Developmental Biology Fundamentals

• Development is how a multicellular organism forms a specific arrangement of different cells and shapes.
• Zygote: A fertilized egg, which is the beginning of development.
• Embryo: The period from fertilization to birth.
• Animals other than colonial flagellates and sponges feature recognizable neurons that can be excited electrically and have long processes.

Fundamental Questions of Developmental Biology

• Pattern formation: Cells and tissues form specific patterns, with heads being anterior and tails being posterior.
• Morphogenesis: The creation of ordered form.
• Morphogenesis happens through cell migrations, changes in cell number and shape, growth, death, and changes in cell membrane composition.
• Cells know when to divide due to growth regulation.
• Reproduction is how germ cells combine to transmit instructions to create an organism.
• Human development is the process by which humans develop, and what can cause issues.
• Regeneration happens when organisms replace body parts; mammals regenerate new structures via stem cells.
• Environmental cues influence development in many organisms, with the genome allowing response to these cues.
• Evolution involves how changes in development create new body forms, considering the constraints needed for survival.

General Steps of Development

• Fertilization: Mature sex cells (gametes) fuse together, starting egg development and giving the embryo its genome.
• Cleavage: Mitotic divisions occur after fertilization.
• Zygote cytoplasm divides into smaller cells called blastomeres.
• By the end of cleavage, blastomeres form a sphere, which is called a blastula.
• Blastomeres are cells that are derived from cleavage in an early embryo.
• A blastula is an embryonic stage which is composed of blastomeres.
• Blastocyst: A mammalian blastula
• Blastoderm: A single layer of embryonic epithelial tissue that makes up the blastula
• Blastocoel: The cavity within the blastula
• Blastopore: The invagination where gastrulation begins
• Gastrulation: Cell layer rearrangements lead to the formation of three germ layers, forming the gastrula.
• Organogenesis: The embryo contains three germ layers.
• After layers are established, cells interact and move to form tissues and organs.
• Chemical signals are exchanged between cells to form specific organs at specific sites.
• Larval stage: Young organism before sexual maturity.
• Maturity occurs, followed by gametogenesis.

Methods of Morphogenesis

• Morphogenesis: How cells create an ordered form.
• Invagination: A region of cells folds inward, like poking a soft rubber ball.
• Involution: An outer layer expands and turns inward, spreading over the internal surface.
• Ingression: Individual cells migrate from the surface into the embryo's interior.
• Delamination: One cellular sheet splits into two parallel sheets.
• Epiboly: Epithelial sheets (usually ectodermal cells) move and spread as a unit to enclose deeper layers.

Cell Fate Commitment

• Commitment: Cell biochemistry and function differences are preceded by a process assigning each cell a certain fate.
• Specification (reversible) and determination (irreversible) are the two stages where commitment can be divided into.
• Cell fate is specified when it can differentiate autonomously in a neutral environment.
• Commitment to cell identity is liable at the reversible stage, so a transplant will change the fate of the host population.
• Autonomous specification occurs when each blastomere receives specific cytoplasmic determinants, acting without interactions.
• In autonomous specification, the position of cells does not matter.
• Conditional specification: Cells achieve fates by interacting, including cell contacts, secreted signals, and physical properties.
• In conditional specification, the position of cells matters.
• Syncytial: Cytoplasm that contains multiple nuclei
• Within syncytial blastoderm, future cells' identity is achieved across the entire embryo's A-P axis.
• Opposing gradients define axial position in the syncytial nucleus, where position matters.
• Determination is when a cell or tissue can differentiate autonomously even when placed in another region.

Term Definitions

• Blastomeres: Cells derived from cleavage in an early embryo.
• Blastocoel: The cavity within the blastula.
• Blastoderm: A single layer of embryonic epithelial tissue which makes up the blastula.
• Blastula: An embryonic stage composed of blastomeres.
• Blastocyst: A mammalian blastula
• Invagination is where gastrulation begins
• Regions which are competent to become neural tissue are established in the egg cell.
• An amphibian eggs, poles are divided into animal, being anterior to becomes the nervous system, and the epidermis while vegetal pole, being posterior, becomes the gut structure.
• Amphibian and mammalian cleavages have various differences, for example mammals divide slower
• After fertilization, egg cells divide repeatedly and results in blastomeres and surround a fluid filled cavity called blastocoel
• Blastomeres dont divide at the same time
• Blastoderm: The group of cells above blastocoel
• A frogs appearance is spherical during the blastula stage
• Birds blastula appears as flattened sheet of cells
• Two laters of cells termed epiblast on top, the precursor of the embryo and the hypoblast which gives rise to the yolk sac, is required to pattern the epiblast to establish the foetus.
• Vegetal pole of ooctye (Oocyte) Maternal VegT and Vg1 accumulate
• (Late blastula) Transcribed Vg1 protein induces Wnt antagonists (Dickkopf) and transcribed VegT activates transcription of Nodal protein
• P-Smad2 upregulates Eomesodermin and Brachyury triggers cells above cells to accumulate p-Smad2
• In mesoderm, VegT transcription is triggered in the mesoderm to lock in fate
• Dorsal blastopore lip (DBL): An area that will be critical in establishing the ectoderm
• Ectoderm ecloses embryo.
• Endoderm internalized.
• Mesoderm in the middle.
• Loss of blastocoel happens
• Primitive streak: Forms the longitudinal axis of the embryo and this is where cells will migrate inward during gastrulation.
• A transient group of cells that provides signals for the ectoderm is called the Hensen's node"

Germ Layers

• Ectoderm derivatives
• Epidermis
• Nervous System
• Cornea
• Lens of the eye
• Mesoderm derivatives are the:
• Skeleton
• Muscle tissue
• Circulatory system
• Dermis
• Endoderm derivatives are the:
• Liver
• Pancreas
• Epithelial lining of the digestive tract

Ectoderm

• Surface ectoderm (epidermis): BMP levels high.
• Neural crest: BMP levels moderate.
• Will generate pigment cells (melanocytes) and the PNS (sensory neurons)
• Neural plate/neural tube: BMP levels low, Sox transcription factors is expressed.
• Moves into the body to form neural tube, the precursor of the CNS- brain + spinal cord.

Neurulation

• Neurulation is when the neural plate, neural groove, begin and continue until there are neural folds Neurula: An embryo undergoing neurulation.

• Neurulation is the movement of the presumptive nervous cells into the interior of the embryo, leading to the formation of the nervous system.

• Ectoderm gives rise to the neural plate-> precursor of the CNS + ONS.

• Convergence-> neural plate buckles at midline to form neural groove.

• Closure of the dorsal neural folds forms the neural tube.

• The entire CNS develops from the walls of the neural tube; PNS develops from the neural crest.

• Primary neurulation: Cells surrounding neural plate direct neural plate cells to proliferate, invaginate into the body, + separate from the surface ectoderms to form an underlying hollow tube

• Secondary neurulation: Neural tube arises from the aggregation of mesenchyme cells into a solid cord that subsequently forms cavities that coalesce to create hollow tube.

• Primary neurulation forms anterior portion of the neural tube

• Secondary neurulation forms posterior portion of neural tube

• Secondary neurulation begins at a level of sacral vertebrae of the tail.

• Neural tube is complete when these two separately formed tubes join together +the size of the transition zone between the primary and secondary varies among species

• Closure of neural tube occurs at multiple points

• Paracrine factors: Diffusible proteins are synthesized by one cell and diffuse inducing changes to neighboring cells

• Closure 1 (cranio-rachischisis)

• Closure 2 (anencephaly)

• Closure 5 (spina bifida)

• Structures:

• Noggin formation regulates hinge point.

• BMPs are expressed in cells of the surface ectoderm (green), Noggin is expressed in the dorsal neural folds (blue), and Shh is expressed ventrally, in the notochord and floor plate (orange).

Primary and Secondary Vesicles

• Structures that arise from the three primary and five secondary vesicles can be listed as:
• Prosencephalon
• Telencephalon: Cerebrum
• Diencephalon: Thalmic areas
• Mesencephalon
• Mesencephalon
• Midbrain
• Rhombencephalon
• Metencephalon
• Pons and cerebellum
• Myelencephalon
• Medulla oblongata

Development Stages

• Mouse + Human development are marked by theiler stages and carnegies stages
• Gastrulation begins at TS09 and CS09
• Chick development is staged using Hamburger and Hamilton scales
• Primary vesicles visible at HH 10
• Secondary vesicles visible at HH 11
• Embryo begins to turn as HH 13
• At zebrafish, gastrulation begins at 50% epiboly
• Nueral plate is present at approximately 90% epiboly
• Brain vesicles are seen at the 14-16 somite stage.
• At frog development the Nieuwkoop and Faber is where the
• Blastula present at 4 hrs post fert (stage 7)
• Gastrulation begins at 7-10 hrs post fert (stages 10-12)
• Neurula begins at stages 13-22
• At Fruit fly
• gastrulation occurs in stages 6-7
• cns forms in embryo, which continues through the larval stage

Cell Communication

• Cell communication in the developing embryo happens through juxtacrine or paracrine signaling.
• Juxtacrine involves direct contact, while paracrine uses ligands across distances.

Paracrine Signaling Families

• Paracrine factors induce numerous organs by binding to receptors and initiating changes in target cells.
• Fibroblast Growth Factors (FGFs): Ligand -> RTK -> GEF -> Ras -> MEK -> ERK -> transcription factor -> transcription.
• Ligand -> receptor -> JAK -> STAT -> STAT dimerization -> transcription.
• Hedgehog Family:
• Hedgehog processing and secretion involves: Autoproteolytic cleavage.
• Addition of lipids: palmitic acid and cholesterol.
• Without Hedgehog binding to Patched, Smoothened degrades and the transcription factor Ci/Gli is connected to microtubules.
• When Hedgehog is present, additional co-receptors fosters strong binding of Patched, proteins shape changes, endocytosis is activated.
• Smoothened releases Ci/Gli that allows it to enter the nucleus where it acts as a transcriptional activator.
• Wnt Family:
• Active Wnt is associated with glypican, Wnt associates with Frizzled.
• Notum cleaves off lipids on Wnt, releasing it.
• Negative Feedback Regulation of Wnt: Active Wnt signaling leads to Upregulation of Notum, secreted and interacts with glypicans, also binds palmitoleic potions of Wnt proteins
• TGF-B superfamily:
• TGF-ẞ superfamily ligand -> receptor II -> receptor I -> smad activation -> smad dimerization -> new transcription.

Neural Induction

• Neural induction is the process of changing undifferentiated tissue to turn into neural tissue.
• Timing is important for neural induction.

Seminal Experiement

• Hans Spemann and Hilde Mangold's experiment showed nervous tissue is induced via the dorsal blastopore lip.
• Established the concept of induction and the discovery of DBL (Organizer), where cell fate is determined by signals from other cells.
• Grafted blastopore cells can induce neural issues from an area that wouldn't give rise to that tissue normally.
• When intact animal caps without any mesoderm tissue are removed and placed in placed in culture -> epidermal tissue forms
• When intact animal caps include mesoderm tissue, neural tissue forms (DBL)
• To find mesoderm inducers, growth factors from TGF-beta and FGF families were added to cultures of dissociated animal caps
• Animal caps treated with enzymes to break cells apart -> dissociation
• Treated cells then reaggregate and take on cell fate
• Short dissociation period= epidermal
• Long dissociation period- neural tissue
• Observations suggest that longer dissociation period presumptive
• contact-mediated or secreted signals from animal caps were no longer at
• concentration that was sufficient to prevent neural formation
• Without signal neural tissue formed with signal epidermal
• General idea that neural tissue can form when specific signal is
• missing
• Research involved activin to see effect on mesoderm
• Some experiments found animal caps without mesoderm gave rise to epidermal tissue, it was suggested there was another tissue

Noggin and Follistatin In Relation To Neural Tissue

• Noggin (an identified protein) induced a normal body axis and neural tissues in UV-treated embryos.
• Noggin directed the neural tissue.
• Follistatin could bind to +inactivate activin in its region
• Follistatin is present in Xenopus gastrula stage
• It induces the expression of neural markers but that mesoderm would not form if tissue does not form
• In summary, Follistatin could control neural induction and inhibit activin. Additional experiments yielded similar affects and it was determined to be a third direct pathway
• BMPs induced epidermal markers while suppressing neural markers _BMP2 + 4 are expressed throughout ventral regions of frog embryo but are restricted to regions of ectoderm associated with epiderm _In cultures of animal cap cells dissociated for 3 or more hours treatment with BMP2 or BMP4 led to formation of epidermal tissu
• BMP4 at high concentration inhibited ability of chordin, noggin, and follistatin to form neural cells + instead induced epidermal tissue
• BMP signaling was required for formation of epidermal tissue + blocking BMP is necessary for formation of neural tissue _ The DBL secretes BMP antagonists noggin, follistatin, and chordin that then diffuse into dorsal regions of the embryo where they inhibit exogenous BMP signals _ Ectoderm closest to the node gets least amount of BMP signaling + forms neural tissue _ Ventral ectoderm is not exposed to sufficient levels of inducers so BMP signaling occurs + epidermis forms

Influences on SMAD Transcription

• BMP binds to serine/threonine kinase receptors.
• This phosphorylates a serine or threonine on the type 1 subunit.
• The phosphorylated subunit phosphorylates transcription factors (SMAD1 + coSMAD), which translocate to the nucleus where activate target genes that induce epidermis.
• Transcription factors phosphorylated post-BMP binding include those of the SMAD family.
• Due to chordin, follistatin, and noggin interfere with BMP, signaling SMAD1 and transcription never occurs.
• Signaling molecules thought to contribute to neural induction include FGFs and Wnts.
• FGFs have multiple roles at different stages of neural induction.
• In the hypoblast, FGFs regulate which cells remain at the epiblast surface during gastrulation.
• In organism that continue on, FGFs help determine which cells are going to become neutral.
• 4 vertebrate tyrosine kinase receptors that can dimerize and cross phosphorylate molecules

Nervous System Development

• The nervous system develops along two planes: Anterior-Posterior (A-P) and Dorsal-Ventral (D-V).
• In the A-P direction, forebrain regions (p1-p6) develop posterior->anterior while hindbrain (r1-r7) is anterior->posterior direction
• Neuromeres transiently limit cell movement until they have been exposed to signals to help with development -Neuromeres: Prosomeres and Rhombomeres
• Although neuromeres correspond to general anatomical areas found in the adult nervous system, the purpose of segmentation at such early stages is not to designate rigid boundaries of the adult structures
• the transient boundaries serve important functions, such as limiting the movement of cells until they have been exposed to signals critical to their subsequent development and specialization

Secondary Vesicals

• Anterior Neural Ridge (ANR) -> specifies telencephalon

• Zona Limitans Intrathalamica (ZLI) -> diencephalon

• Midbrain-Hindbrain Border (MHB)/Isthmus -> influences development of future midbrain, pons, and cerebellum

• Notochord -> hindbrain and spinal cord regions

• prechordal plate -> thickening that prevents movement of the notochord further anteriorly

• Activation-transformation model

• Proposes that the signal for neural induction- the “activator” induces competent ectoderm to form anterior brain regions with progressively higher concentrations

• of "transforming” signals converting the tissue into hindbrain and spinal cord

• These signals (morphogens) diffuse to establish concentration gradients.

• Signals are not produced in a wave but at multiple points and at different times

• Some signals transform previously induced areas into new areas.

• Some signals inhibit (antagonize) those signals to prevent transformation.

• Cells of the hypoblast (cell later that is displaced by the endoderm) specify the anterior neural ridges (ANR) via FGFs.

• ANR then regulates forebrain-specific genes.

• Genes directly important for prosomeres.

• Otx2 -> expressed anterior to the isthmus.

• Lack otx (no head structure formation). Pax6 -> expressed telencephalon and diencephalon.

• Lack of Pax -> small/absent eyes + ears. Six3 -> expressed p3-p6. Irx3 -) expressed p2 (rhomomeres). Restricted domains of the six and irx families are maintained b/c these two proteins suppress each other. Irx3 expression also influenced by wnt. -Loss of wnt causes loss of irx3 expression whereas increasing wnt levels leads to expansion of irx3 expression into more anterior regions (wnt + irx needed to pattern posterior diencephalon structures regions and delineate them from more anterior six3 expressing regions)

• Blocking endogenous wnt signaling leads to larger brain regions and forebrain regions extending to midbrain. -Specification of the forebrain versus midbrain regions where the levels also depends on the levels of neural induction and Wnt signaling.

• If the wnt is overexpressed in the presence of neural inducer -> genes and structures associated with anterior brain regions are suppressed.

• If wnt expression is inhibited in the midbrain regions, the anterior brain regions will expand posteriorly

• Wnt signaling is normally inhabited in forebrain regions

Midbrain

• Isthmus organizer (IsO) produces multiple molecules that pattern the midbrain and anterior hindbrain regions, as well as signals that prevent the spread of signals that prevent the spread of signals originating in the forebrain and posterior hindbrain
• Many of the genes for midbrain patterning can be detected as early as gastrulation
• Two important genes
• Otx2 -KO of Otx -> areas anterior to the isthmus take on posterior characteristics -Gbx2 -KO of Gbx -> areas posterior to the isthmus take on mesencephalon characteristics -*loss of either gene= the adjacent area is able to expand -Wnt -Wnt is necessary to maintain expression of En, a loss of Wnt likewise disrupts development of the midbrain and anterior hindbrain. Wnt, FGF8, and En2 also work together to repress the expression of genes associated with more anterior regions, including Pax6 and Otx2.. -FGF8
  • Most characterized in A/P patterning -> FGF8 -FGF8 - Causes cerebellar induction and hindbrain induction -Lower concentrations = midbrain development -Higher concentrations = cerebellar development 2 isoforms = FGF8a (midbrain induction); FGF8b (cerebellar induction) En2

Hindbrain Structure Signalling

• Rhombomeres: Transient swellings that form segments that extend from the midbrain to the spinal cord
• Segmentation genes are a group that includes the gap, pair-rule, and segment polarity gene classes. Gap genes are the first to be activated and will then establish the large boundaries of the head, thorax and abdomen.Combinations of gap genes that control pair-rule genes which divide 3 segments, these segmented patterns regulate segment polarity genes which establish individual cell characteristics (All three regulate expression of HOX genes)
• Hox genes: are expressed in distinct, overlapping patterns within each rhombomere. Hox code is the collection of that process

Hox Genes

• Hox genes are transcription factors containing a DNA binding domain (homeodomain) and their expression is important for spinal cod and hindbrain patterning.
• Rhombomeres are distinguished by the expression of Hox genes
• RA is taken up by the target cell and translocated to the nucleus via cellular RA-binding proteins (CRABPs), degraded via Cyp26 enzymes.

1. Each Hox gene responds preferentially to a given RA concentration. In the hindbrain, Hox genes located at the 3' end require lower amounts of RA which are closer to the 5' end. These structures are then developed along the A/P axis, structures in the more anterior regions of the hindbrain develop

Hindrbain RA

• Increased RA leads to the expansion to posterior, decreasing formation of anterior structures and Cyo26 express along the spinal cord
• Loss of Cyp26 increases RA and is vital for anterior patterning.
• In odd numbered rhombomeres adhere better
• Cell surface molecules are expressed in even versus odd numbered rhombomeres.
• Enphrin (ligand) is present in the ever numbered where its receptor Ehp the is tyrosine kinase
• There is a factor involved call krox 20:
• It is keeps the expression of Hoxb1 in r4
• The general requirement for Cdx genes in spinal cord patterning species. -Zebrafish lack this gene, in general. -When the is not the abov gene, the hindbrain expand into into parts of the anterior

D-V Patterning Anatomical Landmarks

• Nectochord as signaling center
• Roof region specialized ghilia to create this area
• Notochord is mesodermal structure that spans body
• Alar plate
• Dorsal horn
• Basal plate
• Ventral horn
• Intermediate zone Motor neurons and interneurons divided by the pools each of which do several regions
• DI 1-6

Neural Tube Compoditions

_ Neurla tube composed of groups of factor expressed on based on transcription that the

1. regions that where the tube goes that determine
2. there are various steps and what that impacts and so for not to make the region correctly -Floor Plate -Motor Plate Loss results loss if the area expands

Neural Tube Shh

• Shh diffuse
• Floor Plate high
• Motor high
• interneurons
• Loss of shh is the less of ventral
• Concentration matters: 1.High floor plate

2. Low = motor
3. Very low interneurons There are factors involved that are for transption

• There are expression of molecules thur down or are not down What and the last on some transcription What it's not or is it's that BMP and and they're in a big influence as well

BMP at it on the roof later

• The high and is is what is in the same the region of the the _The inter neuron's divided into A and B and we all the the region as the The signals form the and are you looking at the that they are There is BMP and Shh antagonize Cerebellum arises and if plate influence In the cell and there are loss what can that due to those regions is to be

Cell Historical Neuron Theories

• Different glial different
• Neurons layer of with no connected work

Radial Glial Cells

Radial glia cells the where things come in from the neuron:

• From there can create various layers
• There's more layers in for layer more cells to the move thither there has to be the the things for -There's different cell structures -But not out to so many the
  • And there's multi potent cells and there's multi potent the multi

Nuclea Region

Cell location moves to suface the process occur during division And there stage is the cell to cycle is prepared -Daughter either do this or there the next cycle And it's only a atricular the region

Different is the cell is

• the Radial Multi for