Lecture 4 (Animal Development) PDF

Announcements: Labs begin next week!! Farq Rm 109 Midterm: Thursday Feb 13th 1  Differentiation: Developmental Commitment Differentiation: process of formation of specialized (distinct) cells swap...

Announcements: Labs begin next week!! Farq Rm 109 Midterm: Thursday Feb 13th 1  Differentiation: Developmental Commitment Differentiation: process of formation of specialized (distinct) cells swap buflable During development uncommitted cells become committed autonomously into cells we recognize (i) Unspecified, (ii)Ideveloped Specified and then may become something else (iii) Determined Or labile influenced Stemcells devel autonomously byenvironed non labile winds cell very specific type  cape ype stable Tests for Fate, Specification and Determination orthotopic graft b isolated orthotgraft inheterotopic groff  Hierarchy of specification cells originator Potency: range totyolent of possible cell pluripotent types impotent nature  Difference genes spression Modes of Cell Type Specification Autonomous Specification – Acquisition of cytoplasmic molecules. Teterminants Conditional Specification – Interactions between cells. 5  Autonomous Specification -cytoplasmic molecules, located in specific part of cell Symmetrical cell division non identical delemundits to one go  cell only (i) Autonomous Specification Determinants extract cytoplasm Mfinker Ectopic head arlo 1 Identify mRNA protein (ii) Conditional specification inductive 8  Work in Dev. Biol: growth – I.D. inducing factors, determinants, transc. factors etc., that defines states of developmental commitment.  Conditional specification How can cells influence fate of neighbour? Direct interaction adhesion Morphogen inducing factor works like a gradient  Ca dependent adhesion molecule Adhesion Cadherin- mediated cell Mesynch adhesion cells don't have cadherin but epithel E codherm epithelial do cell N cadherin neural cells 11 homophilic adhesion all w E cool only interact w other cells w E cool Differential cell affinity neuralation (amphibian embryo) Ectodermal pigmented embryo E cod Mesodermal unpigmented Ncad up 12  Morphogen gradients inducing factor Erm morphogen low pIeast morphogen cell type 13  How does one identify a morphogen? – the molecule must be there of themorphogen – demonstrate that cells respond to that compotent to tomorphogenrespond molecule If a molecule thenresponse fails – response of those cells depend on the concentration of the molecule Zebrafish embryos Nodal: long-range morphogen head mesoderm notochord muscles genes activated by Nodal 16  Possible alternatives? – Cascade of inductions (Nodal does not do anything at lower concentrations) Negative controls confirm that Nodal is a morphogen normal cells with Nodal receptor mutated transplanted cell 18  Possible alternatives? – Cascade of inductions (Nodal does not do anything at lower concentrations) – Daughter cell with source of Nodal migrated to position further away Negative controls confirm that Nodal is a morphogen t control mildtype control cells with Nodal receptor mutated transplanted cell 20  Possible alternatives? – Cascade of inductions (Nodal does not do anything at lower concentrations) – Daughter cell with source of Nodal migrated to position further away – Injection causes response Negative controls confirm that Nodal is a morphogen cells with Nodal receptor mutated transplanted cell 22  Positive control(s)?? use Nodal test for technique in case of negative result use of dye showed injection done properly (since no response) 23  Recap Develop. commitment either labile (specification) or stable (determination) Cytoplasmic determinant: comm. to dev. pathway of cells that inherit it Inducing factor can alter dev. pathway of cells exposed to it Embryo is created by immature cells 24 Cell Differentiation Notes created on January 16, 2025 at 10:04 AM by Minutes AI Determining Feed Map (00:00 - 09:55) Labeling a cell or region of the embryo is essential for understanding animal form. Following the label log during development helps track cell differentiation. Differentiation of Cells Differentiation process starts from an originator cell to a mature cell. This process is referred to as differentiation until terminal differentiation is Cells begin as uncommitted and have a broad ability to become different tissues. They become more committed during development. Stages of Differentiation **Unspecified Cells (Stem Cells)** Stem cells are undifferentiated and self-renewing. **Specified Cells** These cells can develop autonomously into recognizable cell types but remain **Determined Cells** These cells develop autonomously to a definite cell type, stable and on a path to Experimental Methods Experimental embryology determines when cells become specified or determined. Protocols include fate specification and determination experiments. **Orthotopic Graft** These notes were taken with Minutes AI (https://myminutes.ai)  Tissue from one embryo is placed in the same location of another embryo to **Heterotopic Graft** Tissue is placed in a different part of the embryo to observe its development Developmental Specification (09:55 - 19:53) Normal development observed in orthotopic grafts. Tissue can become specified but not determined. Depends on tissue type and developmental stage. Heterotrophic grafts show that tissue can be determined regardless of graft location. Stages of Specification Specification starts broad and narrows over time. Stem cells can become various tissues initially. As development progresses, potential becomes more limited. Originator cells are referred to as "Tobin token." Totipotent or pluripotent stem cells at the top of the hierarchy. Mature cells at the bottom express different genes and proteins. Methods of Specification Two methods for specification: autonomous and conditional. **Autonomous specification**: Cells acquire cytoplasmic molecules **Conditional specification**: Cells interact with neighboring cells, altering their Asymmetrical Cell Division Asymmetrical cell division leads to non-identical daughter cells. One daughter cell may be smaller and contain different cytoplasmic These notes were taken with Minutes AI (https://myminutes.ai)  Asymmetrical division is crucial for autonomous specification. Identifying cytoplasmic determinants is a major goal in developmental biology. Cytoplasm Extraction and Ectopic Head Formation (19:53 - A needle is used to extract cytoplasm from an embryo. The extracted cytoplasm is inserted into a second area to observe development. This can lead to the formation of an ectopic head. The cytoplasm contains components sufficient to produce head structures. Identification of Factors in Cytoplasm After cytoplasm extraction, the focus is on identifying messenger RNA or proteins. Working with messenger RNA is easier than working with proteins. Once a factor is identified, it can be produced in vitro for further experimentation. Specification Types Autonomous specification involves the influence of cytoplasmic components. Conditional specification involves cell-cell interactions. Example: Vegetal pole of the embryo influences nearby cells to become Inductive Factors in Development Inductive factors can include transcription factors and growth factors. Cells can influence their neighbors through: Direct interactions (adhesion). Secretion of signaling molecules (morphogens). Cell Adhesion Mechanisms These notes were taken with Minutes AI (https://myminutes.ai)  Cadherin proteins mediate cell adhesion. Cadherins are calcium-dependent adhesion molecules. Cadherins interact to facilitate cell-to-cell connections. They span the cell membrane and interact with the cytoskeleton. Cadherins and Cell Adhesion (29:53 - 39:52) Cadherins link cytoskeletons of adjacent cells. Different cell types express different cadherins (e.g., epiderin in epithelial cells, Homophilic adhesion occurs between cells with the same type of cadherin. Cells with E-cadherin interact only with other E-cadherin expressing cells. Cells with N-cadherin interact only with other N-cadherin expressing cells. Epithelial vs. Mesenchymal Cells Epithelial cells are tightly adhered and work as a unit. Mesenchymal cells are loose and scattered. Mesenchymal cells do not adhere to one another. They lack cadherins. Experimental Observations Cells can be manipulated by altering pH. Increasing pH disrupts cadherin interactions, causing cells to become loose. Decreasing pH allows cells to re-aggregate based on cadherin expression. Morphogens and Their Gradients Morphogens are produced by one cell and influence neighboring cells. Concentration of morphogen affects cell specification. These notes were taken with Minutes AI (https://myminutes.ai)  The gradient of morphogen concentration leads to different cell types. High concentration results in one cell type (e.g., blue), lower concentration leads There are thresholds in morphogen concentration. Cells adopt specific types based on whether they are above or below these Experiment with Drosophila (39:52 - 49:44) Transplantation of leg cells to antenna region Cells taken from mid part of leg Transplanted to distal tip of antenna Possible outcomes of transplantation Could form mid part of leg Could form distal tip of antenna Actual result: became distal part of leg Key Findings Cells were specified to become a leg Not determined, but specified Responded to morphogen gradients Same morphogen affects both antenna and leg development Different responses due to varying concentrations Morphogen Identification Criteria Presence of the protein Must be at the right place and time Competence of affected cells Cells must respond to the morphogen These notes were taken with Minutes AI (https://myminutes.ai) Morphogen Functionality (49:44 - 59:38) Inhibition of the molecule results in no response. Response is dependent on concentration. Morphogens work as a gradient. Differentiation of cells is influenced by concentration. Zebrafish as a Model Organism First identified morphogen was Nodal. Zebrafish are tropical freshwater fish, easy to grow in aquariums. They are vertebrates with a vertebral column and specific tissue organization. Nodal Experiments Techniques to demonstrate Nodal presence: In situ hybridization for identifying mRNA. Immunostaining for protein detection. Nodal's role in embryonic development: Injecting nodal mRNA into embryos to observe responses. Nodal influences anterior-posterior determination, especially in the dorsal part. Gene Expression Analysis Genes analyzed in response to Nodal: Goosecoid: expressed in head-forming tissues. Floating head: establishes midline spinal part. No tail: forms the posterior part and associated muscles. Concentration effects on gene expression: High concentrations lead to goosecoid expression. These notes were taken with Minutes AI (https://myminutes.ai)  Intermediate concentrations lead to floating head expression. Low concentrations lead to no tail expression. Extracellular Protein and Cell Interaction (59:39 - 1:09:34) An extracellular protein influences cells by interacting with receptors. High concentrations of nodal lead to expression of goose coin. Lower concentrations still bind to normal receptors, causing cell signaling for Determinants and Morphogens Determinants can be transcription factors, but not all are. Morphogens are a class of determinants that are extracellular. Experimental Procedures In situ experiments are conducted for each gene. Messenger RNA is injected to observe gene expression. Multiple genes can be analyzed simultaneously. Review and Hypothesis Challenges A paper was rejected due to skepticism about the hypothesis. Reviewers doubted the effectiveness of nodal at low concentrations. Suggested that cell interactions might cause the observed effects. Experimental Validation Cells with mutated nodal receptors were used to test the hypothesis. No response to nodal was observed in mutated cells. Further experiments showed that normal cells could still respond to nodal. These notes were taken with Minutes AI (https://myminutes.ai)  Proper expression of floating head and no tail was observed despite some cells Additional Explanations and Controls Concerns about daughter cells migrating were addressed. Transplanted labeled cells showed expected responses to nodal. The importance of positive controls was discussed. Positive controls ensure experimental techniques are functioning correctly. Positive Control in Experiments (1:09:35 - 1:12:44) Importance of positive control Essential for validating negative results and ensuring proper technique. Use of controls in experiments LOLA was the first morphogen without a good positive control. Normal can be used as a positive control. Adding dye can confirm proper injection and cell viability. Specification and Determination Development commitments Types include lay well specification and stable determinant. Types of specification Autonomous specification due to cytoplasmic determinants and asymmetric cell Conditional specification influenced by inducing factors like morphogens. Timing in Embryonic Development Importance of timing Factors influencing cells must be considered at specific stages of specification. These notes were taken with Minutes AI (https://myminutes.ai)  Immature cells in embryos Embryos consist of immature cells that are not yet differentiated. Proper induction timing is crucial for development. These notes were taken with Minutes AI (https://myminutes.ai)