BIO543

24 August 2023 13:35

Developmental Biology

1st August’23

Totipotent stem cell- can develop into any cell type.

Qs to be explored in this course:

Qs1- How do you get an entire organism from a single cell?

Qs2- How do cells differentiate? Cell differentiation gives rise to diversity. Example: Leukemic patients die not due to the
tumour itself, but due to inability of blood cells to differentiate to produce fully functional cells. Hence, the main question.

Qs3- How do organs arise?

Qs4- How do cells know when to stop growing?

Qs5- How is this blueprint for developing an organism transmitted?

Qs6- How is the development of an organism integrated into its habitat?

Tools to study dev. bio

1. Anatomical- studying anatomy and embryos, comparative embryology, evolutionary embryology, mathematical modelling
of anatomical parts.

2. Experimental- Experiments with model organisms like c. elegans, frogs and zebrafish.

3. Genetic- study genes involved in development, transcriptomics during development.

Embryos as tools

Darwin observed that resemblance in embryos reflects resemblance in descent.

JV Thomson observed that larval barnacles are identical to larval crabs and so classified them as arthropods rather than
mollusks.

3rd August’23

Embryonic homologies

-Homologous organs are those which arise from common ancestral structures. Eg: wing of bird and human forelimb. Function
is not same but descended from common precursor organ. Embryos of humans and chicken look quite similar.

-Analogous organs have similar function but do not arise from a common ancestor. Eg: wings of butterfly and bird.

-Sometimes homologous organs may adapt to different functions, based on environment; gill arches of fishes became jaw
bones for humans.

Developmental defects

-2% of humans are born with a developmental deformity, also called malformations.

-Caused by external agents (called teratogens, e.g., Alcohol, tobacco, etc). The study of how environmental agents cause
developmental defects is called teratology. Eg: thalidomide was a painkiller in the 60s, caused birth defects in children ,

(Thalidomide was used to treat morning sickness. Exposure of the fetus during this early stage of development resulted in
cases of phocomelia, a congenital malformation in which the hands and feet are attached to abbreviated arms and legs).
Genetic Alliance; District of Columbia Department of Health. Understanding Genetics: A District of Columbia Guide for Patients and Health Professionals. Washington (DC): Genetic Alliance; 2010
Feb 17. Appendix D, Teratogens/Prenatal Substance Abuse. Available from: [Link]

Mathematical modelling

- attempts to define ‘laws’ of development.

- DA Thomson showed that ratio of widths between 2 whorls of a spiral shell could be calculated mathematically. Most
mollusks have shells that spiral at an angle of 80-85 degrees. Such symmetrical growth is called isometric growth.

-Asymmetrical growth is called allometric growth. Eg: Our limbs grow faster than our head after birth.

Steps in the cycle of life

-Mitosis results in cell cleavage formation.

-In initial phase, cytoplasmic volume does not increase, but cell size decreases.

-Cells divide rapidly. Eg: in Xenopus (clawed frog) egg, 37,000 cells are made in 43 hours. The cells in this cleavage stage are
called blastomeres. Energy requiring process, generated by ATP from mitochondria present in cytoplasm. Hence, within the

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called blastomeres. Energy requiring process, generated by ATP from mitochondria present in cytoplasm. Hence, within the
same cytoplasmic space, thousands of cells are made within a short time period.

-As cytoplasmic components are used up, nucleus begins to synthesize them. The embryo now enters mid blastula transition
(MBT) phase. This is characterized by biphasic cell division.

Images showing stages of development- Gilbert’s textbook pg 47

Role of nucleus in development

-Acetabularia is a single cell rhizoid which has cap, stalk and base.

-Nucleus is located near the base and can be easily removed.

-J Hammerling in 1930s exchanged the nuclei of 2 species, A. crenulate and A. mediterannea. Both are distinguished by
their cap shape.

- the shape of the cap was determined by the donor nucleus. Thus it was inferred that the nucleus controlled cell shape.

-the nucleus contains info that specifies the type of cap produced.

-if the nucleus was removed, the cap was still produced. Thus, this material required for cap development enters the
cytoplasm much earlier than cap production.

- this info in the cytoplasm is not used for several weeks and lies dormant. The info is transmitted into the cytoplasm as
mRNA. This was postulated 30 years before the mRNA was actually discovered.

How to approach experimental embryology?

-the defect experiment: destroy a whole or a part of the embryo and see what effect it has.

Read about an interesting article: can a biologist fix a radio? ( [Link]

2802%2900133-2)

-the Isolation expt: Isolate parts of the embryo and study them. (Driesch’s Experiment)

-Recombination expt: replace different parts of an embryo from different embryos.

-Transplantation expt: transplant parts of an embryo to another embryo.

Roux’s Pre-determined Cell-Differentiation experiment(1880)

• took a fertilized frog egg
• waited until 2-celled stage
• took a hot needle and killed one of the cells
• found out that other cell developed normally.
Conclusion: frog embryo is a mosaic of self-differentiating parts. Differentiation is predetermined.
08-08-2023
Driesch’s Isolation experiment (1891)

-removed fertilisation envelope (zona pellucida) and separated the four cells of pluteus (shield mushrooms) at the 4 -cell
stage.
-Plutei developed from 4 separate cells.
-they developed abnormally.
Conclusion: ?

John Gurdon's experiment showed that differentiated adult cells could be induced to an undifferentiated state, where
they could once again become multiple cell types. Gurdon's experiment disproved the theory that differentiated cells could not
be undifferentiated or dedifferentiated into a new type of differentiated cell.

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Sheep Cloning Experiment (Single cell nuclear transfer) Pg 77 (pdf)

The donor cells were sychronized till they reached G1 phase before fusing the cell with the receptor’s cytoplasm (enucleate
cell).
Conclusion: Something in the Oocyte’s cytoplasm caused the transformation of the Udder cells into Totipotent cells.
Problems associated with Dolly (the clone)
Dolly aged a lot faster but successfully gave birth to a fertile offspring.
The tendency of sterility increased with successive generations.

Histone Modifications (the gatekeepers of gene access):

Enhancer Elements: Pg 85
Pausing of transcription with the help of NELF (pauses) and TEC (Elongates) transcription factors.
Mid-blastular Transition in Zebrafish

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The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal
and tumorigenesis in stem cells.

Silencer Elements:

A cell’s fate can be differentiated by two ways:

What’s in the cell
Position

Control of Transcription: Methylation

Eg: The shift of activation of Globin family genes during development. This shift depends on the site of

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 Eg: The shift of activation of Globin family genes during development. This shift depends on the site of
haematopoiesis and the point of time in the gestation period.

Inheritable methylation patterns (Dnmt3/ de-novo, Dnmt1/ perpetual ergo inheritable)

Genomic Imprinting (next class)

10-08-2023
Genomic Imprinting
Theory of mendel’s gene expression probability. Each of two alleles has an equal probability to express.
Exceptional case - Imprinted genes. E.g., IGF-2 (Embryo needs the father’s copy of IGF-2 and the mother’s gene is always
Imprinted) and IGF-2r is Paternally Imprinted (only the Maternal gene expresses, and the paternal gene is imprinted to be
methylated).
Eg. H19
Read up on negative and positive selection.
Why are imprints there? Why has it evolved that way?
Think of what if an organism needed to inherit essential genes only from the Y chromosome. The absence of the gene in
females leads to them not surviving. Imprinting is a mechanism that allows only one side of genes to express on an autosome.

Note: Example of methylation not silencing a gene is Methylation of H19 Promoter (as above) which prevents the
transcription inhibitor CTCF from binding, thereby as a consequence activates IGF-2.

Differential RNA processing (Alternate Splicing)

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Embryo’s do not require a nucleus

There is a need to regulate RNA expression (post-transcriptional regulation) because you don’t need them to translate 24*7.

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There is a need to regulate RNA expression (post-transcriptional regulation) because you don’t need them to translate 24*7.
A known mechanism for this is miRNA silencing.

Most of the miRNAs come from the maternal cell. That is why an embryo won't graft itself onto the endometrium of a mother
from a different species. (Quiz question)

One mi RNA can regulate more than 1 gene. Similarly, one gene is regulated by more than one miRNA.
Only 7 nucleotides actually bind completely with the recognition site of its complementary mRNA despite it being 20-25 nts
long.
miRNAs act more like a regulator of a fan’s compared to a binary switch. siRNAs on the other hand is binary in output.
Dosage Compensation
The need for both X’s in a gene is only in germinal cells therefore in other somatic cells, one of the X chromosomes are
inactivated, usually by RNA based silencing (he didn’t dive into the mechanism in detail)
Psuedogenes - a gene that has two copies (over 90% similarity) but neither of them translates to any protein. Today it is
known that these genes make RNAs and they act as the mop of miRNAs.
Guo X, Zhang Z, Gerstein MB, Zheng D. Small RNAs originated from pseudogenes: cis- or trans-acting?. PLoS Comput Biol.
2009;5(7):e1000449. doi:10.1371/[Link].1000449
Development of C. elegans (pg 265)
The ideal organism to study development.

Why C. elegans ?
According to Sydney Brenner:
○ Every cell and gene could be identified and studied.
○ The embryogenesis is 16hrs so, the whole development can be studied in a single day.
○ The worm is a hermaphrodite.

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Anterior Posterior (AP) axis formation
P granules (ribonucleoprotein complexes) migrate to the posterior end and polarise the cell.
Control of Blastomere Identity
C. elegans exhibits both conditional (extrinsically determined) and autonomous modes of differentiation.
The P1 cell develops independently and can make all its embryos without an AB cell. An experiment on this property proved
that only P1 cells are stem cells.
The differentiation of an AB cell is conditional and needs the descendants of the P1 cell.
Mechanisms of Cell Fate Determination

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Gastrulation

H.W.
• Calculate the number of WBCs and RBCs in an average human.
• Take 24hrs and 48 hrs as an average population doubling time.
• Find out how many doubling does it take.
• How much time does it take to reach that number?

24-08-2023 (Part -1 | 08:00 -0 9:30 ) (C_Trivedi)

Stem cells-: Have potential to self-renew and to differentiate into different lineages for the lifetime of an animal.
Exhaustion assay-at what point does stem cell activity get exhausted?
What is the stem cell count in a sample? Determined by spleen colony-forming assay.
Definitions of potency- page 217, Gilbert. Few examples given below:
Embryonal stem cells (totipotent)
Fetal tissue stem cells (pluri or multipotent)
Cord blood stem cells (pluri or multipotent)
Adult stem cells (pluri ot multi)

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Adult stem cells (pluri ot multi)
Embryonal carcinoma cells (pluripotent), tumor arising in embryo
Teratomas- mass of undifferentiated cells.
Hw- find the name of father of stem cell biology, was on the cover of TIME magazine.
Ans- James Thomas
Yamanaka- pioneer of concept of induced pluripotent stem cells, won nobel prize

Only hematopoietic stem cell can be identified with a single marker protein
Reprogramming of adult fibroblast cells by changing a few transcription factors (KLF4, SOX2, c-MYC, nanog, OCT-3/4,
LIN-28). This changed the cells into pluripotent cells (resembled embryonic stem cells).

24-08-2023 (Part -1 | 08:00 -0 9:30 ) (V_Mistry)

Stem cells: Stem cells are a unique type of undifferentiated cell with the remarkable ability to develop into various specialized
cell types in the body. can divide and self-renew
Exhaustion assay - Esmail D. Zanjani
Spleen colony forming assay: The Spleen Colony-Forming Unit (CFU-S) assay is a classic experimental technique used to
study hematopoietic stem cells (HSCs) and their ability to generate colonies of blood cells in the spleen of a mouse. This
assay was first developed by Till and McCulloch in the 1960s
What are stem cells: characterized by potential to self-renew and differentiate into several different lineages for the lifetime
of an animal.
Totipotent refers to a type of cell that has the ability to differentiate into any type of cell, including those required for the
development of a complete organism. These cells are often found in the early stages of embryonic development. E.g. zygote
Pluripotent stem cells are a type of cell that can differentiate into many different types of cells, but not all. They are able to
differentiate into cells from all three germ layers of the embryo, but not into cells required for the development of a complete
organism like totipotent cells. Pluripotent stem cells are often found in the inner cell mass of a blastocyst, which is the early
stage of embryonic development. E.g. HSC
Multipotent can only make a few cell types. E.g. monocyte
In humans:
ES cell from 5-7 day embryo toti
EG cell from 6-week embryo pluri
Fetal tissue stem cell pluri/multi
Cord blood SC, Placental SC pluri/multi
Adult SC (HSC, Mesenchymal SC) pluri/multi
Embryonal carcinoma cell (teratocarcinoma- germ cell tumor) Pluri
ES cell- james thomson
iPSCs- activated copies of four genes that encoded some of these critical transcription factors, nearly any cell in the adult
mouse body could be made into an induced pluripotent stem cell (iPSC) with the pluripotency of an embryonic stem cell.
These genes were Sox2 and Oct4 (which activated Nanog and other transcription factors that established pluripotency and
blocked differentiation), c-Myc (which opened up chromatin and made the genes accessible to Sox2, Oct4, and Nanog), and
Klf4.

24-08-2023 (Part -2 |13:00-14:30 )

Clinical relevance of CD34 molecule

CD34 is a transmembrane glycoprotein expressed on early lymphohematopoietic stem cells, progenitor cells, and endothelial
cells. Also, embryonic fibroblasts and some cells in fetal and adult nervous tissue are CD34-positive.
( [Link]
20glycoprotein,nervous%20tissue%20are%20CD34%2Dpositive.)

Epitope is just the subset of an Antigen.

Monoclonal antibodies recognise a single epitope of an antigen. Polyclonal antibodies recognise different epitopes of a single
antigen.
ISHAGE protocol for CD34 enumeration
Side scatter is the measure of granularity while doing Flow Cytometry.

Criteria to identify Hematopoietic Progenitor cells: (4 points)

They express the CD34, 133, 45
CFU assays
Size and Granularity distinctive - Flow Cytometry
Immunophenotypic profiling of surface proteins - Flow Cytometry

Mesenchymal Stromal Cells (MSC)

Differentiate into osteogenic, chondrogenic and adipogenic lineages
Non-haematopoietic cells
Summary of criteria to identify MSCs (ISCT criteria).
»95% positive for CD105, CD73 & CD90

Embryology: Animated (YT video)

Early Embryogenesis

Trophoblasts are more differentiated than embryoblasts.

Blastulation is when the Zona pellucida dissolves and a blastocoel is formed.

Wnt Signalling Pathway.

[Link]

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[Link]
20organogenesis%20during%20embryonic%20development.

The dissolution of zona pellucida is essential for implantation.

Richard Harland (UC Berkeley) 2: The Cellular Basis of Gastrulation

The first time a heartbeat is audible is at around the 21st day.

Vegetal Plate -

Diploblastic and Triploblastic animals (by number of germinal layers)

Ectoderm is usually areas which is exposed (skin, hair, mammary glands)

endoderm - the gut, the tracheae, Thyroid, Lungs,
Mesoderm ( Everything that is in between - blood, muscles, bone, heart, the urinogenital system)

Ectodermal tissue is of three lineages - Peripheral Epidermis, Neural crests (NC) (Gives rise to the PNS), Neural Tube (gives rise to the
CNS - Brain and Spinal cord).

Neurulation (Chapter 13 - Fig 13.2)

1° Neurulation -
2° Neurulation -

The notochord tells the cells where is the mid point of the neural plate. That forms a point of attachment for the invaginating Neural Plate
via medial hinge point cells (MHP).
The hinge point cells (medial and Dorsolateral) are necessary for bending the walls and join to form a hollow tube like structure.

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Cartilage cells are the hardest to engineer (natural or synthetic) because it is extremely flexible and tensile which are two properties
engineers have failed to create in a single substance, even synthetically.

Note - Skull is mesodermal by origin while the brain is ectodermal by origin.

Notochord is Mesodermal in origin.

Bone is mesodermal but the Dentine is Ectodermal (from neural crests) and both are calcified tissue.

Only facial cartilage is Ectodermal while all the other cartilages are mesodermal.

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Secondary Neurulation

@inproceedings{FilasMechanismsOB, title={Mechanisms of Brain Morphogenesis 3 Primary Neurulation , Chicken A Ectoderm

Neural Plate Neural Groove Brain Anterior Spinal Cord Secondary Neurulation , Chicken Multiple Hinge}, author={Benjamen
A. Filas and Gang Xu and Larry A. Taber}, url={[Link] }

From <[Link]
Xu/2f1d060bc56c136c5b36b90c591a52712bbbc07f>

02-08-2023

Closure of the Neural Tube

BMP - Bone Morphogenetic Protein - Usually follow's the TGF-beta signalling pathway.
BMP prevents the fold from happening.
A BMP ligand inhibitor noggin ensures the closure of the neural tube.

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Types of Promoters - Constitutive, Inducible, Conditional, X promoters.

Cobble stone epithelial cells (the Neural epithelium) How does these flat cells act as a hinge?
Wedge cells allow the hinge function that in turn helps fold the neural tube. The formation of hedge cells requires a normal signalling of
BMP, but not too much.

How is this controlled?

BMP is responsible for the formation of a wedge cells and overall regulation

Shh - Sonic hedge hog gene

[Link]

Secondary Neurulation

Mesenchymal to Epithelial Transition (MET)

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o0gyb6

TGFβ functions as a tumour suppressor by mediating its antiproliferative effects in a large variety of cell types.

Chaudhury A, Howe PH. The tale of transforming growth factor-beta (TGFbeta) signaling: a soigné enigma. IUBMB Life.
2009;61(10):929-939. doi:10.1002/iub.239

From <[Link]

Wnt gene in Stem cell differentiation. - Read Up

Importance of Morphogen Gradients

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Importance of Morphogen Gradients

Neural Crest Development (Chapter 15)

Development of the nervous system can be divided into 4 different stages:

1. Specification of neural identity
2. Outgrowth of axons to their target
3. Formation of synapses with target cells which can be other neurons muscles or glandular cells
4. Refinement of Synaptic connections through elimination of axon branches (cell death)

(05/09/2023)

Development of Mesoderm

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Most of the notochord disintegrates while some of it eventually get localised into the nucleus pulposus of the vertebral discs.

Somite - Trunk
Facial Cartilage come from the Neuro ectodermal cells.

Hox genes control the differentiation of Paraxial Mesoderm.

Fig 17.2

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Somite formation

Hairy gene - Somites are blocks of cells that arise from the paraxial mesoderm, which organise themselves into whorls called
somitomeres, which gets further compacted and surrounded by an epithelial layer and separate from the paraxial mesoderm.

Epithelialization
Mesenchymal to Epithelial transition (MET)

Epithelialization of Somite Boundary

Read up on knee replacement surgeries

Axial Specification

Somites are Pleurepotent

07-09-2023
Development of the Heart (Pg 592)
(Skipping Kidney Development)

• The Heart arises from the Splanchnic System.

• It is the first functional unit in the embryo and the Heart is the first functional organ.
• The endoderm and the primitive streak also specify some of the cardiac cells to become cardiac muscle cells.
• The mesoderm cells migrate on both sides of the primitive streak and form cardiac cells.

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The splanchnic mesoderm is closer to the endoderm and a small bit of it integrates with the endoderm to form a tissue called the
Splanchnopleure which gives rise to the Facial Cartillage.
Somatopleure, similarly is the integrates of ectoderm and mesodermal tissue.

The conversion of cardiac muscle cells are regulated by transcription factors (tf) Cerberus &Nkx2-5 and possibly BMP-4.
These tf activate the expression of cardiac muscle specific genes like alpha myosin, cardiac actin etc.

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Rightward Twist = Dextral Looping

Endocardial Cushion Cells cushions the inside the heart while looping. Gives rise to valves and septa.

Septum Primum forms the atrial wall and Septum Secundum grows parallel to the primum and forms the Foramen Ovale as a flap of
tissue allowing flow from the right atrium into the left atrium.

Before birth, the foramen ovale allows blood flow to bypass the lungs (a foetus gets the oxygen it needs from the placenta, not the lungs).
That way, the heart doesn't work hard to pump blood where it isn't needed. When new-borns take their first breath, a new flow direction
happens.

From <[Link]

The Aorta and the Pulmonary artery are formed from a single tube called the Truncus Arteriosis.

Maternal and Foetal blood don't mix.

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Maternal and Foetal blood don't mix.
"Cardiac Development' by Lisa McCabe for OPENPediatrics

Development of the Heart (3D)

Development of the ventricles and large arterial vessels

Heart Embryology Animation

12-09-2023

The Formation of Blood Vessels

Regulated by three pathways; Wnt, BMP and Notch.
and is constrained by three parameters.
Physiological constraints; BV follow the embryonic development pattern of other organs.
Evolutionary Constraint; The embryo extends BVs into the yolk sac even though there's no yolk inside.
Physical constraints; The size of the vessels decreases, the resistance to the flow of blood increases,

Vasculogenesis vs Angiogenesis
Eoiblast, Mesoderm, (BMP)->, Hemangioblast->->(Plueripotent HSC: Angioblast) ->-> (Blood SC & Lymphocyte SC: Endothelial
cells)

VEGF (Vasculo-Endothelial Growth Factor) Receptor

Four Steps;
Vessel Formation.
Branching
Network Formation.
Maturation & Remodelling.

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Ang 1 - Angiopoetin ligand

Mesendoderm
Initial blood formation is extra-embryonic. Yolk sac angiogenesis and vasculogenesis.
intraembryonic -
HSCs form the RBCs first.

The aorta-gonad-mesonephros (AGM) region [Just below the dorsal side of the Aorta] develops from the para-aortic splanchnopleure and
produces HSC.

From <[Link]
&sourceid=chrome&ie=UTF-8>

Blood Cells form first and the endothelium forms around it.

14-09-2023

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Figure 18.21 VEGF and its receptors in mouse embryos. (A) Yolk sacs of a wild-type mouse and a littermate heterozygous for a loss-of-
function mutation of VEGF-A. The mutant embryo lacks blood vessels in its yolk sac and dies.

Tip cells - The tips of already formed vessels.

Tip cells express DLL4 ligand. This DLL4 ligand prevents adjoining cells from responding to VEGF via Notch Signalling.

What are the receptors for angiopoietin 1?

(A) Angiopoietin-1 is secreted by mesenchymal/fibroblast/mural cells and binds to the Tie-2 receptor located on endothelial cells. This
receptor activation triggers the release of factors from the endothelium that cause a chemotactic attraction of mesenchymal cells.

From <[Link]
0ahUKEwjc1tqW0amBAxUoTmwGHeqGAEoQ4dUDCBA&uact=5&oq=Angiopoetin+1
+receptor&gs_lp=Egxnd3Mtd2l6LXNlcnAiFkFuZ2lvcG9ldGluIDEgcmVjZXB0b3IyBhAAGB4YDTIIEAAYHhgNGA8yCBAAGB4YDRgPMggQABgIGB4YDTIKEAAYCBgeGA0YDzII
EAAYigUYhgMyCBAAGIoFGIYDMggQABiKBRiGA0j1K1CyBlijKXABeAGQAQCYAd4BoAGmD6oBBTAuNS41uAEDyAEA-
AEBwgIKEAAYRxjWBBiwA8ICChAhGKABGMMEGAriAwQYACBBiAYBkAYI&sclient=gws-wiz-serp>

Anti-Angiogenesis
There has to be a stop signal for blood vessels in the developing embryo.
Some tissues like the Cornea is avascular. Cornea regulates angiogenesis in two ways.
• It prevents the release of VEGF from ECM.
• Second, It secretes a soluble form of VEGF receptor to trap the VEGF ligand.
Excessive Secretion of soluble VEGF-R can cause Preclampsia, a condition characterised by Hypertension and poor renal filtration.

Haematopoesis

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Evolution of HSC

26-09-2023
Assignment: Design a new life form using Principles of Development (example physical constraints of capillary formation).
Written (2 pages)

Morphogens, Rate of transcription/translation.

What to write? What principles are you using?
E.g. Human with a tail. Why is it that we don't have tails? What are the tf that covers tail development? Justify the evolutionary
advantage to the tail.

Limb Development
Brain, heart, circulatory system, ...

Opposable thumb - evolutionary advantage of hominids

Patten formation, Organ development,
The forelimb and hindlimb are different, how?
Which cells are involved?

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Limb formation happens in the Proximal-distal axis.

The three regions (Stylopod, Zeugopod and Autopod) comes from three cell lines.

Apical Ectodermal Ridge (Pg 615)

The distance for development begins from this ridge (somites) via morphogen gradients (like the process during somite formation).

Scanning electron micrograph of an early chick forelimb bud, with the apical ectodermal ridge in the foreground. (A after Stocum and
Fallon 1982; C courtesy of J. Streicher and G. Müller; D courtesy of K. W. Tosney.)

The bones originally start as cartilage and then become bones.

It is so because it is not easier to reverse calcification of bones.

Microenvironment conditions along with Physical forces like pressure plays a role in bone formation.

Rules for Limb Development

• Common to all tetrapods
• Limb should function in a 3d co-ordinate system. Therefore limbs are arranged into axes. The axis formation is governed by
β-FGF.
• Shh regulates the anterior posterior axis, while dorsoventral axis is regulated at least partially by Wnt7a.
• In all tetrapod embryos, only 4 limb buds are formed. Why?
For supporting speed and balance.
Why do humans have short forelimbs?

Discussion: Selection for economical bipedal walking in Australopithecus and endurance running in Homo likely contributed
to the shift toward relatively smaller distal forelimbs across hominin evolution, with modern human proportions attained in
Pleistocene Homo erectus and retained in later species.
From <[Link]

• The position of limb bud is controlled by the level of Hox gene expression in vertebrates. The hox expression along the a-p axis
determines the limb bud development even though original somites may differ.
• Similarly lateral plate mesoderm will induce myoblasts to migrate out from somites and enter the limb bud.
• Retinoic acid is critical for limb bud outgrowth.
• A gradient of retinoic acid along the a-p axis, activate homeotic genes in specific cells.

The hindlimb development differs from forelimb development (e.g. Birds).

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Apical Ectodermal Ridge
Mesenchymal cells in the limb region secretes factors that induces the ectodermal cells to form AER. These factors are proteins from the
FGF family.

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 03-10-2023

Germ Cells
The only cells that undergoes meiosis.
A lot of cells cannot undergo meiosis because of their higher rate of mutation associated with rapid division (e.g. dermal cells, intestinal
epithelium, etcetera).
• Egg development relies on factors synthesized by other cells. E.g., yolk proteins in birds and amphibians are made in the liv er cells
and carried to the ovaries.
• Egg cells are usually larger than body cells.
• Certain genes in egg and sperm are imprinted.
• More than 80 genes have been identified as imprinted.
The sex is determined at fertilization.
the gonads are very similar initially and the basic form of genital organs is female.

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SOX9 is not synthesized from the Y-chromosome.

The reason the Y chromosome is able to direct testis formation even when more than one X chromosome is present may be a matter of
timing. It appears there is a crucial window of opportunity during gonad development during which the testis determining factor (now
known to be the product of the Sry gene) can function. If the Sry gene is present, it usually acts during this duration to promote testis
formation and to inhibit ovary formation. If the Sry gene is not present (or if it fails to act at the appropriate time), the ovary-forming
genes are the ones that will function (Figure 6.2B; Hiramatsu et al. 2009; Kashimada and Koopman 2010).

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Wolffian duct -> Vas deferens

Transcription Factors in development of gonad

nnn

β-catenin is antagonistic to Sox9

Sox9 determines testes formation

Rspo1 acts as a marker for somatic cells that undergo meiosis.

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Sry determines cell migration

Sox9 and Sry is responsible of the migration of germ cells (mesodermal) into the gonads.

Development of Plant Genitalia

The germ cells are specified only when flowers develop.

Any meristem can give rise to germ cells of either sex and there are no sex chromosomes.
(Double fusion in plants).

05-10-2023

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Regeneration in different organisms.

• Salamander's capability to regenerate limbs and tail

• Many insects can also regenerate appendages.

• Zebrafish can regenerate the heart after removal of part of a ventricle.

• Planaria have remarkable abilities to regenerate.

Epimorphosis

• Regeneration due to growth of a new correctly patterned structure such as limbs.

• Regeneration of an organ merely by repatterning of existing tissue without growth is called morphallaxis.
• In epimorphosis new positional values are linked to growth from the cut surface.
• In morphallaxis, a new boundary region is created near the cut first and then the new positional values are specified relativ e to this cut.

Regeneration of the mammalian liver

• Liver has the highest regenerative capacity in mammals.
• Even in humans, the amount of liver regenerated is equal to the amount of liver removed (around up to 75%)
• The liver regenerates by proliferation of existing tissues.
• There is a return to the embryonic state during liver regeneration. Foetal transcription factors are synthesized, although no t all embryonic liver
features are present.

Cancer as a Developmental disorder

Liver regeneration follows a non-hierarchical model of regeneration unlike that of HSCs.

Dev Bio Page 31

 05/10-10-2023

Dev Bio Page 32

 10-10-2023
A "Head inhibitor" prevents secondary axis formation in recipients whose head is intact.
Grafting is done with micro-sutures.
But if that was the case, Case (C) would conflict.

Planaria (Flat Worm)

Neoblasts
Has self renewal and cell differentiation abilities

Dev Bio Page 33

Head to Tail polarity

Wnt controls tail specification in Planaria

Two Models of Neoblast Differentiation

Notum is required for head specification in Planaria

Dev Bio Page 34

Salamander regenerate using de-differentiation of cells

In both cases the limb formation is complete within 72 days

Regenerative Medicine
• Bone regeneration offers many opportunities in the area of regenerative medicine. While fractured bones can heal they do not grow well as age
advances & are more likely to leave gaps.
• Both the stem cells and the environment plays an important part in this.
• Growth factors like BMP-2 give limited regenerative abilities to the healing bone.
• A combination of newly synthesized materials, mesenchymal stem cells and growth factors released over a long time is most eff ective in bone
regeneration.

Causes of Ageing
• Oxidative damage due to reactive oxygen species.
• Genetic instability and ability to fix mutations.
• Telomere shortening

17-10-2023

Congenital Disorders (Chapter 24)

Inborn errors of metabolism form a large part of genetic diseases involving congenital disorders of metabolism.

Specific development disorders are disorders in which development is delayed in one specific area or areas, and in which basically all other areas of
development are not effected.

It is estimated that half of two thirds of all human conceptions do not develop to term.

Many of these embryos that express abnormality fail to implant. Others implant but do not develop into a successful pregnancy.

A major cause of this is chromosome al abnormalities that interfere with developmental processes.

Developmental Errors lead to Cancer

Throughout development, morphogenetic processes drive the formation of highly specialized organs.

Once development is completed, homeostasis takes over; notably, the repair, and maintenance, of adult tissues such as bone, muscle, liver and skin
also uses developmental signalling pathways and stem cells.

Dev Bio Page 35

also uses developmental signalling pathways and stem cells.

It is perhaps not surprising that ectopic activation of the signalling pathways that control normal development and homeostasis can lead to
hyperproliferative conditions, resulting in cancers.

During development, cell polarity directs the formation of tissue structures and specifies cell fates through the asymmetric distribution of
determinants. Loss of cell polarity is commonly observed in advanced tumours.

Down's Syndrome

Pleiotropy

The production of several effects by one gene or several genes is called pleiotropy.

Mosaic pleiotropy is when effects are produced independently as result of being important in different critical functions

E.g., c-kit gene (a receptor gene that can be shed. This mechanism is used to mop up free ligands without transducing a signal. Sometimes the
complex can by taken up by the cell which transduces another kind of signal) is critical for hematopoietic stem cells, pigment stem cells & germ
stem cells. Mutations in this gene causes anaemia, sterility and albinism.

Relational pleiotropy is when a defective gene in one part of the embryo causes a defect in another part even when the gene is not expressed in this
tissue. E.g., in small eye syndrome, the defect is in the mitf gene that is expressed in pigmented retina. However the lens & the cornea are smaller
even though they do not express this gene.

19-10-2023
Genetic and Phenotypic Heterogeneity

When similar phenotype is produced by defect in different genes its called genetic heterogeneity, E.g. defect in c-kit or stem cell factor produces the
same phenotype.

When the same mutation produces different phenotype in different individuals, it is called phenotypic heterogeneity. This is a result of different
networks being disrupted by the same gene.

E.g. The same mutation in FGFR3 gene showed different phenotypes in 10 unrelated human families. These ranged from mild anomalies to
potentially lethal malformations.

Another example: The same CoViD vaccine had different responses in different people.

Phenotypic heterogeneity in women can also be caused by variations in the X-chromosome inactivation.

Environmental Assaults of Human Development (Teratogens)

Environmental factors like pesticides can also disrupt development

Rachel Carson reported that DDT was destroying bird eggs and causing
reproductive defects in several species.

In the same year thalidomide was shown to cause limb and ear abnormalities.

Viruses like Rubella can also cause developmental defects.

Preventing Developmental Diseases

Developmental diseases cab be prevented by thorough screening and family history

during pregnancy.

Advances in Ultrasound and Non-invasive imaging methods also enable early

detection of developmental disorders.

Prenatal Diagnosis and Preimplantation Genetic Diagnosis (PGD) also help in

prevention of developmental diseases.

Chorionic Villus Sampling

CVS involves taking a sample of the placenta for genetic diagnosis.

Foetal cells can also be captured using amniocentesis.

A combination of IVF & PGD can be used to prevent developmental

disorders.

16/11/2023

End Sem - (25 marks/90minutes)

5 questions, probably essay like questions or 5 MCQs in a question.
One direct question.
One to answer based on one's understanding of a concept (like that of limb development.)
Some question based on correlation of two concepts.
Formulate an experiment if this happens under these conditions.

Dev Bio Page 36

 Formulate an experiment if this happens under these conditions.
There will be keywords.
Choice of choosing 5 questions from a bigger lot.

Discussed Quiz 3
There will be a quiz 4, Thursday 23rd, November

Is cancer a developmental disease

Injection of tumour cells into a developmental embryo does not lead to tumours.
Why?

Stroma plays an important role in carcinogenesis

The numerator is Epithelium

The denominator is stroma

N-methylnitrosourea (NMu) or just the control vehicle (VEH)

Role of Shh in carcinogenesis

Dev Bio Page 37

 21-11-2023
miRNA as a means of cancer therapy

miR 138 is specifically expressed in Glioma Stem cells (GSCs)

Chan, X. H. D., Nama, S., Gopal, F. E., Rizk, P., Ramasamy, S., Sundaram, G. M., Ow, G. S., Vladimirovna, I. A., Tanavde, V., Haybaeck, J.,
Kuznetsov, V. A., & Sampath, P. (2012). Targeting glioma stem cells by functional inhibition of a prosurvival ONCOMIR-138 in malignant
gliomas. Cell Reports, 2(3), 591–602. [Link]

miR138 is a novel therapeutic target for glioblastomas

Expression of exonic miRNA or linked Open Reading Frame (ORF) in context specific
physiologic states.
Sundaram, G. M., Common, J. E., Gopal, F. E., Srikanta, S., Lakshman, K., Lunny, D. P., Lim, T. C., Tanavde, V., Lane, E. B., & Sampath, P.
(2013). ‘See-saw’ expression of microRNA-198 and FSTL1 from a single transcript in wound healing. Nature, 495(7439), 103–106.
[Link]

EGF driven microcircuitry hijacks the wound healing switch

Dev Bio Page 38

10.1084/jem.20170354
From <[Link]

Developmental Therapies
• How can the knowledge of development be used to treat diseases?
• Understanding angiogenesis helped develop a myriad of anti-caner treatments that were anti-angiogenic.
• Antibodies against VEGF and specific VEGF receptors have been used to develop specific therapies against different tumours.

CAM assay (Chorion Allantoid Membrane assay)

Tissue regeneration using embryonic stem cells

• Many tissue can be regenerated using stem cells like embryonic stem cells and induced pluripotent stem cells.
• For e.g. ES cell derived dopaminergic neurons can be transplanted in Parkinson's disease patients. Understanding neuronal dev elopment can
make this process more efficient.
• Similarly ES cells & iPS cells can be differentiated into pancreatic β-islets that can be useful from treating diabetes.

Tissue regeneration using stem cells

ES cells can be programmed to give rise to blood cells. These can be useful for treating a variety of haemetopoetic disorders like B-thalassemia,
haemophilia, sickle-cell anaemia, etc.

In 2007 , Jacob Hanna made iPS stem cells from tail fibroblasts of a sickle cell anaemia mouse. He then electroporated ....

Adult stem cells and regeneration

MSCs showed great promise for cardiac therapy especially in acute myocardial infarction. However subsequently it was established that these results
didn't hold out in larger studies utilizing MSC for treating acute myocardial infarction. Also we know now, that MSC form blood vessels & do not
differentiate into cardiomyocytes. MSC have also been used extensively for bone regeneration. Various factors like BMP-4 have been identified to
aid bone formation.

MSC have also been used extensively for bone regeneration. Various factors like BMP-4 have been identified to aid bone formation.

Direct transdifferentiation
It may not be necessary to go through a stem cell route to achieve differentiation into stem cells.
Kajiyama transferred Pdx-1 gene into human adipose derived stem cells.

Genome-editing Technologies
• Homing Endonuclease
• Zinc Fingers
• TALENs
• CRISPR-Cas9

Stem Cells and genome editing

Genome editing can be carried out in stem cells to achieve long lasting corrections of mutated genes.
Many different diseases from thalassemia to diabetes to HIV resistance have been tackled using this approach.

Dev Bio Page 39

16-21/11/2023
22 November 2023 01:03

16/11/2023

End Sem - (25 marks/90minutes)

5 questions, probably essay like questions or 5 MCQs in a question.
One direct question.
One to answer based on one's understanding of a concept (like that of limb development.)
Some question based on correlation of two concepts.
Formulate an experiment if this happens under these conditions.
There will be keywords.
Choice of choosing 5 questions from a bigger lot.

Discussed Quiz 3
There will be a quiz 4, Thursday 23rd, November

Is cancer a developmental disease

Injection of tumour cells into a developmental embryo does not lead to tumours.
Why?

Stroma plays an important role in carcinogenesis

Dev Bio Page 40

N-methylnitrosourea (NMu) or just the control vehicle (VEH)

Role of Shh in carcinogenesis (Shh acts as a mitogen for some cell types like HSCs and
cerebellar granule neural progenitor cells).

21-11-2023
miRNA as a means of cancer therapy

Dev Bio Page 41

miR 138 is specifically expressed in Glioma Stem cells (GSCs)

Chan, X. H. D., Nama, S., Gopal, F. E., Rizk, P., Ramasamy, S., Sundaram, G. M., Ow, G. S.,
Vladimirovna, I. A., Tanavde, V., Haybaeck, J., Kuznetsov, V. A., & Sampath, P. (2012).
Targeting glioma stem cells by functional inhibition of a prosurvival ONCOMIR-138 in
malignant gliomas. Cell Reports, 2(3), 591–602. [Link]

miR138 is a novel therapeutic target for glioblastomas

Expression of exonic miRNA or linked Open Reading Frame
(ORF) in context specific physiologic states.
Sundaram, G. M., Common, J. E., Gopal, F. E., Srikanta, S., Lakshman, K., Lunny, D. P., Lim, T.
C., Tanavde, V., Lane, E. B., & Sampath, P. (2013). ‘See-saw’ expression of microRNA-198 and
FSTL1 from a single transcript in wound healing. Nature, 495(7439), 103–106.
[Link]

EGF driven microcircuitry hijacks the wound healing switch

10.1084/jem.20170354
From <[Link]

Dev Bio Page 42

From <[Link]

Developmental Therapies
• How can the knowledge of development be used to treat diseases?
• Understanding angiogenesis helped develop a myriad of anti-caner treatments that were
anti-angiogenic.
• Antibodies against VEGF and specific VEGF receptors have been used to develop specific
therapies against different tumours.

CAM assay (Chorion Allantoid Membrane assay)

Tissue regeneration using embryonic stem cells

• Many tissue can be regenerated using stem cells like embryonic stem cells and induced
pluripotent stem cells.
• For e.g. ES cell derived dopaminergic neurons can be transplanted in Parkinson's disease
patients. Understanding neuronal development can make this process more efficient.
• Similarly ES cells & iPS cells can be differentiated into pancreatic β-islets that can be
useful from treating diabetes.

Tissue regeneration using stem cells

ES cells can be programmed to give rise to blood cells. These can be useful for treating a variety
of haemetopoetic disorders like B-thalassemia, haemophilia, sickle-cell anaemia, etc.

In 2007 , Jacob Hanna made iPS stem cells from tail fibroblasts of a sickle cell anaemia mouse.
He then electroporated ....

Adult stem cells and regeneration

MSCs showed great promise for cardiac therapy especially in acute myocardial infarction.
However subsequently it was established that these results didn't hold out in larger studies
utilizing MSC for treating acute myocardial infarction. Also we know now, that MSC form blood
vessels & do not differentiate into cardiomyocytes. MSC have also been used extensively for
bone regeneration. Various factors like BMP-4 have been identified to aid bone formation.

MSC have also been used extensively for bone regeneration. Various factors like BMP-4 have
been identified to aid bone formation.

Direct transdifferentiation
It may not be necessary to go through a stem cell route to achieve differentiation into stem cells.
Kajiyama transferred Pdx-1 gene into human adipose derived stem cells.

Genome-editing Technologies
• Homing Endonuclease
• Zinc Fingers
• TALENs
• CRISPR-Cas9

Stem Cells and genome editing

Genome editing can be carried out in stem cells to achieve long lasting corrections of mutated
genes.
Many different diseases from thalassemia to diabetes to IV resistance have been tackled using
this approach.

Dev Bio Page 43