animal tissues. Notably, polarized 3DSIM elucidated the orientation of actin filaments. Furthermore, the implementation of diverse deconvolution algorithms further enhances three-dimensional resolution. The DMD-based 3DSIM system presents a rapid and reliable methodology for investigating biomedical phenomena, boasting capabilities encompassing 3D superresolution, fast temporal resolution, and polarization imaging.

cell biology↗

bioRxiv2023

Comparative analysis of methodologies for detecting extrachromosomal circular DNA

Extrachromosomal circular DNA (eccDNA) is crucial in oncogene amplification, gene transcription regulation, and intratumor heterogeneity. While various analysis pipelines and experimental methods have been developed for eccDNA identification, their detection efficiencies have not been systematically assessed. To address this, we evaluated the performance of 7 analysis pipelines using three simulated datasets, in terms of accuracy, similarity, duplication rate, and computational resource consumption. We also compared the eccDNA detection efficiency of 7 experimental methods through twenty-one real sequencing datasets. Our results identified Circle-Map and CReSIL as the most effective pipelines for eccDNA detection through short-read and long-read sequencing data, respectively. Moreover, third-generation sequencing-based Circle-Seq showed superior efficiency in detecting copy number-amplified eccDNA over 10 kb in length. These results offer valuable insights for researchers in choosing the most suitable methods for eccDNA research.

cancer biology↗

bioRxiv2023

Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse embryos

During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory towards the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.

developmental biology↗