Lecture 12: Metamorphosis PDF

🥽 Lecture 12: Metamorphosis LO: Define metamorphosis in animals Metamorphosis: a rapid change in form from one developmental state to another It is a post-embryonic process where a larval or immature stage...

🥽 Lecture 12: Metamorphosis LO: Define metamorphosis in animals Metamorphosis: a rapid change in form from one developmental state to another It is a post-embryonic process where a larval or immature stage transforms into an adult stage. Involves dramatic tissue remodelling. In metamorphosis, larval tissues and organs are completely replaced by adult structures. Eg) amphibians (tadpole to frog), insects (butterfly metamorphosis from larval stage) Metamorphosis in amphibians Stages of amphibian metamorphosis: Lecture 12: Metamorphosis 1  Embryo: Fertilised egg contains an embryo, which after embryogenesis hatches into a tadpole. Tadpole: Lives in water, has external gills and developing mouth. Feeds on plant material. Later stages: Hind limbs develop first, followed by forelimbs. Frog: The tail resorbs and hind/forelimbs fully develop. The animal transitions from an aquatic lifestyle to a semi- terrestrial lifestyle, with partly aquatic adult frogs. Three types of extensive morphological change during metamorphosis: Apoptosis and Resorption of Larval Structures: Resorption of gills and tail that allow tadpoles to breathe and swim in water. Growth and Differentiation of Adult Structures: Development of limbs and lungs for terrestrial life. Tissue Remodelling of Larval to Adult Organs: Some organs are retained but undergo significant remodelling (e.g., intestine). Includes developmental maturation of certain enzymes like hemoglobin and eye pigments. Lecture 12: Metamorphosis 2  LO: Understand the hormonal control of amphibian metamorphosis Metamorphosis is regulated by both hormonal and environmental factors Sequence of Events Leading to Metamorphosis: (1) Environmental cues Temperature, light, and photo period act as environmental cues These cues signal the hypothalamus in the brain. (2) Hypothalamus response Hypothalamus releases corticotropin-releasing hormone (CRH). (3) Pituitary response CRH stimulates the pituitary gland to release: Prolactin (which tends to delay metamorphosis but isn't the focus here). Thyroid-stimulating hormone (TSH), which is the key hormone promoting metamorphosis. (4) Thyroid gland activation TSH travels through the bloodstream to the thyroid gland (located in the throat in humans). The thyroid gland releases thyroid hormones - T4 (thyroxine) and T3 (triiodothyronine), collectively referred to as T8. (5) Thyroid hormone action T3 and T4 initiate and promote metamorphosis. These hormones also provide feedback to the brain, encouraging the continued release of thyroid hormone, thereby sustaining the metamorphic process. Lecture 12: Metamorphosis 3  Thyroid hormones regulate metamorphosis 2 main types of thyroid hormones: T3 (triiodothyronine) and T4 (thyroxine) → they are tyrosine-based hormones, meaning they are derived from the amino acid tyrosine. T3 is the active form of the hormone, while T4 serves as a prohormone. In mammals and other animals, T4 is primarily produced and then converted to the active form, T3, in target tissues. The conversion of T4 to T3 is facilitated by an enzyme called diodinase, which removes one iodine group from T4. Both T3 and T4 are small, iodine-carrying molecules, and sufficient iodine is crucial for proper thyroid function. Iodine deficiency in humans can result in thyroid dysfunction and lead to conditions such as goiter (swollen thyroid gland). In amphibians, T3 and, to a lesser extent, T4 regulate the metamorphosis process. Lecture 12: Metamorphosis 4  The thyroid hormones trigger various changes in gene expression and cellular behavior, promoting the transformation from the larval to the adult stage. Confirming the role of thyroid hormones (1) Removal of Thyroid Hormone (Thyroidectomy): Excising the thyroid gland lead to metamorphosis being inhibited ⇒ results in the production of a giant tadpole that is unable to undergo metamorphosis into a frog. (2) Addition of Thyroid Hormone: Feeding tadpoles with thyroid extract induced precocious metamorphosis, meaning metamorphosis occurs earlier than it normally would. In axolotls, an amphibian that is naturally stuck in the larval stage (a condition called neoteny) The lack of thyroid-stimulating hormone (TSH) from the pituitary gland prevents axolotls transformation into a salamander. However, by administering thyroid hormone, these axolotls can be induced to transform into salamanders. Thyroid hormone and TH receptors throughout the stages of amphibian development: Context: Radioactive iodine uptake (RAIU) is used to track the activity of the thyroid gland, which relies on iodine to produce thyroid hormones (T3 and T4) (1) Pre-metamorphosis: This phase involves cell proliferation, brain development, and limb bud formation. Lecture 12: Metamorphosis 5  Thyroid hormone receptors are expressed early, but thyroid hormone levels are still very low. The thyroid gland is small, as indicated by low uptake of radioactive iodine. (2) Pro-metamorphosis: During this phase, limb buds start to differentiate and grow. There is a gradual rise in thyroid hormone levels, which triggers pro-metamorphic changes like the growth and differentiation of limbs. The thyroid gland becomes more active, with increasing radioactive iodine uptake. (3) Climax: The limbs are now fully developed, and major internal changes occur, such as gill resorption, organ remodeling, and body weight loss. Thyroid hormone levels peak during this phase, and the thyroid gland reaches its largest size, indicated by substantial radioactive iodine uptake. This peak is crucial for completing metamorphosis. (4) Post-metamorphosis: After the climax phase, thyroid hormone levels drop, leading to the final transformation into an adult frog. Different events triggered by different TH levels Lecture 12: Metamorphosis 6  Elevated levels of thyroid hormone (TH) trigger amphibian metamorphosis Thyroid hormone receptors (TR) are present in tadpoles (before T3) Thyroid size and function increases throughout metamorphic phases: ^ Thyroid follicles (pink stained pockets) also increase in numbers and size, correlating with increased T4 and T3 (TH) production Thyroid Hormones Receptors (TR) and Thyroid Hormone (TH) Mechanism of Action Lecture 12: Metamorphosis 7  Thyroid hormone receptors (TRs) are DNA-binding transcription factors TRs typically form a heterodimer complex with retinoic acid X receptors at specific thyroid hormone-responsive elements in DNA. In the absence of thyroid hormone, TRs form a complex with co- repressors that inhibit gene expression In the presence of thyroid hormone (T3 & T4; T4 is converted to T3 by a diodinase enzyme in tissues), TH binds to TR and dislodges the co- repressor, T3 recruits co-activators, transforming the complex from a transcriptional repressor to a transcriptional activator Recruited coactivators induces histone acetylation → leading to transcription Thyroid Hormone (TH) Action in Metamorphosis Lecture 12: Metamorphosis 8  In pre-metamorphosis, there is → low levels of TSH from pituitary, low TH, low TRs TR binds with retinoid receptors and co-repressors, leading to repression of thyroid receptor gene activation. Consequently, low levels of TR are produced. In pro-metamorphosis, there is → environmental input triggers increase in TSH, increased TH & TR production TH binds to TR, dislodges co-repressors, and recruits co-activators. This promotes increased transcription of the TR gene, leading to more TR production. The increased TR binds with TH, perpetuating a positive feedback loop. In climax, there is → high TSH from pituitary, high levels of TH and TR High levels of TH and TR lead to further activation of more genes. Co-activators are recruited, stimulating the transcription of genes required for completing metamorphosis. Dominant Negative TR Blocks Metamorphosis Dominant Negative Thyroid Receptor (DNTR): DNTR binds to DNA but cannot activate it; thus, it cannot respond to thyroid hormone → thus, Lecture 12: Metamorphosis 9  blocking metamorphosis Experiment: Wild Type Tadpoles: Given thyroid hormone (T3), they regress their tails and produce collagenase, which breaks down the tail, facilitating metamorphosis. Dominant Negative Transgenic Tadpoles: Even when given T3 or thyroxin, they cannot undergo metamorphosis due to the dominant negative mutation in the receptor. They grow larger but do not regress their tails, and collagenase production is absent. LO: Describe the role of thyroid hormone in regulating re- modeling of the intestine during metamorphosis in the frog, Xenopus laevis Thyroid hormone (TH) induces intestinal re-modeling through direct and indirect effects upon intestinal epithelial cells and induces: (1) Epithelial cells apoptosis Direct Effect: T3 (active thyroid hormone) induces apoptosis directly in some epithelial cells. Lecture 12: Metamorphosis 10  Indirect Effect: T3 acts on fibroblasts in the underlying connective tissue, which then send signals to the epithelial cells to undergo apoptosis. (2) De-differentiation to form adult stem cells Direct Effect: T3 acts directly on select epithelial cells to induce de- differentiation into adult stem cells. Indirect Effect: T3 affects fibroblasts in the connective tissue, which then signal specific epithelial cells to de-differentiate into stem cells. Mechanism of Action: T3 directly affects both fibroblasts in the connective tissue and larval epithelial cells. Direct Effects: T3 induces apoptosis in some epithelial cells and de- differentiates others into stem cells. Indirect Effects: T3 acts on fibroblasts which, in turn, influence the epithelial cells to undergo apoptosis or de-differentiation. Lecture 12: Metamorphosis 11  LO: Understand the cell autonomous actions of thyroid hormone during metamorphosis, and how the same hormone has different effects in different tissues Regional Specificity: Different body regions respond to varying amounts of thyroid hormone (TH) and thyroid hormone receptors (TR) at different times. The type of response depends on intrinsic factors within the cells themselves. The same TH stimulus can result in different outcomes. Type of response: Apoptosis: Cell death in specific regions (e.g., tail regression). Proliferation: Cell growth in other regions (e.g., muscle development in legs). Differentiation: Development of adult structures in certain areas Organ-Specific Transplants Tail Transplants: If a tail from one tadpole is transplanted to another, it still undergoes normal regression. This indicates that the fate of the tail is determined by its own intrinsic response, not its new location. Optic Cup Example: When an optic cup (eye) from one tadpole is transplanted into the tail region of another, the optic cup remains unaffected by the normal tail regression, highlighting that its response is independent of the surrounding tissue changes. Lecture 12: Metamorphosis 12  TH Controls Limb Development During Metamorphosis TH controls limb development early in Xenopus metamorphosis - and so - the use of methimazole (a TH antagonist that binds to but does not activate the receptor) blocks both metamorphosis and limb development. Research Questions: Does TH control a single cell type that then influences other cell types indirectly, or does it act on all cell types simultaneously? Experiment: using transgenic tadpoles carrying cell-type specific Dominant Negative Thyroid Receptor (DNTR) to blocks TH effects in nerve vs cartilage vs muscle cell precursors Because DNTR is nuclear-located, effects must be cell- autonomous - ie. DNTR affects gene expression directly within Lecture 12: Metamorphosis 13  the cell where it is expressed, it will only disrupt thyroid hormone signaling in that specific cell and does not impact or require communication with neighboring cells. Results: Differentiation of each of the major limb cell types (eg. nerve, cartilage, muscle) is under cell autonomous control of TH Nerve-Specific Dominant Negative: When DNTR is driven specifically in nerves using Neural-β-tubulin, the tadpole shows normal limbs but has disrupted nerves, leading to paralysis. Muscle-Specific Dominant Negative: When DNTR is driven specifically in muscles using a cardiac actin promoter, the limbs develop normally, but the muscles are absent, resulting in paralysis. TH has direct effects on each major cell type involved in metamorphosis: Nerves: Modulates DNA synthesis and cell cycle regulators specific to nerve cells. Muscles: Directly affects muscle cell development. Cartilage: Activates cell-type-specific genes like SOX9, which is involved in cartilage development. LO: Understand the role of programmed cell death during metamorphosis During metamorphosis, the thin skin of tadpoles is replaced by thicker, water-resistant adult skin. This process is regulated by thyroid hormone Lecture 12: Metamorphosis 14  (TH). Larval skin has thin epidermis & expresses larval keratin (mRNA for larval keratin). Adult skin has thick epidermis & expresses adult keratin. The thin larval skin undergoes apoptosis and replaced by basal cells of the epidermis, which develop into the adult skin → TH drives the differentiation and thickening of the skin. Experimental Approach: Keratin promoter drives dominant negative TR (DNTR) in larval epidermis, making it non-responsive to TH Results: Control Tadpoles: Normal metamorphosis: transition from thin epidermis to thick adult skin. Keratin changes from larval to adult type. Increased apoptosis in the larval epidermis (marked by active caspase 3). Underlying collagen and fibroblasts continue to remodel the skin. DNTR Tadpoles: Lecture 12: Metamorphosis 15  Fail to undergo normal apoptosis in the epidermis. Larval epidermal marker (gene 19) is retained. Underlying collagenase still breaks down collagen, but epidermal layer does not transition properly. Less pigmentation change (melanin). Conclusions: The experiments demonstrate the cell-autonomous action of TH on the epidermis TH directly affects epidermal cells, driving their transformation and apoptosis. Underlying connective tissue can still remodel, but the epidermal layer remains unchanged if TH action is blocked. LO: Describe gut re-modelling during metamorphosis (incl. genes involved) Intestinal Re-modelling During Xenopus Metamorphosis (Aquatic Herbivore → Semi-Terrestrial Omnivore) Pro-Metamorphosis Stage: the larval or tadpole intestine is convoluted and coiled, simple tube structure, there’s a single fold Lecture 12: Metamorphosis 16  Climax Stage: during this stage, significant apoptosis of the larval epithelium occurs, new stem cells emerge in the epithelial layer, shorter epithelium compared to the larval stage End of metamorphosis - adult intestine: adult intestine is shorter & not coiled, many epithelial folds, thicker connective tissue and muscle layer s The adult intestinal epithelium resembles a more mammalian-like epithelium, with increased folds and stem cells in the troughs between Xenopus system is an ideal model for human intestinal development Mammalian intestinal development also involves thyroid hormones, TH deficiency leads to impair intestinal development. Xenopus Advantage: Develops outside the maternal body - so Xenopus intestine development can be studied in vitro, using T3 - allowing easier manipulation and study of TH effects. Tissue-tissue Interactions and Stem Cell Specification Cell-cell interactions involving hedgehog (Shh) and BMP signalling - showing that the stem cell niche is essential for formation of adult intestinal epithelium TH Effects: Acts on both epithelial cells and connective tissue cells to form adult stem cells. Lecture 12: Metamorphosis 17  TH activate Sonic Hedgehod (Shh) pathway in metamorphosing Xenopus intestine, expression of Shh and its receptors peak during climax of metamorphosis Shh regulate BMP4 signalling, upregulating BMP4 in the connective tissue Shh and BMP4 acts on stem cells to express markers like LGR5 → this interaction creates a stem cell niche, leading to the de-differentiation of some epithelial cells into adult stem cells and the subsequent differentiation of these stem cells into the adult intestinal epithelium. LO: Understand the concept of gene switching during metamorphosis During metamorphosis, cells in various tissues (e.g., liver, tail, skin) shut off larval genes and turn on adult genes in the same cell - undergoing gene switching Gene switching is defined as the reversible on/off switching of gene expression Example: Fibroblasts in the Tail Larval Stage → Fibroblasts produce collagen, contributing to the development of the notochord (the rod-like structure along the backbone). Lecture 12: Metamorphosis 18  Metamorphosis → TH causes fibroblasts to switch from producing collagen to producing collagenase & collagenase breaks down collagen, leading to the breakdown of the notochord and tail. Experimental Evidence: In Situ Hybridization: Control Animal: Collagen is expressed (purple staining), and collagenase is minimally expressed. Shows normal notochord structure with collagen present. After 2 Days of TH Treatment: Collagen remains present, but collagenase expression increases. Collagenase begins degrading the tail. After 4 Days of TH Treatment: Collagen gene expression is switched off (no collagen present). Collagenase expression remains high, leading to continued tail degradation. Gene switching mechanism In the presence of TH, fibroblasts switch gene expression from collagen to collagenase → this shift results in the breakdown of larval structures and contributes to metamorphosis. Lecture 12: Metamorphosis 19 Lecture 12: Metamorphosis 20