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Similar to Dima Chaar Senior Thesis presentation
Similar to Dima Chaar Senior Thesis presentation (20)
Dima Chaar Senior Thesis presentation
- 1. Genetic Fate Mapping of Hoxa5-Expressing Cells in Mouse Embryo Somites Dima Chaar Lab Mentor: Professor Mansfield Barnard College, Columbia University Professor Romeo
- 2. What are Hox genes? • Hox genes encode Transcription Factors • Transcription factors are proteins that bind and control the activity of other genes • The combination of Hox genes expressed in embryonic musculoskeletal and neural tissues specifies regional identity of those tissues along head-to-tail axis • Hox mutations lead to homeotic transformations
- 3. Role of Hox genes in Development • During development, • Most of the musculoskeletal system arises from somites • The brain and spinal cord arise from the neural tube • Somites compartmentalize and their derivatives differentiate • Hox genes are expressed in the somites and neural tube, where they regulate regional identity Dermomyotomeà • Dermis • Muscle • Muscle Connective Tissue • Adipocytes Sclerotomeà • Chondrocytes (cartilage) • Perichondrium • Ligaments • Tendons Neural Tubeà • Neurons
- 4. My focus: Hoxa5 • Hoxa5 expression occurs in the cervical-to-thoracic transition in somites • Hoxa5 is required for regionalization of the musculoskeletal and nervous systems in the same area • I will be focusing on Hoxa5’s role in somite differentiation
- 5. • We know: • Where Hoxa5 is expressed • What roles Hoxa5 performs in vertebral development from studies of loss-of-function mutations • We don’t know: • How Hoxa5 plays its crucial role in vertebral development • Which tissues are derived from Hoxa5-expressing cells and when is Hoxa5 expression necessary? • Transcriptional targets
- 6. • My research focused on determining the cell fates of Hoxa5-expressing cells and their descendants at different time points •Research Questions 1. Which tissue types do Hoxa5 descendants become? 2. Is there a particular spatial distribution of Hoxa5 descendants within a tissue? 3. How do these Hoxa5-expressing cells behave differently than others?
- 11. Experiment 1: Generate embryos in which the Hoxa5 - expressing cells and their descendants are labelled with Tdtomato Inject Tamoxifen at specific dose (9, 12 or 15 mg/40mg) Harvest and embed embryos in OCT Section with cryostat Immuno- histochemistry Microscopy and Photography At e8.5 At e14.5 Choose Tamoxifen dose with • Adequate Tdtomato labeling • Embryos survive
- 12. Generating embryos in which the Hoxa5-expressing cells and their descendants are labelled with Tdtomato X Hoxa-5 Cre/+ Male Tdtomato /Tdtomato Female Hoxa-5 Cre/Tdtomato embryo
- 18. Experiment 2: Fate Map Hoxa5 descendants in somites from e9.5, e12.5 and e13.5 Generate embryos in which the Hoxa5 - expressing cells and their descendants are labelled with Tdtomato Inject optimal dose of 9mg/40g of body weight Harvesting and OCT embedding of embryos Sectioning with cryostat Immuno- histochemistry Microscopy and Photography At e9.5, e12.5 & e13.5 At e14.5 or e15.5
- 19. Method: Timeline Embryonic Stages (days) e7.0 e8.5 e9.5 e10.5 e11.5 e12.5 e13.5 e14.5 e15.5 Hoxa5 expression begins Tamoxifen Injection Hoxa5- expressing cells labelled with Cre ERT2 Tamoxifen Injection Tamoxifen Injection Harvest HarvestHarvest Hoxa5 Endogenous Expression
- 22. e9.5 e12.5 e13.5 Muscle Actin A5 descendants Muscle Actin A5 descendants Muscle Actin A5 descendants
- 23. e9.5 e12.5 e13.5 Muscle Actin A5 descendants Muscle Actin A5 descendants Muscle Actin A5 descendants
- 24. Summary: Hoxa5-expressing cells show tissue-type restriction • Transgenic Cre line and the Cre-ERT2 line revealed tissue-specific restriction of cells that express Hoxa5 • My Cre-ERT2 results suggest that Hoxa5 expressing cells give rise to chondrocytes in a time-dependent manner Dermomyotomeà • Dermis • Muscle • Muscle Connective Tissue • Adipocytes Sclerotomeà • Chondrocytes • Perichondrium • Ligaments • Tendons ✓ ✓ ✓ ✓ ✓= Hoxa5 descendants contribute to these tissues Neural Tubeà • Neurons ✓
- 25. Tissue Types e9.5 e12.5 e13.5 Chondrocytes No Yes Yes Perichondria Yes Yes Yes Muscle Fibroblasts Yes Yes Yes Dermis Yes Yes Yes Adipocytes Yes Yes Yes Neurons Yes Yes Yes Tamoxifen Injection Time Points • Hoxa5-expressing cells gave rise to dermis, perichondria and adipocytes at e9.5-13.5
- 26. Tissue Types e9.5 e12.5 e13.5 Chondrocytes No Yes Yes Perichondria Yes Yes Yes Muscle Fibroblasts Yes Yes Yes Dermis Yes Yes Yes Adipocytes Yes Yes Yes Neurons Yes Yes Yes Tamoxifen Injection Time Points • The absence of descendants in the vertebral body at e9.5 suggests that chondrocytes do not express Hoxa5 until after e10.5.
- 29. Implications • Studying the role of Hoxa5 is crucial to understanding • Complexities behind embryological development • How early mutations could lead to physical abnormalities and health defects • With this information, future doctors could potentially treat or prevent physical abnormalities using gene therapy
- 30. • National Science Foundation • Howard Hughes Science Pipeline Project • My Lab mentor, Professor Mansfield • My thesis mentor, Professor Romeo • Fellow N&B majors • Past and Present Students of the Mansfield Lab • Biology & Neuroscience Depts. • Friends and Family! J