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2021

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Full-Text Articles in Developmental Biology

Analysis Of Spore Shape Determination In Streptomyces, Ning Sun Dec 2021

Analysis Of Spore Shape Determination In Streptomyces, Ning Sun

Electronic Theses and Dissertations

Streptomycetes are Gram-positive, soil-dwelling bacteria that possess a complex life cycle with the alternation of vegetative mycelium, aerial mycelium, and spores. Streptomyces. coelicolor spore maturation is a complex process that involves spore shape metamorphosis from cylindrical pre-spores into ellipsoid spores, but the details of this process have remained enigmatic. Previously, our lab identified a novel gene ssdA that might play a role in spore shape determination using a transposon-based insertion mutagenesis in S. coelicolor. In this study, I isolated a S. coelicolor ssdA-null mutant that showed increased colony hydrophobicity and misshapen spores in sizes and shapes, confirming the …


The Effect Of Twist1b And Twist2 Knockdown On Tgf-Beta Signaling Expression In Tendon Progenitors, Marinia N. Bishay Dec 2021

The Effect Of Twist1b And Twist2 Knockdown On Tgf-Beta Signaling Expression In Tendon Progenitors, Marinia N. Bishay

Honors Scholars Collaborative Projects

Somites are important mesoderm structures that form in regularly timed intervals from the anterior end of the paraxial mesoderm at the end of gastrulation during embryogenesis. Cells of the ventromedial portion of the somite, the sclerotome, undergo epithelial to mesenchymal transition, surround the notochord, and give rise to connective tissues of the axial skeleton including the vertebrae, tendons, and ligaments. We are interested in understanding how the sclerotome is compartmentalized into the region that makes tendons. Genes expressed in the sclerotome, twist1 and twist2, are likely involved in the downstream differentiation of sclerotome to tendon progenitors. Previous studies have shown …


The Effects Of Chronic Ethanol Exposure On Caenorhabditis Elegans Germline, Andrea Zegarra Dec 2021

The Effects Of Chronic Ethanol Exposure On Caenorhabditis Elegans Germline, Andrea Zegarra

Seton Hall University Dissertations and Theses (ETDs)

Ethanol is the most commonly abused drug in the world. Alcohol consumption increases a number of health risks, morbidity and mortality, and chronic and acute diseases. Some health risks include high blood pressure, stroke, liver disease, cancer, and can include mental health and social problems. Chronic alcohol consumption can lead to learning and memory problems, along with alcohol dependence. Additionally, alcohol consumption has an impact on ovarian reserve, steroid hormone production, sperm quality, fecundity, and fertility treatments. The purpose of this study was to analyze the effects of ethanol exposure on fertility of the N2 wild type as measured by …


Co-Option Of The Yolkless Oocyte Receptor For Crispr/Cas9 May Induce Transient Sterility In Female Drosophila Grimshawi Flies, Bronwyn Miller Dec 2021

Co-Option Of The Yolkless Oocyte Receptor For Crispr/Cas9 May Induce Transient Sterility In Female Drosophila Grimshawi Flies, Bronwyn Miller

LSU New Orleans Theses and Dissertations

Evolutionary developmental biology has historically used generalist model organisms like Drosophila melanogaster. The Hawaiian Drosophilidae radiation, which occurred just 25 million years ago, is an ideal lineage for work in Evo-Devo. Hawaiian flies make up 25% of the world’s Drosophila species and extreme diversity is seen throughout the group. D. grimshawi is a generalist Hawaiian picture-wing fly that has served as a model for the Hawaiian Drosophilidae radiation. However, D. grimshawi’s power and use as a model organism is constrained by our lack of ability to manipulate its genome. In this work, I attempted to edit the genome of …


(Non)Parallel Developmental Mechanisms In Vertebrate Appendage Reduction And Loss, Samantha Swank, Thomas Sanger, Yoel E. Stuart Nov 2021

(Non)Parallel Developmental Mechanisms In Vertebrate Appendage Reduction And Loss, Samantha Swank, Thomas Sanger, Yoel E. Stuart

Biology: Faculty Publications and Other Works

Appendages have been reduced or lost hundreds of times during vertebrate evolution. This phenotypic convergence may be underlain by shared or different molecular mechanisms in distantly related vertebrate clades. To investigate, we reviewed the developmental and evolutionary literature of appendage reduction and loss in more than a dozen vertebrate genera from fish to mammals. We found that appendage reduction and loss was nearly always driven by modified gene expression as opposed to changes in coding sequences. Moreover, expression of the same genes was repeatedly modified across vertebrate taxa. However, the specific mechanisms by which expression was modified were rarely shared. …


Developing Inside A Layer Of Germs—A Potential Role For Multiciliated Surface Cells In Vertebrate Embryos, Ryan R. Kerney Oct 2021

Developing Inside A Layer Of Germs—A Potential Role For Multiciliated Surface Cells In Vertebrate Embryos, Ryan R. Kerney

Biology Faculty Publications

This paper reviews current research on the microbial life that surrounds vertebrate embryos. Several clades are believed to develop inside sterile—or near-sterile—embryonic microhabitats, while others thrive within a veritable zoo of microbial life. The occurrence of embryo-associated microbes in some groups, but not others, is an under-appreciated transition (possibly transitions) in vertebrate evolution. A lack of comparable studies makes it currently impossible to correlate embryo-associated microbiomes with other aspects of vertebrate evolution. However, there are embryonic features that should instruct a more targeted survey. This paper concludes with a hypothesis for the role of multiciliated surface cells in amphibian and …


Regulatory Non-Coding Rnas Modulate Transcriptional Activation During B Cell Development, Mary Attaway, Tzippora Chwat-Edelstein, Bao Q. Vuong Oct 2021

Regulatory Non-Coding Rnas Modulate Transcriptional Activation During B Cell Development, Mary Attaway, Tzippora Chwat-Edelstein, Bao Q. Vuong

Publications and Research

B cells play a significant role in the adaptive immune response by secreting immunoglobulins that can recognize and neutralize foreign antigens. They develop from hematopoietic stem cells, which also give rise to other types of blood cells, such as monocytes, neutrophils, and T cells, wherein specific transcriptional programs define the commitment and subsequent development of these different cell lineages. A number of transcription factors, such as PU.1, E2A, Pax5, and FOXO1, drive B cell development. Mounting evidence demonstrates that non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), modulate the expression of these transcription factors directly by binding …


Analysis Of Tri-Lineage Collagen Reporter Mice Growth Plates Through Light Sheet Microscopy, Olivia Ferrigno Oct 2021

Analysis Of Tri-Lineage Collagen Reporter Mice Growth Plates Through Light Sheet Microscopy, Olivia Ferrigno

Holster Scholar Projects

The growth plate is an active area of cartilage in the long bones of children. Its main function is to enable limb lengthening, but is a relatively weak structure that is prone to fractures and injuries. These injuries can have severe long term ramifications, as they can create premature bone fusion, also known as a bony bridge, that causes stunted or crooked growth. Dr. Liisa Kuhn’s Lab has developed an accurate, reproducible animal model that mimics this same growth plate behavior and can be used to further analyze the growth plate structure, healing process, and new treatments. This animal model …


Insect Developmental Plasticity: The Role In A Changing Environment, Lindsey A. Barnes Sep 2021

Insect Developmental Plasticity: The Role In A Changing Environment, Lindsey A. Barnes

The Cardinal Edge

Climate change has been recognized as a severe threat to biodiversity. In the rapidly growing collection of literature on the consequences of global change, researchers have recently noticed a dramatic decrease in insect populations in a wide range of habitats. Insects are extremely susceptible to climatic change, especially with regard to fluctuations in moisture and temperature. However, insects often exhibit phenotypic plasticity, where organisms will express different phenotypes when presented with a specific environmental stimulus. In developmental plasticity, environmental stimuli at the larval stage can determine adult phenotypes. This review focuses on case studies of developmental plasticity in insects, with …


A Literature Review On The Development Of Upper Limbs In Humans, Anh T. Phan Sep 2021

A Literature Review On The Development Of Upper Limbs In Humans, Anh T. Phan

The Cardinal Edge

The development of tetrapod upper limbs shares an evolutionary origin and has been adapted and specialized for different functions for different species, such as flight in birds, swimming and balance in sea mammals, and coordination and grabbing objects in humans. The basis of tetrapod limb development has common developmental patterns, starting with the formation of the limb bud via Sonic hedgehog (Shh) signaling, where later developmental steps are modified for specialized functions. This review covers the basic developmental patterns of mammalian tetrapod development seen in humans, beginning with the formation of the limb bud, to the axis development of the …


Dentinogenesis Imperfecta: The Genetic Causes And Outcomes, Sydney P. Johnson Ms. Sep 2021

Dentinogenesis Imperfecta: The Genetic Causes And Outcomes, Sydney P. Johnson Ms.

The Cardinal Edge

Dentinogenesis imperfecta (DI) is a genetic disorder characterized by dentin discoloration, tooth development irregularities, and decreased tooth strength. This autosomal dominant disorder is identified in individuals of all ages. There are three classifications of dentinogenesis imperfecta, each with varying presentations and causes. This report covers normal tooth development (odontogenesis), DI development, DI classifications, and the genes involved in this genetic disorder.


Inner Ear Development And Advances In Inner Ear Organoid Formation, Paige Avery Sep 2021

Inner Ear Development And Advances In Inner Ear Organoid Formation, Paige Avery

The Cardinal Edge

The inner ear houses the cochlea which contains hair cells responsible for the transduction of sound waves. The process of forming these hair cells responsible for hearing is a complicated process consisting of many signaling factors that allow ectoderm to form into the otic placode, the otic vesicle, and finally prosensory cells such as hair cells (Schlosser, 2006). Loss of these hair cells contributes to deafness and hearing loss throughout life, and regeneration of these cells may serve as a therapeutic agent for those with irreversible damage. This review looks at the development of the otic placode and hair cells …


Mechanisms Of Prenatal Ethanol Exposure On Causing Developmental Defects Associated With Fetal Alcohol Syndrome Disorders, Jordan M. Powell Sep 2021

Mechanisms Of Prenatal Ethanol Exposure On Causing Developmental Defects Associated With Fetal Alcohol Syndrome Disorders, Jordan M. Powell

The Cardinal Edge

No abstract provided.


Metamorphosis In Amphibians And The Role Of Thyroid Hormone, Hallie Maxwell Sep 2021

Metamorphosis In Amphibians And The Role Of Thyroid Hormone, Hallie Maxwell

The Cardinal Edge

No abstract provided.


The Effects Of Alcohol On The Developing Drosophila Nervous System, Erica E. Hassoun Sep 2021

The Effects Of Alcohol On The Developing Drosophila Nervous System, Erica E. Hassoun

The Cardinal Edge

Ethanol is the most common human teratogen, contributing to fetal alcohol syndrome (FAS) when effects are the most severe. Key effects of fetal alcohol syndrome are observed in the nervous system. The high prevalence of prenatal alcohol exposure necessitates novel treatment and prevention methods. However, ethical issues prevent researching humans in utero. For this reason, the fruit fly Drosophila melanogaster has emerged as a model organism for studying FAS. Because Drosophila is a small and non-placental organism, its environment can be easily controlled, allowing for specific doses and time periods of ethanol exposure to be studied. This review discusses findings …


The Effects Of Ppal-1 In Arabidopsis Gamete Development, Amanda J White, Susana Perez-Martinez, Mark P. Running Sep 2021

The Effects Of Ppal-1 In Arabidopsis Gamete Development, Amanda J White, Susana Perez-Martinez, Mark P. Running

The Cardinal Edge

Prenylation is a type of post-translational modification in which a 15- or 20-carbon lipid is added to the carboxyl (C) terminus of the protein. Arabidopsis thaliana contains the PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL) gene, which encodes a protein with homology to the α-subunits of the three known prenylation enzymes, PFT, PGGT, and Rab-GGT. We previously identified two mutations in PPAL, one of which is ppal-1, which contains a T-DNA insertion in the fourth intron. We have previously observed that self-fertilizing heterozygous ppal-1 plants produce progeny in which homozygous ppal-1 is underrepresented. This project attempts to ascertain …


Developmental Programming Of Dna Methylation And Gene Expression Patterns Is Associated With Extreme Cardiovascular Tolerance To Anoxia In The Common Snapping Turtle, Ilan Ruhr, Jacob Bierstedt, Turk Rhen, Debojyoti Das, Sunil Kumar Singh, Soleille Miller, Dane A. Crossley Ii, Gina L. J. Galli Sep 2021

Developmental Programming Of Dna Methylation And Gene Expression Patterns Is Associated With Extreme Cardiovascular Tolerance To Anoxia In The Common Snapping Turtle, Ilan Ruhr, Jacob Bierstedt, Turk Rhen, Debojyoti Das, Sunil Kumar Singh, Soleille Miller, Dane A. Crossley Ii, Gina L. J. Galli

Biology Faculty Publications

Background

Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia …


Investigation Of Notch Signaling In Cone Fate Specification In Vertebrate Retina, Xueqing Chen Sep 2021

Investigation Of Notch Signaling In Cone Fate Specification In Vertebrate Retina, Xueqing Chen

Dissertations, Theses, and Capstone Projects

In the vertebrate retina, cone photoreceptors are crucial for high acuity color vision. Several retinal diseases lead to loss of cones and there is a need to identify the normal developmental genesis of these cells to inform the development of stem cell-based therapies. Cone genesis has previously been shown to be repressed by Notch signaling, however, the mechanism by which Notch signaling controls cone fate determination is still unclear. It has been identified that cone photoreceptors are formed from multipotent retinal progenitor cells (RPCs) that first generate genetically-defined, restricted RPCs with limited mitotic and fate potential to preferentially form cones …


The Slit-Binding Ig1 Domain Is Required For Multiple Axon Guidance Activities Of Drosophila Robo2, Lafreda J. Howard, Marie C. Reichert, Timonthy A. Evans Sep 2021

The Slit-Binding Ig1 Domain Is Required For Multiple Axon Guidance Activities Of Drosophila Robo2, Lafreda J. Howard, Marie C. Reichert, Timonthy A. Evans

Biological Sciences Faculty Publications and Presentations

Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N-terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. While Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles …


Identification Of Phenotypic Defects In The Zinc Finger Transcription Factor Ztf-29, Trae Dunn Aug 2021

Identification Of Phenotypic Defects In The Zinc Finger Transcription Factor Ztf-29, Trae Dunn

Symposium of Student Scholars

Aging and many aging-associated diseases such as Alzheimer’s disease and cancer are characterized by a progressive decline in physiological functions and a decline in the ability to respond to stress. The underlying causes for many aging-associated diseases are unknown. Identifying genes that control normal aging will advance our understanding of the molecular changes that underlie the aging process and might help treat age-associated diseases. In order to get a better understanding of the molecular mechanisms behind these diseases, we utilize the nematode Caenorhabditis elegans as a model organism to examine defects in physiology and aging.

The human PRDM genes code …


Temperature Changes Seen In Lower Extremities After Cpn Local Anesthetic Block, Lora Asberry Aug 2021

Temperature Changes Seen In Lower Extremities After Cpn Local Anesthetic Block, Lora Asberry

Symposium of Student Scholars

Many older patients often suffer from walking issues such as Drop Foot. Drop Foot is caused by the malfunction of nerves in the foot, resulting in the loss of control of the front foot muscle. Within all of our patients, there has been a common fibular palsy, caused by the entrapment of the peroneal nerve. Due to this, they cannot lift up their foot. Some cases are permanent, while others are temporary. In the temporary cases, we have indicated a Phoenix Sign. The Phoenix Sign indicates that a nerve, presumed to be dead, has the capability to be recessed back …


Growth, Maturation, And Weight Status: Insights From A Longitudinal Cohort Of Nebraska Youth, John William Masker Aug 2021

Growth, Maturation, And Weight Status: Insights From A Longitudinal Cohort Of Nebraska Youth, John William Masker

Kinesiology and Sport Sciences Theses, Dissertations, and Student Creative Activity

Pediatric obesity continues to be a major public health concern in the United States, with well-known short-term and long-term consequences. In efforts to combat pediatric obesity and identify children at high-risk for potential health problems, physicians and health professionals widely practice screening and classifying weight status using age-and-sex-specific body mass index (BMI) percentiles. However, some studies suggest the use of BMI for establishing weight status in relation to health risk in youth is problematic, especially during the period of the adolescent growth spurt. More importantly, maturation-related misclassification may result in overestimations of overweight prevalence among early-maturing youth, and underestimations of …


Vitamin D Modulates Rett Syndrome Phenotypes And Underlying Cellular Pathways In An Mecp2-Mutant Mouse Model, Mayara De Cassia Ribeiro Aug 2021

Vitamin D Modulates Rett Syndrome Phenotypes And Underlying Cellular Pathways In An Mecp2-Mutant Mouse Model, Mayara De Cassia Ribeiro

Dissertations - ALL

Rett syndrome (RTT) is a progressive and severe X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MECP2. There is currently no effective treatment for RTT. Female RTT patients develop relatively normally during the first 6-18 months of life, after which they undergo a period of rapid regression, losing the ability to talk, walk and purposefully use their hands, in addition to suffering from deceleration of head growth, and onset of repetitive, autistic behaviors. RTT symptoms can be partially reversed by the re-expression of Mecp2 in adult mice, suggesting the potential for post-symptomatic therapeutic intervention. Among the many dysfunctions …


Human Ipsc Tissue-Engineered Cartilage For Disease Modeling Of Skeletal Dysplasia-Causing Trpv4 Mutations, Amanda R. Dicks Aug 2021

Human Ipsc Tissue-Engineered Cartilage For Disease Modeling Of Skeletal Dysplasia-Causing Trpv4 Mutations, Amanda R. Dicks

McKelvey School of Engineering Graduate Student Theses & Dissertations

Cartilage is essential to joint development and function. However, there is a variety of cartilage diseases, ranging from developmental (e.g., skeletal dysplasias) to degenerative (e.g., arthritis), in which treatments and therapeutics are lacking. For example, specific point mutations in the ion channel transient receptor potential vanilloid 4 (TRPV4) prevent proper joint development, leading to mild brachyolmia and severe, neonatally lethal metatropic dysplasia. Tissue-engineered cartilage offers an opportunity to elucidate the underlying mechanisms of these cartilage diseases for the development of treatments. Human induced pluripotent stem cells (hiPSCs) are an improved cell source option for cartilage tissue engineering given their minimal …


Neuronal Migration In Developmental Hyperserotonmia: Assessment Of Vesicular Glutamate In The Raphe Nuclei, Trey M. Shupp Aug 2021

Neuronal Migration In Developmental Hyperserotonmia: Assessment Of Vesicular Glutamate In The Raphe Nuclei, Trey M. Shupp

Graduate Theses/Dissertations

The neurotransmitter serotonin is involved in the early development of the central nervous system and the organization of neurons throughout the cerebral cortex and cerebellum. It is proposed that serotonin indirectly interacts with cells in the marginal zone of the cerebral cortex known as Cajal-Retizus (CR) cells. These cells secrete the extracellular matrix protein reelin, which is known for its role in neuronal organization and migration during early neural development. It has been observed that low levels of serotonin are associated with similarly low levels of reelin during development and have been reported to result in disorganization of neurons in …


Regulation Of Lung Mesenchymal Cells By Epithelial Wnt Ligands, Odemaris Narvaez Del Pilar Aug 2021

Regulation Of Lung Mesenchymal Cells By Epithelial Wnt Ligands, Odemaris Narvaez Del Pilar

Dissertations and Theses (Open Access)

The mesenchyme comprises of heterogeneous cell populations that support neighboring structures and are integral to intercellular signaling. Despite such importance, mesenchymal cells are poorly defined morphologically and molecularly. Their signaling regulation is not well understood, lagging their counterparts in the epithelial, endothelial, and immune lineages. Leveraging single-cell RNA-seq, three-dimensional imaging, and lineage tracing, first, we delineated different lung mesenchymal cell types and classified them into three-proximal-distal axes in the neonatal and adult murine lung. Each axis is associated to the structure they support – the endothelium, epithelium and interstitium. We also identified two subpopulations of neonatal lung myofibroblasts – ductal …


The Effect Of Sub 5% Oxygen Tension On In Vitro Human Embryo Development, Bradley James Link Aug 2021

The Effect Of Sub 5% Oxygen Tension On In Vitro Human Embryo Development, Bradley James Link

EVMS School of Health Professions Theses & Dissertations

Objective: To determine the impact of < 5% O2 tension on the in vitro development of human pre-implantation embryos. It was hypothesized that the optimal O2 tension would differ from the commonly utilized 5%, as shown by beneficial effects after the implementation of a more physiological O2 tension such as 2% for either the partial or full duration of in vitro culture.

Aim: To conduct a randomized controlled study to determine the effect of O2 tension during in vitro human embryo culture on the production of reactive oxygen species (ROS), apoptosis, gene expression and embryo development.

Background: The …


Bone Morphogenetic Protein Signaling Requirement In Centripetal Migration In Drosophila Melanogaster Oogenesis, Sheila Mosallaei Aug 2021

Bone Morphogenetic Protein Signaling Requirement In Centripetal Migration In Drosophila Melanogaster Oogenesis, Sheila Mosallaei

UNLV Theses, Dissertations, Professional Papers, and Capstones

Cells interact with each other via signals to coordinate migration for proper tissue development and maintenance, immunity and tissue repair. Signals regulate migration via changes to cytoskeletal components or transcriptional regulation. Here, I used Drosophila melanogaster to investigate whether and how a transcriptional signal, Bone Morphogenetic Protein signaling, drives a cell migration, centripetal migration. The migrating cells, centripetal follicle cells, consist of two subsets of cells, “leading” and “following” centripetal follicle cells, that although adjacent to each other, migrate differently. The differences in modes of migration correlate to differential gene expression in the centripetal follicle cells. The leading centripetal follicle …


Analysis Of The Role Of Akirin Interactions With Nuclear Body Proteins During Myogenesis, Laura Yorke Jul 2021

Analysis Of The Role Of Akirin Interactions With Nuclear Body Proteins During Myogenesis, Laura Yorke

Master of Science in Integrative Biology Theses

Akirin is a highly conserved, small nuclear protein of indeterminate structure ubiquitously expressed in non-fungal eukaryotic species. It was first discovered in the innate immune response, but many other functions have since been found, including embryonic muscle patterning and myogenesis. Animals with either nonfunctional or missing Akirin have aberrations in embryonic muscle patterning, along with other defects. Akirin interacts with transcription factors such as Twist to coordinate development through interfacing between Twist and other complexes, such as the Brahma Chromatin Remodeling Complex (BRM). Therefore, Akirin likely plays a general role in transcription during early development, interfacing with other transcriptional machinery …


Insights Into The Molecular Evolution Of Fertilization Mechanism In Land Plants, Vijyesh Sharma, Anthony J. Clark, Tomokazu Kawashima Jun 2021

Insights Into The Molecular Evolution Of Fertilization Mechanism In Land Plants, Vijyesh Sharma, Anthony J. Clark, Tomokazu Kawashima

Plant and Soil Sciences Faculty Publications

Land plants have evolved successive changes during their conquest of the land and innovations in sexual reproduction have played a major role in their terrestrialization. Recent years have seen many revealing dissections of the molecular mechanisms of sexual reproduction and much new genomics data from the land plant lineage, including early diverging land plants, as well as algae. This new knowledge is being integrated to further understand how sexual reproduction in land plants evolved, identifying highly conserved factors and pathways, but also molecular changes that underpinned the emergence of new modes of sexual reproduction. Here, we review recent advances in …