Systems Physiology

Superior (Cranial) and Inferior (Caudal)

Superior = toward the head

Inferior = away from the head

Anterior (Ventral) and Posterior (Dorsal)

Anterior = towards or at the front of the body

Posterior = toward the back

Medial, Intermediate, and Lateral

Medial = toward or at the middle of the body

Lateral = away from the middle

Intermediate = between a more medial and more lateral structure

Proximal and Distal

Proximal = closer to the origin of the body part

Distal = farther from the origin of the body part

Superficial (External) and Deep (Internal)

Superficial = toward or at the body surface

Deep = deep beneath the body’s surface

Midsagittal Plane and Parasagittal Plane

Midsagittal plane = cut was made perfectly on midline from back to front.

Parasaggittal plane = cut was off-centred, not on midline

Frontal (Coronal) Plane

Cuts body vertically from sides, right to left. Produces a frontal or coronal section.

Transverse Plane

Cuts body horizontally, produces a cross section.

4 Principles of Karl Ernst von Baer

• General features appear before specialised features

• General features progress into specialised features

• Embryos from more evolved animals diverge more than embryos from primitive animals

• Embryos of higher animals resemble embryos of more primitive animals (but not the adult)

Evolutionary Biology

Compares embryonic development across species.

Teratology

Studies embryonic defects to infer normal development

Modern embryology discovered that…

The amorphous zygote undergoes gradual changes as opposed to being a miniature already formed.

Hans Spemann Transplantation Experiment

Demonstrated that the dorsal blastopore lip of an amphibian embryo acts as an "organizer". By transplanting this tissue to a new location on a host embryo, they induced the formation of a second, complete embryonic body axis, proving that specific tissues dictate cell fate in development.

Alan Turing Reaction-Diffusion Model

Chemical basis of morphogenesis: the initial symmetry in embryos can be broken by the interplay between two diffusible molecules (activator and inhibitor), whose interactions lead to the formation of patterns.

John Gurdon Experiment

Used SCNT (Somatic Cell Nuclear Transfer) to prove that specialized adult cells retain the full genome needed to create an entire organism, fundamentally challenging the belief that cellular differentiation is irreversible. By transferring the nucleus of a mature frog intestinal cell into an enucleated egg, he created cloned, swimming tadpoles.

Shinya Yamanaka Discovery

Found that the Yamanaka factors when put into a somatic cell can reprogram it into a stem cell-like state

What are the Yamanaka factors?

SOX2, KLF4, and OCT4

3 Types of Embryonic Stem Cells

Totipotent, pluripotent, and multipotent.

Totipotent Stem Cells

Can generate all cell types including extra-embryonic tissue (ex. placenta).

Pluripotent Stem Cells

Can generate all cell types and is the derivative of the 3 germ layers but can’t generate extra-embryonic tissue.

Multipotent Stem Cells

Can generate multiple related cell types.

How is pluripotency maintained?

By transcription factors that activate pluripotency genes and repress differentiation programs.

Cellular Tools for Embryology

Fate mapping to determine cell fate: dye and genetics approaches – transplant experiments (chimera) experiments (1960/70’s).

Molecular Tools for Embryology

• Forward and reverse genetics (2000’s)

• Genome editing (crispr cas9) (2013) - transgenesis (transient vs stable) 2010’s revolution

• Gain of function and Loss of Function, Knock in, Fluro Tags cellular and molecular

Steps of Transcription

Initiation, elongation, splicing, 3’ processing, and termination.

Basal Transcriptional Machinery

RNA Polymerase II (synthetize RNA strand) + mediator, integrator and elongation protein complexes = micro machine that produce RNA.

Transcription Factors

Are the switches for the micro machines and are a class of protein that binds (and sometime bend) DNA. They function to induce cell lineage specification and maintain cell type identity. They also to turn on and off gene expression and modulate the process of transcription.

What do transcription factors bind to?

Enhancer and promoter regions

Enhancers

Enhancers are distal, orientation-independent sequences that bind activator proteins to significantly boost transcription rates.

Promoters

Promoters are DNA sequences located immediately upstream of a gene that initiate transcription by binding RNA polymerase, acting as an essential on/off switch.

Process of Transgenic Animal Making

Plasmids are added via electroporation to embryonic stem cells which are then added to embryos before they are implanted into a parent . These animals will then express the desired protein.

How are gain or loss of function animals made?

1. A mouse is engineered so the target gene is flanked by LoxP sites (“floxed” gene).

2. A second mouse expresses Cre in a specific tissue (e.g., liver or brain).

3. The mice are crossed.

4. In cells where Cre is expressed, the gene between the LoxP sites is deleted → gene is knocked out only in that tissue.

The opposite happens for gain of function where the gene will become activated.

Pregnancy Stages

Duration is 38 to 40 weeks. 1st trimester = embryo and after that = fetus.

Two Types of Germline Cells

Oocyte and Sperm and have 23 chromosomes each.

Oocyte

Female germ (egg) cell, X or X chromosome, 100um, rich in cholesterol (for week 1 nutrients).

Zona Pellucida

A layer of glycoprotein which prevents the egg from attaching to an ectopic site, also provides species-specific sperm binding.

Sperm

Male germ cell, X or Y chromosome, the head is 5.1x3.1um.

Chromosomal Disjunction

Homologous chromosomes in meiosis I or sister chromatids in meiosis II separate ensuring each daughter cell receives a complete set of chromosomes.

Chromosomal Nondisjunction

Failure of homologous chromosomes or sister chromatids to separate properly during cell division.

Spermatogenesis

The process of making sperm cells.

Oogenesis

The process of making an oocyte or egg cell in females.

Arresting of Oocytes

All the eggs (around 1 million) will arrest at prophase I shortly after birth. Each menstrual cycle some oocytes will continue meiosis and arrest at metaphase II until fertilization.

Causes of Female Chromosomal Malformations Due to Meiosis

• Maternal age and chromosomal defect: dramatic increase beyond age 35 in trisomy 21 (Down syndrome).

• Loss of cohesion complex may be responsible for abnormal meiosis

Main Events of Early Embryonic Development

• Week 1: fertilisation to blastocyst formation

• Week 2: implantation to bilaminar formation

• Week 3: gastrulation to form 3 germ layers

• Week 4: neurulation

Week 1 of Early Embryonic Development

Ovulation, fertilization, cleavage, morula, zona hatching to form blastocyst (end of week 1), implantation.

Fertilization

Sperm from a male meets an oocyte from a female to forms a zygote via cell proliferation (termed cleavage).

Blastocyst Formation

Once the zygote loses the zona pellucida there is a sodium and water infiltration which divides the cell type into 2 (inner cell mass and trophoblasts) to from a blastocyst and once this happens it can implant

What do the trophoblasts and inner cell mass form?

Trophoblasts → placenta

Inner cell mass → embryo

Week 2 of Early Embryonic Development

Implantation and Bilaminar Formation

Implantation

• The blastocyst travels closely to the uterus wall and L-selectin on the surface of the trophoblasts attaches to the uterine epithelial cells 

• The trophoblasts secrete enzymes that degrade the epithelial cells and allow the blastocyst to infiltrate the uterine wall to implant

Bilaminar Formation

The inner cell mass becomes 2 layers which are the epiblast and hypoblast. The epiblast becomes the embryo and the hypoblast is a yolk sac rich in cholesterol that functions in embryo patterning, nutrient transport, and are primitive red blood cells.

Primitive Streak

A groove-like structure that appears in the epiblast during gastrulation marking the beginning of the process defining the future anterior-posterior axis of the embryo.

Week 3 of Early Embryonic Development

Gastrulation occurs to form the 3 germ layers including mesoderm differentiation into paraxial and splanchnic components, heart tube formation, and paraxial mesoderm derivative formation.

Gastrulation

Epiblast further divides into the 3 primary germ layers which includes the endoderm (epithelial cells), mesoderm (mesenchymal stem cells), and ectoderm.

Notochord Formation

Occurs during week 3 and is a rod-like structure made of a cartilaginous substance derived from the mesoderm. Its role is to guide proper development of vertebra and differentiation of neural tube.

Mesoderm

The most abundant germ layer in the human body and gives rise to the musculoskeletal system (bone, cartilage and muscle), cardiovascular system (heart, blood and blood vessels), and connective tissues found throughout our bodies.

Differentiation of Mesoderm

Differentiates into paraxial mesoderm (forms skeletal muscle) and splanchnic mesoderm (forms cardiac muscle and smooth muscles of internal organs).

Molecular Markers on Cardiac and Skeletal Muscle

Skeletal: Myf5 and Pax7

Cardiac: Nkx2-5 and Gata4

Neurulation

The neural tube will develop into the central nervous system. The cranial neuropore also closes around day 24, and the caudal neuropore closes around day 28.

Neural Crest Cell (Ectomesenchymes) Formation

Occurs during week 4 and develop from neural ectoderm via epithelial to mesenchymal transition and form:

• Teeth

• Skull bones (anterior skull and facial bones)

• Melanocytes

• Peripheral nervous system: peripheral neurons, glia cells

Ectoderm

Gives rise to the nervous system (central and cranial), skin and oral mucosa, cranial bone via neural crest cells, and teeth.

Endoderm

Gives rise to the colon, stomach, intestines, liver, pancreas, lungs, thyroid and parathyroid.

Morphogenesis

Cells form the shape and physical structure of the organ. They do this via cell proliferation, differentiation, migration, extracellular matrix deposition, tissue folding and convergence, and apoptosis.

Heart Development

The heart is the first major organ to develop because there is an urgent need for a blood supply to provide oxygen and nutrients to the growing embryo.

Genes Involved in Heart Development

Over 500 genes identified with cardiovascular development and its not fully understood because no single transcription factor/morphogen is responsible for cardiogenic specification or heart development.

What regulates heart development?

Genetic and epigenetic factors, transcription factors, morphogens, apoptosis, cell-cell adhesion, extracellular matic, and mechanical factors including cellular migration and hemodynamics (blood flow).

Days and Thickness of Embryo

• Days 0-6 formation of blastocyst

• Days 7-12 embryo becomes bilaminar and implanted fully

• Days 13-18 embryonic disc becomes trilaminar: gastrulation with three germ layers (ectoderm at the top, mesoderm in the middle, and endoderm at the bottom)

• Lateral plate mesoderm (the side bits of the mesoderm) subdivides into somatic, coelom (cavity), splanchnic, and extra-embryonic

Cardiogenic Specification

Begins on day 18 (end of 3rd week) where the lower region (splanchnic mesoderm) will develop into the heart tube. Includes the specification of distinct regions that will eventually form different parts of the heart via the formation of 2 heart fields.

Morphogen Gradients

A key mechanism in embryonic development that allows for precise spatial and temporal regulation of gene expression. Expressionainhibition zones spatially restrict cardiogenic mesoderm.

What do the morphogens Mesp1 and Mesp2 control?

Migration of the cardiac progenitor cells into the cranial region of the embryo.

What do the morphogens BMP2, BMP4, TGFβ, and Activin control?

They specify cardiogenic progenitors in the lateral mesoderm.

What are BMP2, BMP4, TGFβ, and Activin inhibited by?

The release of chordin and noggin from the notochord and release of Wnt1/3a/8a from the forming neuroectoderm.

What morphogens inhibit Wnt?

Crescent (FRZB2) and Dickkopf (DKK1)

Formation of the 2 Heart Fields

Cardiac progenitor cells form the first heart field and second heart field (pharyngeal mesoderm) located medial to the first heart field.

Genes that specify cardiogenic progenitors in the first heart field include…

Nkx2.5, Gata4, Tbx Family (Tbx5), FGF8, SHH

Genes that specify cardiogenic progenitors in the second heart field include…

Increased Nkx2.5 and Gata4, Hes-1, Islet-1 (Isl1) and additional inhibitors e.g. Notch.

What do the heart fields go on to form?

1st Heart Field: LV, atria, aorta

2nd Heart Field: sinus venosus wall, RV myocardium, atrial septa, outflow tract and the myocardial wall of the RA, LA

Left to Right Specification of the Heart

The symmetry to specify left and right is initially formed by morphogen gradients. Where there is basically extra transcription factors on the right that inhibit stuff that would be happening on the left on the right.

Right Heart Specification

• Activin signal on the right side of Hensen's node

• Local repression of sonic hedgehog (SHH) expression

• FGF8 expressed on the right side, can inhibit Pitx2 via Snail Related (SnR2)

Left Heart Specification

• SHH restricted to the left side of Hensen's node

• Allows activation of NODAL expression on the left side of the embryo

• NODAL -> Pitx

• Pitx2c is a key regulator of left asymmetry

• SHH, Nodal and Pitx-2 genes on L

• Left sided specification starts prior to heart tube formation

What processes are involved in folding and heart tube formation?

Lateral folding, cranial-caudal (head to tail, starts to curve) folding, and ventral folding.

Lateral Folding

1. Days 20-21: the sides are coming together

2. Day 22: the heart tube fuses and the pericardial cavity also fuses to form the epicardium. The myocardium secrete cardiac jelly

3. Day 24: the heart tube ‘beats’ in a peristaltic motion

Blood Flow Through the Heart Tube

From placenta via the umbilical vein (oxygenated), through heart tube, connected to dorsal aorta. The blood comes in to the heart through the sinus venosus and out through the truncus arteriosus.

What are the layers of the heart tube?

Endocardium (inner), cardiac jelly, myocardium

Cardiac Jelly

Secreted by myoblasts in the myocardium and it assists with cell migration, proliferation, and differentiation.

Cranial-Caudal Folding

Folding of the cranial and caudal parts down and back (for the front). Occurs from day 22-26.

What does the aortic sac of the heart tube become?

The pharyngeal arches/great vessels

What does the truncus arteriosus of the heart tube become?

Ascending aorta and pulmonary artery

What does the bulbus cordis (conus) of the heart tube become?

Outflow track and valves of the right ventricle

What does the primordial/primitive atrium of the heart tube become?

The left and right atria

What does the sinus venosus of the heart tube become?

Large veins and pulmonary veins, visceral pericardium, gestational conduction system, and coronary sinus.

Contribution of the Secondary Heart Field During Tube Formation

While the primitive tube forms primarily from the primary heart field, the secondary heart field continuously adds progenitor cells to the arterial and venous poles. This elongation is what later forces the tube to loop.

What occurs after folding and heart tube fusion?

Looping and chamber specification

What drives heart looping?

• Rapid elongation of the heart tube (especially via secondary heart field addition)

• Constrained space within the pericardial cavity

• Differential growth across regions

3 Stages of Cardiac Looping

1. C-shape

2. D-shape

3. S-shape

C Looping

The truncus arteriosis and bulbus cordis move to the right and the right ventricle moves down and right.

D Looping

Bulbus cordis and primitive ventricle grow rapidly causing the tube to bend ventrally and rightward. This is called D-looping (dextral looping).

S-Shaped Configuration

The left ventricle moves and left to twist, the pulmonary artery and the sinus venosus move back and up.

What regulates chamber specification?

Morphogen gradients, retinoic acid (contributes to atria specification), and the expression of retinaldehyde dehydrogenenase2 (Raldh2).

Morphogen gradients that regulate chamber specification

• Tbx5 specifies atrium and SV

• Irx4 specifies ventricles and outflow tracks (OFT: BC, TA and AS)

• Combination of both specifies the LV