Lecture 8 BIO3147 (2024) PDF

Figure 4.25 Sonic Hedgehog Head of a cyclopic lamb born of a ewe that ate Veratrum californicum early in pregnancy, caused by ingestion of the alkaloid cyclopamine. In some parts of Idaho, up to 25% of newborn lambs were affected at some ranches during the end of Summer Figure 4.25 Sonic Hedgehog Ce...

Figure 4.25 Sonic Hedgehog Head of a cyclopic lamb born of a ewe that ate Veratrum californicum early in pregnancy, caused by ingestion of the alkaloid cyclopamine. In some parts of Idaho, up to 25% of newborn lambs were affected at some ranches during the end of Summer Figure 4.25 Sonic Hedgehog Cell, Vol. 75, 1401-1416, December 31, 1993, Copyright 0 1993 by Cell Press Sonic hedgehog Mediates the Polarizing Activity of the ZPA Robert D. Riddle, Randy L. Johnson, and Cliff Tabin Department of Genetics Harvard Medical School Boston, Massachusetts 02115 Ed Laufer, Summary (A) Sonic hedgehog is shown by in situ hybridization to be expressed in the nervous system (red arrow), gut (blue arrow), and limb bud (black arrow) of a 3-day chick embryo The zone of polarizing activity (ZPA) is a region at the posterior margin of the limb bud that induces mirrorimage duplications when grafted to the anterior of a second limb. We have isolated a vertebrate gene, Sonic hedgehog, related to the Drosophila segment polarity gene hedgehog, which is expressed specifically in the ZPA and in other regions of the embryo, that is capable of polarizing limbs in grafting experiments. Retinoic acid, which can convert anterior limb bud tissue into tissue with polarizing activity, concomitantly induces Sonic hedgehog expression in the anterior limb bud. Implanting cells that express Sonic hedgehog into anterior limb buds is sufficient to cause ZPA-like limb duplications. Like the ZPA, Sonic hedgehog expres- acid is unlikely for ZPA activity ( One of the strong date ZPA morpho retinoic acid, at a tions, induces a gene (the retinoi than that normal 1991). This impli than is required retinoic acid is p lieved that rathe signal, retinoic ZPA. The anterio implant and direc strated to acqui that tissue to an 1983; Wanek e to a ZPA graft d Exogenous retin stream of the ZP One approach fying new signali Figure 4.25 Sonic Hedgehog Sonic hedgehog mutations induce cyclopia (Nature; 1996) Ov=optic vesicle; op= olfactory placode Figure 4.25 Sonic Hedgehog Alkaloids (Cyclopamine/Jervine) from the Veratrum plant inhibits Shh signaling! (Science; 1998) Opt=optic vesicle; olf= olfactory placode; Mx=maxillary; Mn=mandiubluar Figure 4.26 Wnt4 is necessary for kidney development and for female sex determination Wnt4 is necessary for kidney development and for female sex determination. (A) Urogenital rudiment of a wild-type newborn female mouse. (B) Urogenital rudiment of a newborn female mouse with targeted knockout of Wnt4. Kidney fails to develop. In addition, the ovary starts synthesizing testosterone and becomes surrounded by a modified male duct system. Figure 4.27 Notum antagonism of Wnt Lipidation and HSPG (glypican) is also essential for Wnt signaling. Notum creates a negative feedback mechanism for Wnt signaling. Figure 4.28 Wnt signal transduction pathways *Phosphorylation & Ubiquitination are critical for the regulation of Wnt signaling Figure 4.29 Relationships among members of the TGF-β superfamily Figure 4.30 The Smad pathway is activated by TGF-β superfamily ligands Different TGF-beta ligands lead to phosphorylation of different Smads to regulate transcription Figure 4.33 Wnt diffusion is affected by other proteins (12th edition) (A) Diffusion of Wingless (Wg, a Wnt paracrine factor) throughout the developing wing of wildtype Drosophila (above) is enhanced by Swim, a protein that stabilizes Wg and that is made by some of the wing cells. When Swim is not present, as in the mutant below, Wg does not disperse and is confined to the narrow band of Wgexpressing cells. (B) Similarly, Wingless usually activates the Distal-less gene (green) in much of the wild-type wing (above). However, in swim-mutant flies (below), the range of Distal-less expression is confined to those areas near the band of Wg-expressing cells. Figure 4.34 The Fgf8 gradient (A) Image is of a resulting zebrafish gastrula, showing Fgf8 protein being produced by and secreted away from isolated labeled cells (green). On the right is a schematic representation of select cells and the Fgf8 expression. Fgf8 is seen in a gradient in the extracellular matrix as well as being internalized in receiving cells. (B) Quantification of Fgf8 protein at different locations in (A), indicated by “X” marks in the schematic. Manipulation of endocytosis affecting the internalization of Fgf8 by receiving cells causes predictable changes in the range of Fgf8 secretion. Inhibition of endocytosis with the dominant negative GTPase dynamin causes a shallower Fgf8 gradient over a longer distance (green plot) (LOF, loss of function), whereas increased endocytosis with the overexpression of the endosomal protein Rab5c (GOF, gain of function) yields a steeper and shorter Fgf8 gradient (blue plot). Figure 4.34 The Fgf8 gradient (C) Five primary mechanisms for shaping the Fgf8 gradient. (1) The difference in the rate of fgf8 transcription and fgf8 mRNA decay can influence the amount of Fgf8 protein ultimately secreted from a producing cell. Once secreted, Fgf8 can (2) freely diffuse or (3) travel rapidly along HSPG fibers for directed diffusion. Figure 4.34 The Fgf8 gradient (4) In contrast, however, dense areas of HSPGs can also confine and restrict Fgf8 diffusion. (5) The Fgf8-FGFR complex can also be internalized by endocytosis and targeted for lysosomal degradation. Together these different mechanisms result in the displayed gradient of Fgf8, and differential responses in cells that experience different concentrations of Fgf8 signaling (different colored nuclei). Figure 4.35 Filopodia-transported morphogens (Part 1) (A) Cytonemes from the air sac primordium (ASP) extend toward the epithelium of the wing imaginal disc in Drosophila to shuttle the FGF (green) and Dpp (red) morphogens, produced by the wing disc, back to the cell bodies of the ASP. Figure 4.35 Filopodia-transported morphogens (Part 4) (D) Illustration of the Drosophila wing imaginal disc during its interactions with tracheal cells, namely the ASP. Hh-, Dpp-, and FGF-expressing cells are represented as blue, red, and green domains. (E) Magnified cross section of the boxed region in (D). Figure 4.35 Filopodia-transported morphogens (Part 6) In the chick limb bud, long, thin filopodial protrusions have been documented extending both from Sonic hedgehog-producing cells in the posterior region (purple cell with green Shh protein in left image) and from the target cells in the anterior limb bud (red cells). These opposing filopodia directly interact (brackets, left image), and at this point of interaction it is proposed that Shh and its receptor Patch can bind (right illustration). Figure 4.37 Mechanism of Notch activity (A) Prior to Notch signaling, a CSL “CBF1, Suppressor of Hairless, Lag-1) transcription factor is on the enhancer of Notch-regulated genes. The CSL binds repressors of transcription. Figure 4.37 Mechanism of Notch activity (B) A ligand (Delta, Jagged, or Serrate protein) on one cell binds to the extracellular domain of the Notch protein on an adjacent cell. This binding causes a shape change in the intracellular domain of Notch, which activates a protease. The protease cleaves Notch and allows the intracellular region of the Notch protein to enter the nucleus and bind the CSL transcription factor. This intracellular region of Notch displaces the repressor proteins and binds activators of transcription, including the histone acetyltransferase p300. The activated CSL can then transcribe its target genes. Figure 4.38 C. elegans vulval precursor cells (VPCs) and their descendants lin-12=Notch; lin-3=EGF (D) Model for the determination of vulval cell lineages in C. elegans. The LIN-3 signal from the anchor cell causes the determination of the P6.p cell to generate the central vulval lineage (dark purple). Lower concentrations of LIN-3 cause the P5.p and P7.p cells to form the lateral vulval lineages. The P6.p (central lineage) cell also secretes a short-range juxtacrine signal that induces the neighboring cells to activate the LIN-12 (Notch) protein. This signal prevents the P5.p and P7.p cells from generating the primary central vulval cell lineage Figure 5.1 The stem cell concept (A) The fundamental notion of a stem cell is that it can make more stem cells while also producing cells committed to undergoing differentiation. This process is called asymmetric cell division. (B) A population of stem cells can also be maintained through population asymmetry. Here a stem cell is shown to have the ability to divide symmetrically to produce either two stem cells (thus increasing the stem cell pool by one) or two committed cells (thus decreasing the pool by one). This is called symmetrical renewing or symmetrical differentiating. (C) In many organs, stem cell lineages pass from a multipotent stem cell (capable of forming numerous types of cells) to a committed stem cell that makes one or very few types of cells to a progenitor cell that can proliferate for multiple rounds of divisions but is transient in its life and is committed to becoming a particular type of differentiated cell. Figure 5.2 An example of the maturational series of stem cells: differentiation of neurons Figure 5.3 Blood-forming (hematopoietic) stem cells (HSCs) These multipotent stem cells generate blood cells throughout an individual’s life. HSCs from human bone marrow (photo) can divide to produce more HSCs. Alternatively, HSC daughter cells are capable of becoming either lymphoid progenitor cells (which divide to form the cells of the adaptive immune system) or myeloid progenitor cells (which become the other blood cell precursors). The lineage path each cell takes is regulated by the HSC’s microenvironment, or niche Figure 5.4 To divide or not to divide: an overview of stem cell regulatory mechanisms Shown here are some of the more general external and internal molecular mechanisms that can influence the quiescent, proliferative, or differentiative behaviors of a stem cell Figure 5.8 Establishment of the inner cell mass (the ICM, which will become the embryo) in the mouse blastocyst From morula to blastocyst, the three principal cell types— trophectoderm, epiblast, and primitive endoderm Figure 5.9 Divisions about the apicobasal axis Depending on the axis of cell division in the trophectoderm, the trophectoderm layer can be expanded (left), or the inner cell mass (ICM) can be seeded (right) Figure 5.16 Young blood can rejuvenate an old mouse (A) Parabiosis—fusion of the circulatory systems of two individuals—was done using mice of similar (isochronic) or different (heterochronic) ages. When an old mouse was parabiosed to a young mouse, the result was an increase in the amount of vasculature (stained green in the photographs) as well as the amount of proliferative neural progeny in the old mouse. Progenitors that are SOX2+ cell are labelled red in photographs and quantified in graph). (B) Administering GDF11 (typically higher in young mice) into the circulatory system of an old mouse was sufficient to similarly increase both vasculature (green in photographs) and the population of neural progenitors. Figure 5.15 The ISC niche and its regulators (12th Edition) (A) The intestinal epithelium is composed of long, fingerlike villi that project into the lumen, and at the base of the villi, the epithelium extends into deep pits called crypts. The ISC and progenitors reside at the very bottom of the crypts (red), and cell death through anoikis occurs at the apex of the villi. Figure 5.15 The Intestinal Stem Cell (ISC) niche and its regulators (B) Along the proximodistal axis (crypt to villus), the crypt epithelium can be functionally divided into three regions: the base of the crypt houses ISC, the proliferative zone is made of transit amplifying cells, and the differentiation zone characterizes the maturation of epithelial cell types. Pericryptal stromal cells surround the basal surface of the crypt and secrete opposing morphogenic gradients of Wnt2b and Bmp4, which regulate stemness and differentiation, respectively. Figure 5.23 Major sources of pluripotent stem cells from the early embryo Embryonic stem cells (ESCs) arise from culturing the inner cell mass of the early embryo. Embryonic germ cells are derived from primordial germ cells that have not yet reached the gonads. Figure 5.24 Inducing stem cell differentiation from ESCs ESCs in culture can be coaxed with the same developmental factors (paracrine and transcription factors, among others) to differentiate into the cell types of each germ layer. With the inhibition of several growth factors, ESCs can make ectoderm lineages; for mesoderm or endoderm lineages, however, ESCs are first induced to become primitive streak-like cells (PS) with paracrine factors such as Wnt, Bmp4, or activin, depending on the desired differentiated cell type Figure 5.24 Protocol for curing a “human” disease in a mouse using iPS cells plus recombinant genetics (1) Tail-tip fibroblasts are taken from a mouse whose genome contains the human alleles for sickle-cell anemia (HbS) and no mouse genes for this protein. (2) The cells are cultured and infected with viruses containing the four transcription factors known to induce pluripotency. (3) The iPS cells are identified by their distinctive shapes and are given DNA containing the wild-type allele of human globin (HbA). (4) The embryos are allowed to differentiate in culture. They form “embryoid bodies” that contain blood-forming stem cells. (5) Hematopoietic progenitor and stem cells from these embryoid bodies are injected into the original mouse, which has been irradiated to clear out its original hematopoietic cells. This treatment cures its sickle-cell anemia. Figure 5.25 Organoid derivation (12th Edition) – to be continued after midterm 1 (A) Schematic represents the various strategies used to promote the morphogenesis of specific tissue-type organoids. In most cases, a threedimensional matrix (Matrigel) is used.