A History of Developmental Biology

Developmental biology is a branch of life sciences that aims to understand how organisms grow and develop. It provides insights into cell differentiation, tissue formation, and the development of the whole organism from a single fertilized egg.

Molecular genetics was crucial in identifying the genes that regulate development and how these developmental mechanisms were evolutionarily conserved. It also revealed the developmental control genes underlying human diseases, the mechanism of pattern formation, and organogenesis. Integration of developmental biology with stem cell biology, imaging, genomics, and gene editing have helped create disease models, which have improved our understanding of diseases and develop therapies to treat them.¹

Image Credit: Rohane Hamilton/Shutterstock.com

Early Foundations

Origins of Developmental Biology

Aristotle

There were two theories of development during Aristotle’s time: one was preformation where the preformed embryo (complete organism) is contributed by the sire (male) and the space for this organism to grow is provided by the dam (female), and the other theory later known as epigenesis was supported by Aristotle.

According to the epigenesis theory, the single-celled embryo gradually progresses through different stages to form the complete organism. He cracked open the chick eggs on successive days of their 21-day incubation period and observed what was present in them. Aristotle believed that the white of the egg forms the embryo and the yellow provides the nutrients. He also observed a speck of blood that forms the heart, followed by the veins and other organs. By 3 weeks, the complete bird was formed. He delineated the developmental stages and proved that it was epigenesis and not preformation that occurs wherein a single cell gradually develops into a complete organism.^2,3

Karl Ernst von Baer

Karl Ernst von Baer published four laws of animal development, which were a response to the recapitulation theory of Johann Friedrich Meckel. Meckel believed that the different developmental stages of highly complex animals were the adult forms of less complex animals. von Baer believed that animal embryos shared only a few basic forms, and subsequently branched out into organisms with a completely different appearance.

Four laws of von Baer

• The general features of an organism appear before the specialized features.
• The development of an organism starts with a simple and uniform structure that gradually becomes complex and diverse. For example, the presence of the vertebral column appears early in embryonic development, and the specialized features, such as fur on mammals, appear in the later stages.
• Different species of animals appear similar only in the initial few embryonic forms; they diverge as ontogeny progresses. He discussed the embryonic forms of humans, chicks, and fish where they appeared similar in the early stages but looked increasingly dissimilar with continued growth.
• He believed that the embryos of complex animals resemble only those of less complex animals, not their adult forms.⁴

Early Experiments and Findings

Ernst Haeckel

Ernst Haeckel is known for the biogenetic law, which states that an organism’s developmental phases (ontogeny) follow that of its evolutionary history (phylogeny). He supported this theory using the drawings of embryonic fish, chick, pig, and man showing the embryos of these organisms looked very similar in the early developmental stages. The three assumptions of the biogenetic law are as follows:

• The law of correspondence states that every stage of development in higher animals resembles those of the adult stages of lower animals. For example, the gill slits observed in human embryos resembled the adult form of the fish.
• Phylogenesis occurs toward the later stages of the developmental process. Haeckel believed that the similarity observed in the embryos of the different animals in the early stages was caused by developmental constraints. These limitations were absent toward the later stages of development, adding new characters.
• The principle of truncation states that the early developmental phases occur rapidly in higher organisms compared with lower organisms. Otherwise, embryonic development would become much longer than the gestation period due to the addition of new characters when normal ontogeny ends in the extant species.^5,6

Wilhelm Roux

Wilhelm Roux’s most famous experiment is on green fog eggs. Once he showed that the eggs could develop in an artificial environment, the next step was to determine if an entire fertilized egg and its parts could grow in an artificial environment or if the parts depended on each other for proper development.

He pricked one of the two blastomeres in a 2-cell stage egg only once with a hot needle for a lengthier duration than his previous experiment. A light brown color appeared in the area surrounding the egg. When he observed the punctured eggs under the microscope, he noticed that most of them were destroyed, very few undamaged blastomeres survived and only some underwent normal development. The destroyed cells appeared grey indicating cell death.

Roux believed that the cells died due to needle puncture and not because of their inability to interact with the other cells. The half embryo in the eggs that survived developed normally, with the region next to the punctured cell slightly affected. He concluded that a 2-cell or 4-cell stage egg can develop independently in the formation of an embryo, suggesting a predetermined developmental path owing to internal factors operating on the cell.^7,8

Learn more about cell fate determination

Major Milestones

Pre-Mendelian Era

Many researchers were trying to elucidate the underlying mechanism of fertilization. They understood that the chromosome number must be reduced in some manner before fertilization, or else the chromosome number would double every time a sperm fused with an egg. Edouard van Beneden showed that the sperm and eggs of Ascaris had half the number of chromosomes compared with the other cells in the body (somatic cells).

This was due to a process called meiosis. August Weismann’s germ plasm theory suggested two types of cell division where the germ cells (eggs or sperms) were the carriers of heredity while the somatic cells were not, leading to the discovery of meiosis.

Many Greek philosophers believed that individual traits were acquired from the environment and could be inherited by the offspring. Weismann pointed out that hereditary characteristics are transmitted via germ cells, not somatic cells. Later he identified that the hereditary material was present in the chromosomes.

Francis Galton demonstrated that every generation contributed to the entire makeup of the individual. For example, he suggested that the offspring of a tall man and a short woman would be of intermediate height as each parent contributes half of the complete heritage.⁹

Gregor Mendel

Through his pea plant experiments, Mendel presented three laws of inheritance:

• Law of Dominance: A heterozygous offspring with a dominant and a recessive trait will express the dominant trait. The recessive trait is expressed when the offspring inherits the recessive trait from each parent.
• Law of Segregation: During gamete formation, the gamete receives one allele from each parent, which is transmitted to the next generation via random selection.
• Law of Independent Assortment: Alleles from different genes separate independently from one another during gamete formation. This law emphasizes that every trait has a separate gene and segregates independently of other genes.¹⁰

Although Mendel never used the term “gene” in his studies, he did believe that some hidden hereditary determinants were responsible for the inheritance of characteristics. By the 1900s, chromosomes were known and were seen as a basis for Mendel’s hereditary determinants.

Boveri and Sutton postulated the chromosomal theory of inheritance where Boveri demonstrated the individuality of chromosomes and their continuity through cell division, characteristics essential for genetic material. Through the experiments on the spermatogenesis of grasshoppers, Sutton suggested an explanation for Mendel’s laws of independent assortment and segregation.

Although a champion for Mendel’s hereditary principles, Bateson observed the phenomenon of gene linkage, which was a violation of the law of independent assortment. This exception led to the modification of the chromosomal theory of inheritance by Thomas Hunt Morgan. Genes demonstrating linkage were located on the same chromosome, whereas those showing independent assortment were on different chromosomes.¹¹

Thomas Hunt Morgan

He performed ground-breaking research on fruit flies and provided proof for the chromosomal theory of inheritance, gene linkage, and gene mapping. He examined thousands of fruits flies using a microscope and a magnifying glass and confirmed that genes are located on the chromosome.

After breeding fruit flies, he noticed one offspring with white eyes, whereas all others had red eyes. He started breeding the white-eyed fly and observed that the trait was present only in males. Further breeding analysis revealed that the genetic factors that controlled eye color and sex determination were present on the same chromosome. This established that chromosomes possess the genes that allow the offsprings to inherit traits from their parents.

Morgan proposed the crossing over or recombination concept when he observed that linked traits occasionally separated. He suggested that a crossover may occur between two paired chromosomes to exchange information. In addition, he explained that the recombination frequency was determined by the distance between the genes on the chromosome.

Genes located closer to one another were likely to get inherited together while those that were farther apart on the same chromosome were likely to be separated by recombination. Thus, the linkage strength is dependent on the proximity of the genes to one another. This formed the basis of the first gene map created by Morgan’s student Alfred Henry Sturtevant using six X-linked genes. He realized that if the crossover frequency was associated with the distance between the genes, this information would be useful to map the genes on the chromosome.^12,13

Morphogenetic Fields

A fertilized cell divides to produce identical cells, which develop distinct fates and positions to become organized into organ systems. The different classes of cells within an organ work together to maintain organ function, and multiple organs interact with each other to produce a functional organism.

Embryogenesis proceeds with the generation of morphogenetic fields of cells, and the cells within each field possess unique positional information, which they use to generate spatial patterns. This process divides the embryo sequentially, first along the major body axes and division into smaller refined units, such as the organ primordia, followed by further partitioning and pattern development. Thus, every field of cells will give rise to a particular organ even when transplanted to a different part of the embryo.

The studies on newt forelimb development by Ross Harrison gave rise to the concept of morphogenetic fields. He showed that a disk of cells in the lateral plate mesoderm at the early neurula stage had developed the potential to give rise to forelimb when transplanted into another region of the embryo, despite the lateral plate mesoderm comprising a uniform layer of cells.

An interesting property was observed upon complete removal of the disk of cells that give rise to the limb: the surrounding cells had the power to produce a complete limb. This suggested that the morphogenetic field has a gradient that decreases in organ-forming potential as it moves away from the precise location occupied by the region from where the forelimb arises.^14-16

Homeotic Genes

Homeotic genes regulate the anatomical structure development of various organisms during early embryogenesis. Any mutation in the homeotic genes can cause improper development. Edward B. Lewis identified homeotic genes within the antennapedia or the bithorax complex.

The antennapedia complex comprising five genes is responsible for the development of head and thorax segments (the front of the embryo), and the bithorax complex comprising three genes is responsible for the development of posterior segments and the abdomen (the back of the embryo).

Mutations in the antennapedia gene leads to growth of legs on the head instead of the antennae, and inactivation of Ultrabithorax converts the halteres to a second set of wings. Homeotic genes are referred to as Hox genes in vertebrates, comprising four clusters (A to D), and each cluster has 13 genes arranged sequentially from 1-13.

All the different clusters work together to establish the identity of the body segments along the head-tail axis. The genes at the beginning of the cluster are responsible for the development of structures at the head end and those at the end of the cluster are responsible for the structures of the tail end. Mutation of HoxD13 leads to synpolydactyly where extra fingers or toes may be fused.¹⁷

Technological Advancements

Microscopy

Considerable advancements have been made in microscopy techniques, which have enabled us to understand the processes involved in the early embryonic development of organisms. Fluorescent microscopy has provided detailed information on cell structure and the processes occurring within; however, it carries the risk of photodamage. Fluorophore-free techniques are safe on cells and can be applicable in a clinical setting, although the data obtained are small.

Fluorescent Techniques

Two-photon Microscopy

In this method, two or more photons of higher wavelengths (infrared) are simultaneously used for fluorophore excitation. This method has several advantages: decreased photodamage, imaging depth >1 mm, and highly localized excitation. Along with Laurdan dye, this method has been used to capture 3D images of zebrafish embryonic cell membranes, with a high lipid order observed on the apical surface of polarized epithelial cells.

Deep two-photon microscopy showed that the membrane order of the epithelial cells of kidney, gut, and liver duct tissues was remarkably changing during development between 3-11 days postfertilization, with the highest membrane order seen 6 days postfertilization.^18,19

Light-Sheet Microscopy

This method uses a sheet of laser light to illuminate thin sample slices. A wide-field fluorescence microscope is kept perpendicular to the light sheet. The sample is positioned where the illumination and detection axes intersect. The light sheet excites the sample, and the emission is detected using high-speed cameras producing high-quality images. Serial sections are obtained by rotating the sample to alter the imaging plane, providing a 3D representation.

This method was successfully employed to observe the different developmental phases of the zebrafish embryo retina from 1.5-3.5 days postfertilization. The movement and location of the nucleus in wildtype and mutant zebrafish embryos were determined using this method.^18,19

Learn more about microscopy

Second Harmonic Generation

This is a fluorophore-free method where a single photon with a shorter wavelength and double energy is produced from two photons with lower energy. A 3D image of cell behavior during the different cleavage phases of the zebrafish embryo is obtained by combining second and third harmonic generation imaging techniques. Collagen organization was investigated in zebrafish fin wound healing using this method.^18,19

In situ Hybridization

This method is used to obtain gene expression patterns in tissues or embryos. It involves the hybridization of the probe to the mRNA in the organ, which is subsequently fixed, sectioned, and placed on a slide. The radioactive RNA probe is added, which subsequently binds to the complementary mRNA. Finally, autoradiography is performed to visualize the location of the mRNA.

This method has been employed to show Pax6 expression in the developing mouse eye. The Pax6 mRNA is found at the meeting point of the presumptive retina and lens tissues, and as development progresses, in the developing lens, retina, and cornea.²⁰

Whole-mount in situ Hybridization

This relatively recent technique allows the visualization of large areas of gene expression by employing whole organs instead of thinly sliced sections as in the conventional method.

This method was used to identify the Pax6 mRNA in whole chick embryos. The digoxigenin-labeled probe was incubated with the chick embryos, followed by incubation with an anti-digoxigenin antibody. Digoxigenin will be present only where the probe has bound. Then, the secondary antibody covalently linked to alkaline phosphatase was added. Pax6 mRNA was identified as a dark blue precipitate using a dye that is activated by the phosphatase.²⁰

CRISPR/Cas9 System

This gene editing tool is a simple, fast, and precise method to manipulate genes by adding, removing, or altering parts of the DNA sequence. This system comprises the following: a Cas9 nuclease that cuts the dsDNA at a specific location to remove or add small fragments of DNA and a guide RNA located within an RNA scaffold. The scaffold binds to the DNA while the guide RNA directs Cas9 to the precise location on the genome to make the cut.²¹

The CRISPR/Cas9 system has been used in gene therapy and generating in vivo disease models and in vitro cell models. A mouse model was developed for the functional analysis of new missense variants identified in patients with congenital heart disease (CHD). The pathogenesis of CHD caused by GAT4 mutation was studied by combining iPSC and CRISP/Cas9 technology to create a CHD model associated with this mutation.²²

The CRISPR/Cas9 gene editing system has been utilized to correct mutations in Duchenne muscular dystrophy (DMD) animal models. Delivery of a dual adeno-associated virus (AAV) system (Cas9 packaged in ss-AAV and the guide RNA in self-complementary AAV) into the mouse model improved muscle contractility and restored dystrophin expression.²³

What is CRISPR?

Modern Developments

Stem Cell Research

Developmental biology has witnessed remarkable progress in stem cell research. Novel imaging technologies, innovative model systems, and genome editing tools have given valuable insights into the complexities of development and regeneration.

There are three types of stem cells: embryonic stem cells (ESCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs). The use of ESCs raises ethical concerns as they are obtained from human embryos.

This problem was circumvented by iPSCs; they are genetically reprogrammed adult cells that resemble ESCs. iPSCs serve as an excellent tool for disease modeling. They are capable of producing patient-specific disease models and enabling personalized medicine research.

iPSCs are being used as a model for Parkinson’s disease (PD) as they are derived from patients with PD. iPSCs can be differentiated into dopaminergic neurons, and iPSCs-based cell models are used to study the molecular mechanisms of PD. The iPSCs used are derived from patients carrying mutations in synuclein alpha (SNCA), leucine-rich repeat kinase 2 (LRRK2), and many others.

iPSCs-derived neurons carrying the LRRK2 G2019S showed increased SNCA levels. LRRK2 inhibitors reduced SNCA accumulation. Increased mitochondrial DNA levels were observed in iPSCs carrying this mutation compared with those in normal iPSC-derived neurons.²⁴

iPSC-derived cardiomyocytes have recapitulated the pathological hallmarks of hypertrophic cardiomyopathy (HCM). iPSCs carrying HCM mutations exhibit sarcomere disorganization and cardiomyocyte hypertrophy. Mutations in MYH7 have shown hypercontractibility, also observed in iPSCs-derived cardiomyocytes carrying this mutation.²⁵

Regenerative Medicine

This field aims to repair or replace diseased or damaged tissues to reestablish normal organ function. Blindness is frequently caused by age-related macular degeneration. When iPSCs-derived neuronal progenitor cells were transplanted in rats, disease progression was reduced and 5-6 layers of photoreceptor nuclei were generated, reestablishing visual acuity.

The α-1 antitrypsin deficiency deficiency causes chronic obstructive pulmonary disease and liver cirrhosis. A single base pair mutation causes this deficiency, which is fixed upon correcting the mutation in hepatic iPSCs.

Serotonin neurons regulate emotions, depression, sleep, and anxiety. A developmental defect or neuron degeneration can cause brain disorders, such as bipolar disorder or schizophrenia. The manipulation of Wnt signaling in human iPSCs leads to in vitro iPSC differentiation to serotonin-like neurons, which exhibit similar electrophysiological properties and express hydrolase 2. Transplantation of these neurons can cure the abovementioned brain disorders.²⁶

Organogenesis

Organogenesis is a process that comprises cell-cell interactions and cell proliferation, survival, shape, size, and fate, arranging cells into tissues, and finally a functional organ.

Zebrafish have emerged as a useful model to study heart development. The role of a repressive mechanism via retinoic acid signaling in cardiac specification and the presence of a second heart field were identified in zebrafish.

The zebrafish model was used to determine the mechanism of thyroid organogenesis. The generation of stable thyroid reporter lines and double transgenic embryos are useful for live imaging of thyroid development compared with the surrounding tissues. Immobilization of live embryos in low-melting agar was compared with foregut endoderm morphogenesis to monitor thyroid development with epifluorescence microscopy.

Stimulation of embryonic organoids can mimic the important steps in in vivo cardiac development, namely the generation and spatial organization of cardiac progenitors and compartmentalization of the first and second heart field.^27-29

Integration of Developmental Biology with Other Disciplines

Systems Biology

Systems biology has described various developmental aspects from complex intracellular networks to 4D models of organ development. The primary purpose of systems biology is to comprehend the detailed behavior of complex biological processes to develop computational models and predict the effect of various perturbations on a living system. With the increasing availability of experimental data, an in silico model is generated with increasing complexity, completeness, and predictive accuracy, representing the working of an integrated biological process.

Systems biology has a robust set of analytical tools for recording and analyzing embryogenesis, such as intracellular networks, extracellular communication, and multiscale integration. The characterization of intracellular networks is the most common systems-based study to understand developmental processes. The endoderm and mesoderm specification were regulated by a gene regulatory network identified in the embryos of sea urchins.

Large-scale perturbation experiments, computational analyses, and genomic and transcriptomic measurements were conducted to identify the molecular networks that govern embryogenesis. Computational and experimental methods have been integrated to identify the characteristics of transcriptional regulatory sites throughout the genome and the association between transcription factors and their specific binding sites.

Several proteomic techniques have been used to study the role of the extracellular matrix concerning structural support, cell growth modulation, and intercellular behavior in development. This understanding is crucial for cell culture under synthetic conditions.

Tools such as Compucell3D have been used to model skeletal development in a vertebrate limb. An in silico pancreatic organogenesis model was elucidated considering the interactions at the gene and organ level across different time scales.³⁰

Genomics

High-throughput -omics technologies have allowed an unbiased and detailed characterization of molecular states of biological systems. Single-cell genomics has contributed to our understanding of cellular heterogeneity, capturing various developmental stages and lineage tracing.

Single-cell RNA sequencing was applied to mouse embryonic cells from the 2-cell to the 16-cell stage, and an increasingly heterogeneous expression of Oct4 and Sox2 gene targets was identified at the 4-cell stage. Sox21 was jointly regulated by Oct4 and Sox2 and showed heterogenous expression across cells. Sox21 knockdown pushed the cells toward an extraembryonic fate. The observed heterogeneity could push cells toward specific lineages.

In a study on human definitive endoderm cell development, cells were arranged according to the developmental pathway, effectively reconstructing the behavior of established markers. This ordering facilitated the identification of novel regulators, such as KLF8, a driver for definitive endoderm differentiation. This observation was validated by observing the changes in the proportion of differentiated cells after KLF8 knockdown.³¹

Epigenetics

Genetics has played an important role in normal and abnormal development. However, observations suggest that genetics alone is insufficient to regulate development. Environmental factors, including nutrition, stress, and certain compounds, can cause abnormal development. These factors alter normal development to induce developmental mutations but do not promote DNA sequence alterations. Thus, both genetic and epigenetic factors are involved in the developmental process.

Epigenetic factors, such as DNA methylation, histone modifications, chromatin structure, and small noncoding RNAs are involved in regulating gene expression. DNA methylation is shown to be involved in establishing early cell lineages such as stem cells. Chromatin structure and histone modifications regulate gene expression at different stages of development and differentiation.

The critical windows in the early stages of development are susceptible to environmental factors. If this early critical window was affected by an epigenetic event, then later stages of development may differ from normal developmental processes. These alterations in the normal developmental process can contribute to disease etiology.³²

Learn more about systems biology

Current Trends and Future Directions

Emerging Research Areas

Developmental Disorders

Developmental disorders are impairments caused by chromosomal abnormalities, prenatal exposure to substances, infections, injury, malnutrition, or metabolic disorders, affecting a child's mental, physical, learning, or behavioral development.

Neurodevelopmental disorders (NDD) cause abnormal brain function as the development of the central nervous system is affected. Animal models, including mice, zebrafish, and fruit flies, have been used to determine the mechanisms and factors involved in NDDs.

Animal models are unable to recapitulate many of the behavioral deficits in humans accurately; thus, nonhuman primates are considered as models for NDD research. Transient in vivo genetic manipulation using CRISPR/Cas9 technology has allowed the generation of genetically manipulated nonhuman primates and served as a model for Rett syndrome.

Considering that the use of human fetuses and embryos has ethical restrictions, human-specific in vitro models can be used as an alternative approach. 2D neuronal cell cultures of differentiated hESCs and hiPSCs have been used to determine neuronal activity and morphology. iPSCs can also be used to identify suitable drug targets and as a starting material for customized stem cell therapy.³³

Evolutionary Developmental Biology (Evo-devo)

Evo-devo compares the development of different organisms to comprehend the evolution of development. Evo-devo provides an understanding of an organism’s form and function by including details on the developmental pathways, morphogenetic movements, and signaling molecules.

Initially, the tails of swallowtail butterflies were considered as outgrowths or wing extensions. Examination of wing tail development revealed that wing formation was completed by the pupal stage and lacked tails. Tails appear during the late pupal stage after significant programmed cell death removes existing cells. Following the final molt, the resulting shape is recognized as a tail in the adult butterfly.

Evo-devo has revised our understanding of homology. Nonhomologous traits (insect legs, butterfly wing patterns) can possess the same developmental pathways, whereas homologous traits (insect or butterfly legs) can have different developmental pathways.³⁴

Model Organisms

Drosophila melanogaster

Drosophila has served as an effective model organism for over a century, aiding in the study of diverse biological processes, including genetics and inheritance, embryonic development, learning, behavior, and aging. They are easy to breed, reproduce quickly, and possess an external morphology that became the basis for the discovery of mutants and understanding the principles of heredity.

The chromosomal theory of inheritance, sex-linked inheritance, and genetic maps were demonstrated in fruit flies. The homeobox was discovered in Drosophila by cloning the homeotic gene complexes, and it was observed that common developmental pathways exist among diverse species.³⁵

Zebrafish

Zebrafish are easy to breed, produce numerous offspring, and have a short life cycle of 3 months. The early embryos are almost transparent; thus, development can be easily followed under a microscope. Zebrafish are used to study nerve connections using hearing, vision, and touch response assays; etiology of several human diseases; and act as a model for cancer and many developmental disorders.³⁵

Caenorhabditis elegans

C. elegans has been used as a model organism because they are easy to culture, have a short lifecycle of 3-4 days, and one adult can produce many offspring. Modifier screens (suppressors or enhancers) of an already known phenotype have been conducted using GFP as a marker to detect subtle phenotypes. The entire RAS pathway with mutations was defined leading to the multivulva or vulvaless phenotypes. Genetic screens have identified several genes involved in programmed cell death.³⁵

Application in Medicine

Prenatal Diagnosis

Microarrays

Microarray testing can detect aneuploidy and small deletions and duplications with rapid turnaround time and is recommended for evaluating structural abnormalities along with FISH. Patients should be made aware of finding variants with uncertain clinical significance with this test to avoid causing anxiety.³⁶

Preimplantation Genetic Diagnosis (PGD)

PGD can detect chromosomal abnormalities much earlier and is performed after in vitro fertilization. The abnormality can be detected before transferring the embryo back into the mother’s body.^36,37

Noninvasive Prenatal Diagnosis (NIPD)

NIPD involves the analysis of fetal cells (maternal blood) or cell-free fetal DNA (maternal plasma). Cell-free fetal DNA has been analyzed to detect the paternal β-thalassemia allele using RT-PCR for the codon 41/42 (-CTTT) β-thalassemia mutation or restriction enzyme analysis of PCR products for the HbE mutation.³⁷

Genetic Disorders

CRISPR has been used to correct the F508 deletion of three base pairs in exon 10 in patient-derived iPSCs. The correction efficiency is up to 90% with the piggyBac transposase as a selection marker. Another group achieved a correction rate of >20% in patient-derived iPSCs with the F508 mutation via electroporation of CRISPR/Cas RNP. The genetic correction using CRISPR restored CFTR function in airway epithelial cells.³⁸

The FDA has approved Zolgensma and Spinraza for spinal muscular atrophy (SMA). Spinraza is an antisense oligonucleotide that targets SMN2 and alters splicing to restore SMN2 protein function. Spinzara treatment was successful in 84 patients and prevented SMA in the phase III clinical trials. Zolgensma, an intravenous gene therapy for SMA, contains a complete, functional copy of human SMN1.³⁹

Elevidys uses an adeno-associated viral vector (AAVrh74) to deliver a portion of the dystrophin gene “microdystrophin” and is approved for DMD in 4-5-year-old ambulatory patients.³⁹

Hemgenix and Roctavian are approved for hemophilia B and hemophilia A treatment, respectively.³⁹

Regenerative Therapies

The goal of regenerative therapies is to replace or repair the damaged tissue and restore normal function. Regenerative medicine is concentrated in two main areas:

Tissue Engineering:

This involves the implantation of biologically compatible scaffolds at the site where the new tissue is desired. If the scaffold is in the desired shape of the tissue that needs to be generated and the scaffold attracts the cells, the new tissue is formed.

The regeneration of a new kidney from patient-derived cells would benefit patients with kidney disease. Researchers stripped cells from a donor organ and used the remaining collagen scaffold to guide new tissue growth. The scaffold was seeded with endothelial and epithelial cells. The resulting organ tissue removed metabolites, reabsorbed nutrients, and produced urine in vitro and in vivo in rats.⁴⁰

Cellular Therapies

Tissue reconstruction is possible if ASCs are harvested and injected at the damaged or diseased tissue site. These cells can be obtained from fat, blood, bone marrow, and other sources. The only stem cell therapy approved for clinical use is hematopoietic stem cell therapy to treat leukemia, multiple myeloma, and lymphomas. For all other conditions, stem cell therapies are in the experimental phase.

Afamitresgene autoleucel (Afami-cel) is an FDA-approved drug for metastatic synovial sarcoma. It is derived from the T cells of patients, genetically manipulated, and infused back into the patient. The genetically engineered T cell receptor can recognize and bind MAGE-A4 in cancer cells.^40,41

What is bioethics?

Ethical Considerations

The discovery of CRISPR, which has made gene editing easy and accurate, has renewed the ethical concerns about gene editing as editing changes conducted in the germline will be passed on to future generations.

A few ethical concerns are mentioned below:

Safety

Gene editing is associated with two main concerns: mosaicism and off-target effects. Researchers believe that gene editing should not be used for reproductive purposes until its safety has been documented by extensive research. Some researchers believe that gene editing can be useful in cases where PGD will be inapplicable, for example, cases where both parents are homozygous for the disease-causing variant and polygenic disorders. Another concern is using gene editing for human enhancement, which may be controversial. Some argue that gene editing should be approved to cure genetic diseases, with strict regulations for nontherapeutic use.

Informed Consent

As the edits are being made in the embryos, it is a challenge to obtain a true informed consent.

Equity

Like other new technologies, gene editing will be available only to the rich class, increasing the disparities in accessible healthcare.

Genome Editing Research Involving Embryos

People have religious and moral objections to the use of human embryos for research purposes. Federal and NIH funding is not available for research involving human embryos. Some countries have allowed gene editing research on nonviable or viable embryos.

Researchers believe that gene editing in human embryos should be allowed to understand human biology. Concerns regarding its use for reproductive purposes and human enhancement must be regulated via strict policies. Public opinion should be obtained to understand their views on approving germline editing. ⁴²

Conclusion

Developmental biology is an evolving field aiming to understand and examine the growth, development, differentiation, and remodeling of organisms using various cell and molecular biology tools. Developmental biologists have always expressed interest in understanding the formation, shape, and growth of embryos, and it has extended to other areas of stem cell growth and differentiation, organ regeneration, and evo-devo. Different animal model systems have helped to understand systems biology, organogenesis, pattern formation, and regeneration.

Technologies, including CRISPR/Cas9, can be used to manipulate stem cells to fix genetic disorders. Researchers are also working towards improving the safety and efficiency of stem cell therapies.

Understanding the role of evolution in animal development, generating human organoids to model diseases, and using stem cells to understand human development are some of the prospects in developmental biology, which will provide valuable insights into animal development.

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