Drosophila Genetics & Development

Drosophila Overview

• Fruit flies provide a way to identify genes necessary for development via forward genetic screens.
• Genetic screens identify genes necessary for development by removing the function of the corresponding protein from the system.
• Random mutations are created in a population using chemical mutagens, ionizing radiation, transposons, or CRISPR.
• The population is examined for mutations disrupting the biological process of interest.
• The mutated gene is identified via sequencing.
• The normal function of the protein encoded by the identified gene is studied.
• Flies are more similar to humans in development due to three primary body axes, three germ layers, segmented body plan, most major organ systems, many shared behaviors, and amazing conservation of gene function.
• Flies are suited for genetic studies because they are easy to grow, breed quickly, produce many offspring, have a small genome, and develop rapidly.
• Genes are named based on mutant phenotype, e.g., white (eye color) and antennapedia (legs on head).

Drosophila Development

• The life cycle includes fertilization, cleavage, syncytial blastoderm formation, gastrulation, embryogenesis, larval stage, pupation, and metamorphosis to adulthood.
• Flies show a clear segmental pattern along the anterior-posterior (A-P) axis, with cephalic (C), thoracic (T), and abdominal (A) segments.
• Segmentation occurs through progressive waves of gene expression.
• Maternal genes, whose mRNA or protein is stored in the egg, set up an initial gradient, and their mutant phenotype only depends on the mother's genotype such as bicoid.
• Zygotic genes are transcribed from the embryo’s own genome and refine the initial gradient such as hunchback, even-skipped, wingless, or abdominal-A
• Mid-blastula transition marks the switch from maternal to zygotic gene control.
• Fly embryonic patterning genes were identified by larval mutant phenotypes.
• Examples include bicoid (loss of anterior), nanos (loss of posterior), and torso (loss of terminal) mutants.

Major Tissue Movements During Embryonic Development

• During the cleavage phase, the embryo starts as a syncytium and forms a cellular blastoderm.
• Egg and sperm nuclei fuse
• Nuclear division occurs without cell divisions, creating syncytium
• Nuclei migrate to periphery of embryo, and cell boundaries start to form
• Membrane invaginates around nuclei
• A monolayer of cells forms, which is called the syncytial blastoderm.
• Pole cells (primordial germ cells) will separate from somatic cells to form the cellular blastoderm.
• During gastrulation, mesoderm and endoderm move inside.
• A ventral furrow forms, which allows mesoderm to invaginate, moving to the inside of the embryo
• Germ band extension temporarily elongates the embryo along the dorsal side.
• Endoderm got to "get inside" as germ band extends around endoderm
• Mouthparts, thorax, and abdomen are formed when the germ band compresses during segmentation.

Maternal Genes and Initial Axis Patterning

• The micropyle at the anterior determines the AP axis.
• The vitelline membrane surrounds the embryo.
• The ventral side is more rounded, while the dorsal side has respiratory appendages.
• Maternal genes determine the AP axis and fall into anterior (bicoid), posterior (nanos), and terminal (torso) groups.
• bicoid (bcd) mRNA encodes a bi-functional protein that binds nucleic acids and acts as a morphogen, specifying anterior structures. Mutant lacks anterior structures.
• nanos mRNA binds maternal hunchback mRNA and specifies posterior structures without acting as a morphogen. Mutant lacks posterior structures.
• caudal mRNA forms a protein gradient emanating from the embryo’s posterior and acts as a primary posterior morphogen.
• Maternal mRNAs such as bicoid and nanos, and hunchback and caudal localized throughout oocyte.
• Embryo proteins such as Bicoid and Nanos are expressed in shallow gradients
• Hunchback expression inhibited by Nanos
• Caudal expression inhibited by Bicoid
• Bicoid, Caudal, and Hunchback are TFs that regulate zygotic gene transcription.

Localization of Maternal Genes in Oocyte

• Oocyte develops from egg chambers in the ovarian follicle.
• The egg chamber contains the oocyte, nurse cells, and follicle cells.
• The oocyte is localized to the posterior arrested in meiosis I.
• Nurse cells are sister germ cells that form a syncytium with the oocyte and produce maternal mRNAs.
• Follicle cells are somatic cells that secrete the eggshell and source patterning signals.
• The cell with cytoplasmic bridges becomes the oocyte and older egg chambers signal to younger egg chambers to create intrinsic polarity in oocyte.
• gurken mRNA is localized to one side of the early oocyte nucleus and sets axis patterning.
• Gurken protein is localized to posterior of egg chamber, adjacent to follicle cells in oocyte.
• Gurken is a secreted EGF-like signaling protein.
• Torpedo is the receptor for Gurken, expressed in follicle cells and creates complex pattern of microtubule tracks.
• The oocyte is originally localized to the posterior end of follicle by cadherin.
• Gurken is a secreted EGF-like signaling protein.
• Gurken signals twice during oogenesis to set up both AP and DV axes of embryo.

Patterning of AP Axis

• Patterning genes are transcription factors (TFs).
• Gap genes and pair-rule genes are zygotically expressed.
• TFs regulate gene expression through interaction with promoters and enhancers and can be activators, co-activators, or repressors.
• Combinatorial codes of TFs can specify different cell types.
• Gap genes are expressed in large regions along the AP axis.
• Examples include giant, Kruppel, knirps, and tailless.
• hunchback is expressed in a broad concentration gradient from the anterior pole, and mutants lack thoracic segments.
• Kruppel is activated and repressed by Hunchback.
• Gap gene products regulate each other’s expression to refine the pattern.
• Caudal gradient regulates Gap gene expression from the posterior.

Pair-Rule Genes

• Pair-rule genes are expressed in seven stripes corresponding to alternating parasegments.
• Parasegment is the fundamental developmental unit offset from morphological segments.
• even-skipped (eve) is expressed in odd parasegments, and mutants lack odd thoracic and abdominal segments.
• fushi tarazu (ftz) is expressed in even parasegments, and mutants lack even thoracic and abdominal segments.
• Pair-rule expression is dictated by gap genes and neighboring pair-rule expression.
• Each stripe of pair-rule expression is under separate transcriptional control from neighbors, regulated by cis-regulatory modules.
• Transgenes are used to determine which modules correspond to different stripes.
• Multiple TFs bind to an enhancer element for each stripe (activators and repressors).
• Primary pair-rule genes are controlled by gap genes (e.g., even-skipped).
• Secondary pair-rule genes depend on primary pair-rule genes (e.g., fushi tarazu).

Segmentation Genes

• A compartment is a discrete region containing all descendants of a small group of founder cells.
• Segment polarity genes maintain anterior-posterior polarity within each parasegment and turn each parasegment into a compartment.
• Each segment has distinct polarity with denticles in the anterior and naked cuticle in the posterior.
• Segment polarity mutants have all segments present but show a disrupted denticle pattern.
• engrailed (en) is expressed in 14 stripes and is initially under the control of pair-rule genes.
• wingless (Wg) is expressed in the posterior region of each parasegment.
• hedgehog (Hh) is expressed in the anterior region of each parasegment.
• Wg and Hh signaling create the denticle pattern along the AP axis of each segment.
• Segmental pattern of En expression persists throughout the fly's life.

Homeotic Genes

• Segmentation and homeotic selection occur simultaneously.
• Adult body structures are associated with individual segments.
• Gap, pair-rule, and segment-polarity TFs control homeotic selector genes, or Hox genes.
• Hox genes give each embryonic segment a unique identity that determines which adult structures it can form.
• Homeotic transformation is the conversion of one segment or structure into another related one.
• Drosophila has 8 Hox genes organized in two groups along chromosome 3 which are antennapedia and bithorax complex.
• Spatial/temporal co-linearity means the gene order corresponds to their expression patterns and timing along the AP axis.
• Spatial pattern of expression for bithorax complex genes determines unique identity of parasegments 5-14.
• Hox genes regulate each other’s expression and function in a combinatorial way when co-expressed.
• Unique identities of embryonic segments are maintained by Hox genes into adulthood.
• Imaginal discs are larval tissues that give rise to adult structures.
• Hox genes control the activity of genes required throughout development and the pattern of hox gene expression requires chromatin modulation.

Patterning of DV Axis

• Dorsal mRNA forms a gradient that leads to differential expression of genes along the DV axis, specifying distinct tissue types.
• Dorsal gradient is due to regulation of protein regulation and not diffusion from a point source.
• During oocyte development, the oocyte nucleus is localized to anterior-dorsal follicle cells.
• gurken mRNA travels to the dorsal side of the oocyte.
• Gurken/Torpedo signaling restricts expression of Pipe to ventral region of follicle cells
• Pipe modifies unknown substrate that then localizes and cleaves Gd protease.
• Cleaved Gd initiates protease cascade, leading to cleavage of Spaetzle.
• Spaetzle binds Toll, initiating signal transduction in oocyte via Pelle and Tube.
• Toll signaling leads to phosphorylation and degradation of Cactus, allowing Dorsal to enter the nucleus.

Dorsal Signal Transduction Cascade

• The order of the signal transduction cascade was determined through epistasis and injecting pre-cleaved versions of proteases.
• Early members of pathway Toll, spaetzle and pipe show overly dorsal phenotype
• Two signal results are dorsalized and ventralized
• Localization of Pipe allows for differentiation of ventral and dorsal cells.
• DV patterning mRNAs are loaded into the oocyte and are only activated on the ventral side through Pipe signaling.
• Pipe/Toll signaling leads to differential concentrations of Dorsal across the DV axis.
• Genes repressed by Dorsal are expressed in the dorsal region, while genes activated by Dorsal are expressed in the ventral region.
• dpp, tolloid, and zerknullt are dorsal ectoderm and amnioserosa specifying genes repressed by Dorsal, therefore expressed in the dorsal region.
• is activated by Dorsal at low concentrations and is thus expressed in the medio-ventral region.
• twist and snail are ventral mesoderm specifying genes activated by Dorsal at high concentrations and thus expressed in the ventral region.
• Similar system patterns DV axis in vertebrates (but in reverse).

Germ Cell Development

• Germ cells provide continuity of life.
• The zygote produces somatic cells and primordial germ cells (PGCs).
• PGCs give rise to new zygotes.
• Major events in the life of the PGC include specification, migration, and differentiation.
• Specification occurs early in development and often near the embryo's periphery.
• Cell-cell signaling or segregation of germplasm determine the specification
• PGCs relocate from their original location in process known as migration
• PGCs interact with somatic niche cells in the gonads to become germline stem cells in a process known as differentiation.
• Germplasm is segregated through the formation of PGCs at the posterior pole of the fly embryo.
• Pole cells are primordial germ cells, which lay above the surface epithelium.
• The posterior end of the egg contains cytoplasmic factors sufficient to form PGCs.
• Shown through transplantation assay, in which injection of posterior cytoplasm into anterior causes ectopic pole cell formation
• Germplasm contains mRNAs and proteins that assemble in granules.
• Components of germplasm are synthesized by nurse cells and transported to the posterior pole of the oocyte.
• oskar is necessary and sufficient for germplasm assembly.
• germ cell-less (gcl) is necessary for PGC formation; Gcl is required for bud furrow formation.
• polar granule component (pgc) silences transcription in pole cells since repressing transcription in PGCs helps protect from influence of somatic cell fate.
• nanos promotes PGC survival during migration and drives AP patterning.

Segregation of Germplasm in Other Organisms

• In C. elegans, germline granules (P granules) are segregated to posterior daughter cells.
• PIE-1 in C. elegans serves a similar function to Pgc.
• In frogs, germline granules are localized to the vegetal pole.

Migration

• Gastrulation starts the process of germ cell migration.
• Convergent extension and the flip of abdominal structures form an endodermal pocket that allows PGCs to “get inside”.
• Movement of PGCs into the endodermal pocket is coupled with midgut movement during gastrulation.
• After gastrulation, PGCs rely on signaling to form gonads.
• Germ cells "wiggle" into the mesoderm and split into two group
• PGCs split into two streams containing somatic gonad precursor cells—migrate into streams, to form gonads via germline stem cells during gonad coalescence.
• Wunen 1 and Wunen 2 produce a protein signal repelling PGCs in the midgut.
• Columbus encodes a protein highly expressed in gonadal mesoderm with ectopic expression attracts PGCs.

Morphogenetic Movements

• Morphogenesis refers to the generation of shape.
• Patterning influences cell differentiation, which influences morphogenesis, which in turn influences patterning
• Mesenchymal cells involve condensation, cell division, cell death, migration, matrix secretion, and growth.
• Epithelial cells are governed by dispersal, delamination, shape change, migration, division, and control of spindle orientation and division plane.
• Apical vs. basolateral regulation facilitates tissue morphogenesis.
• Tight junctions form a barrier to paracellular diffusion.
• Adherens junctions transmit mechanical forces across tissue, and combine with homophilic cell adhesion with acto-myosin contraction.

Gastrulation

• Future mesoderm invaginates from cellular blastoderm during gastrulation.
• Invagination occurs along the A-P axis, creating the ventral furrow.
• Gastrulation begins with apical constriction of mesodermal cells.
• Myosin concentrates at the apical side.
• Movement depends on contractile networks of myosin and bi-polar minifilaments that undergo rounds of pulsatile contraction and stabilization to shrink apical surface area.

Germ-Band Extension

• Germ-band extension is the convergence and extension of the epidermis that temporarily elongates the fly A-P axis.
• Convergent extension requires mesenchymal cells.
• Cells exchange neighbors.
• Extension is rapid by converging cells forming rosettes of 6-11 cells
• Rosettes result from contraction of supra-cellular acto-myosin cable.

Dorsal Closure

• Dorsal closure occurs when two sides of the epidermis move toward the dorsal midline and fuse.
• Supra-cellular actin cables in epidermis and pulsatile apical constriction of amnioserosa guide dorsal closure.
• Lateral epidermal cells also elongate along the DV axis to close the midline.
• Actin-rich filopodial protrusions from the leading edge help epithelial sheets match up correctly.

Planar Polarity

• Planar polarity is when there are different complements of lipid/proteins on one side of the planar axis, which is orthogonal to the apical-basal axis.
• Can use for morphogenesis
• Can generate uniform movement
• 'Hairs’ occur on the distal side of epithelial sheet cells on the adult drosophila wing.
• Proteins that govern their formation are present in imaginal discs during pupal development.
• Planar polarity studies use mosaic tissues to observe how mutant cells behave with WT cells.
• Mosaic tissues are a mixture of mutant and WT cells.
• GFP expression distinguishes between WT and mutant cells in a sample.
• Advantages of mosaix tissues are the expression of essential genes later in development, internally controlled experiments, and detectable subtle phenotypes, including at the cellular level
• Flp/FRT mitotic recombination generates simultaneous mutant clones and WT clones in proliferation

Cell Autonomy and Genetic Mosaics

• Cell-autonomous mutants manifest the phenotype in the same cells where the gene function was removed such as a transcription factor.
• Non-cell-autonomous mutants manifest the phenotype in WT cells that are adjacent or at a distance such as signaling protein (Hh).

Planar Polarity Genes

• Frizzled (Fz) and Disheveled (Dsh) are involved in the Wg/Wnt pathway and planar polarity, specifying the distal boundary in epithelial cells of wings.
• Fz is the transmembrane receptor for Wnt ligand, while Dsh is a cytoplasmic scaffolding protein that assists in Fz cell signaling.
• Dsh protein domain mediate the planar polarity expression and DIX, PDZ and DEP domains mediate “canonical” Wg signaling.
• Point mutation in Dsh protein only involved in only planar polarity
• In fz mutant clone with mosaic tissue, the hair projects from the center of the cell, and the adjacent hairs point toward mutant cells.
• Dsh and Fz are enriched along proximal-distal cell-cell boundaries in a zig-zag pattern.
• These proteins become specifically enriched on the distal side
• Strabismus (stbm) is a planar polarity gene that encodes a transmembrane protein specifying the proximal boundary in epithelial wing cells with opposing results of fz mutant clone
• Other core planar polarity genes also show enrichment along proximal and/or distal cell boundaries (Diego, Prickle, Flamingo)
• Protein interactions involve:
  • Positive interactions across cell-cell boundaries
  • Mutually inhibitory protein-protein interactions within the cytoplasm
• These interactions segregate Fz and Stbm protein complexes to opposite cell edges to polarize the tissue.
• Core planar polarity genes are also used for hair follicles in the skin, hair cells in the cochlea, and convergent extension.

Overview of C. Elegans

• The C. Elegans fertilized egg undergoes a determinate cleavage stage.
• The embryogenesis stage converts cells into an embryonic worm, enclosed in an egg shell.
• The worm hatches from the shell and undergoes several larval stages (L1, L2, L3, L4) before reaching adulthood (hermaphrodite).
• Worms are comprised of small number of cells such as gastrulation is at 28 cells, L1 is at 558 cells, and the Adult stage has 959 cells.
• Worms can be either hermaphroditic or male.

Advantages to Studying C. Elegans

• Easy to do forward and reverse genetic studies
• First multicellular organism to have genome sequenced
• Ease of RNAi studies
• Simple cellular structure
• Optically transparent
• Invariant/determinate cell lineage
• Allows us to understand where cells are in embryo and when they disappear (apoptosis)

Cell Death in C. Elegans

• Cell death (apoptosis) is a normal part of development.
• Programmed cell death uses regulate cellular number, eliminates abnormal or dangerous cells, creates structures like hands by deleting structures.
• Hallmarks of an apoptotic cell are shrinking/condensing of cell, cytoskeleton collapse, nuclear envelope disassembling and chromatin condensing and fragments.
• An apoptotic stimulus triggers a cascade of activating and inhibitory molecules for apoptosis.
• EGL-1 protein activates the pathway by releasing CED-4 from CED-9.
• CED-9 protein keeps the pathway off by forming a complex with CED-4 to keep it from activating CED-3.
• CED-4 proteins are required for the activation of CED-3.
• CED-3 protein is a caspase that chews up the cell.
• Mutations where cells that should have lived died helped identify negative regulators of the pathway.
• Mutations where cells that should have died lived identified positive regulators of the pathway.
• Pathway for engulfment of apoptosized cell by phagocytosis and degradation has different screening process with recognition of the apoptotic cell.
• Criteria is efficient removal of cellular components and prevents immune response or proliferation of damaged cells
• Core apoptotic proteins are present in all cells at all times and are required for specific signals.
• BMP proteins signal apoptosis to remove webbing from digits.
• Nerve cells cell require specific signals to stay alive so cell number can adjusts to size of target, removes unnecessary energy expenditure.
• Loss of core executioner caspase leads to excess of neurons in the mouse brain.

MicroRNA in C. Elegans

• Heterochronic mutations disrupt normal timing/rate of development.
• Lin-14 encodes a nuclear protein (TF) that has no homologs outside of works.
• mRNA levels stay constant throughout larval development, and the LIN-14 protein forms a temporal gradient.
• Mutations in the lin-14 gene lead to temporal transformations in cell lineage patterns during larval stages.
• GOF in lin-14 causes premature cell death
• Lin-4 is a negative regulator of lin-14 as, lin-14 GOF is phenotypically similar to lin-4 LOF, and LIN-14 protein stays uniformly high in lin-4 mutant larva.
• Molecular basis for repression of LIN-14 translation by lin-4 occurs because, Lin-14 GOF is generated by a small deletion in the 3’ UTR of lin-14 mRNA and Lin-4 encodes small RNA that shows partial complementarity to deleted region.
• MicroRNA which are transcribed by a microRNA gene, is a short RNA transcript that binds to mRNA to block protein assembly for translation

Segmentation in Invertebrates

• Chick gastrulation
• Hensen’s node (Spemann organizer equivalent) initiates gastrulation in the chick embryo.
• The primitive streak is the site of mesoderm and endoderm ingression during gastrulation.
• Ectoderm moves toward the primitive streak, and mesoderm and prospective endoderm migrate inside the subgerminal space.
• Development of specific body structure occurs in anterior to posterior wave
• Henson’s node regresses down AP axis
• Anterior structures develop first, posterior structures last

Somitogenesis

• Presomitic mesoderm vs. somites
• Somites are discrete epithelial balls, whereas presomitic mesoderm is unstructured mesenchyme.
• The timing of somite formation is a cell intrinsic program in anterior presomitic mesoderm.
• Inversion of the presomitic mesoderm causes somites to form in reverse order.
• Oscillations in presomitic gene expression coordinates timing of somite formation.
• C-hairy1 mRNA is expressed in the presomitic mesoderm.
• Waves reach anterior region of presomitic mesoderm, and somite formation occurs.
• Feedback within the Notch pathway produces the intrinsic clock mechanism.
• The determination front in the anterior most cells of the PSM cells are controlled by the concentrations of signaling proteins.
• FGF/Wnt expression continuously moves toward the posterior with the node, forming a high/low concentration gradients.
• When the FGF signal is low enough, signals to PSM cells to make a somite which helps maintains oscillatory signaling
• The direction of the determination wavefront is in the same direction as axis elongation.
• Both Clock mechanism and determination front are needed for somitogenesis.

Retinoic Acid (RA)

• Retinoic acid is expressed in somites and the anterior-most PSM and required for the condensation of PSM mesenchyme into somitic epithelium.
• Secretion of RA by somites forms a gradient along the PSM.
• RA inhibits FGF expression in the anterior PSM and activates Hox genes.
• HOX overexpression is phenotypically similar to the excess RA GOF .

Vertebral Identifies

• Vertebral segments have unique morphology that determined by Hox genes.
• The Hox identity is determined by the transplantation studies
• PSM knows its positional identity along the AP axis before somite formation.
• Temporal activation provides positional information.
• Hox genes display spatial and temporal colinearity for somitogenesis.
• More anteriorly expressed genes occur at the 3’ end of the chromosome and come on earlier.
• More posteriorly expressed genes occur at the 5’ end and come on later.
• Hox genes are named with a letter indicating chromosome and number indicating where on the chromosome is located in either the A, B, C, or D Cluster.
• Spatial colinearity of hox genes occurs in different tissues (both neural tube and mesoderm), but the anterior expression border for a particular gene may not be in the same for all tissues in which is expressed
• Conservation of hox gene function in different vertebrates indicate specific vertebral identity/morphology for that gene.
• Some hox genes specify the same region in different species. c6: typically expressed in cervical to thoracic transition and c9 and a9: typically expressed in thoracic to lumbar transition
• The KO of c8 leads to homeotic transformation from lumbar to thoracic vertebra as an example of full homeotic transformation.

Neural Segmentation

• Hindbrain segmentation involves the Hox genes and neural crest.
• Dorsal mesoderm induces and begins to pattern the neural plate.
• The neural plate folds in on itself to generate a neural tube along the primitive streak in the A to P direction.
• Patterning along the DV axis is due to BMP signaling from ectoderm and Shh signaling from the notochord.
• The neural tube is also patterned along the AP axis to give rise to different brain structures.
• At the posterior end, the rhombencephalon gives rise to the hindbrain, and the floor plate gives rise to motor nuclei.
• Rhombomeres are 8 segments of the hindbrain that are physically restricted from one another.
• Branchial (pharyngeal) arches also show this segmental pattern and are made of epithelial pouches containing all germ layers including neural crest.
• Neural crest occurs at fusion of neural folds.
• Neural crest cells migrate to distant sites around the body, making specific cell types such as melanocytes, sensory, sympatho-adrenal, sympathetic, enteric, and cartilage.
• Tagging neural crest cells contained in the neural tube assists with tracing later in development.
• Pluripotent neural crest stem cells give rise to multipotent neural crest cells, which give rise to cranial and trunk neural crest cells with restricted lineages.
• Bone and cartilage precursors—unique to cranial neural crest cells
• Myofibroblastic precursors—arise from cranial neural crest cells
• Neural and melanocytic precursors—common to trunk and cranial neural crest cells
• Migration pathways for cranial neural crest cells from different brain regions create different head and neck tissues.
• Different brain regions have migration pathways via outward streams to the mid and hindbrain.
• Rhombomeres provide compartments that assist in distinct patterning.
• Hox gene subgroups 1-4 which specify the individual structure giveCombinational Manner of arise from each rhombomere (r1-r8).
• Branchial arches in head and neck structures are patterned by Hox subgroups

Left-Right Axis

• Vertebrates exhibit bilateral symmetry on the outside but left-right asymmetries of the internal organs (e.g., heart, liver, stomach, spleen).
• Early asymmetry arises from gene expression in the lateral plate mesoderm (LPM).
• nodal is expressed on the left side of the LPM.
• The heart is almost entirely made of LPM that forms from fusion of the anterior LPM.

Kartagener’s syndrome

• A birth defect with situs inversus and motility issues
• Cilia cannot flow sperm, cannot clear mucus
• Cilia are microtubule based where either motile or nonmotile
• Axoneme is the microtubule scaffold and basal body anchors axoneme to cell.
• Primary cilium are nonmotile and renal (9+0 arrangement).
• Receptors allow to receive extracellular signals
• Motile Cilia is usually 9+2 doublet microtubules (Dynein and kinesin motors)
• Present in airways, reproductive and the exceptions (nodal and inner ear)

Nodal Cilia

• Nodal Cilia (mouse node with unidirectional clockwise (9+0 motile) cilia, like paddlewheel)

• In iv mice mutation showed it is a disruption to LR axis which shows normal, reversed or nonexistent of Nodal expression

• The dynein motor is needed for that cilia motility

• Position and Clockwise rotation create leftward flow which creates high left Ca2++ levels

• Two hypothesis adjacent - Primary Cilia hypothesis: extra cilia periphery

  • NVP Hypothesis (transporter)

• Artificial Flow restores phenotype and is sufficient Zebrafish Kupffer’s vesicle (DFCs) is a homolog

• No tail (Ntl) gives rise from the expression cell fate

• Ntl Morhpolino, causes LR defects and prevents the formation of KV - Kupffer forms but MO defects - Dyntonin deletion

• In LPM the Nodal released stimulates from peri-nodal region -Nodal: TGF Beta Family and releases itself - Lefty: Restriction Domain - Pitx2: Longer express for morphogen