Morphogenesis Differentiation – 3 Cell-cell communication Lecture 11 Morphogenesis Differentiation – 3 Cell-cell communication

Hedgehog signaling pathway Expression of Patched is a readout of active hedgehog signaling

Hedgehog signaling and cyclopia Cyclopamine is an effective inhibitor of Hh signaling Ventral neural tube patterning and motor neuron specification Sclerotome differentiation Limb patterning Etc.

Wnt signaling pathway Non-canonical Wnt Signaling pathway Wnt and Ca++Signaling pathway Canonical Wnt Signaling pathway Beta-catenin immunoreactivity and TOP-FLASH, TOP-GAL etc. are used as readouts of canonical Wnt signaling

Wnt4 is necessary for kidney development and for female sex determination. (A) Wnt4 mRNA expression is a 14-day mouse embryonic male urogenital rudiment. Expression is seen in the mesenchyme that condenses to form the kidney’s nephrons. (B) Urogenital rudiment of a wild-type newborn female mouse. (C) Urogenital rudiment of a newborn female mouse with targeted KO of Wnt4 shows that the kidney fails to develop. In addition, the ovary starts synthesizing testosterone and becomes surrounded by a modified male duct system.

TGF β superfamily

TGF β signaling pathway P-Smad2,3 is used as readout of TGF beta signaling while P-Smad1,5 as well as BRE-Reporter constructs are used as readout of BMP signaling

Context dependent function of BMP ligands Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, et al. (2006) Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis. PLoS Genet 2(12): e216. doi:10.1371/journal.pgen.0020216 http://127.0.0.1:8081/plosgenetics/article?id=info:doi/10.1371/journal.pgen.0020216

Apotosis During embryonic development we generate three times the number of neurons than what we have at birth!!! By the time I was born, more of me had died than survived. It was no wonder I cannot remember; during that time I went through brain after brain for nine months, finally contriving the one model that could be human, equipped for language. Lewis Thomas, 1992

Figure 1: The principle of TUNEL assay relies on terminal deoxynucleotidyl transferase (TdT)-mediated addition of a modified dUTP (X-dUTP) to 3’-OH ends of DNA fragments that are generated as a result of apoptosis induction. To avoid the loss of fragmented DNA and to allow enzyme and nucleotide entrance, cells need to be fixed and subsequently permeabilized prior to the labeling reaction. Incorporated bromoylated dUTP (BrdUTP) is detected by specific antibody conjugates with a reporter enzyme or fluorescent dye (A). The incorporation of alkyne-containing dUTP (EdUTP) is visualized by Cu(I)-catalyzed alkyne-azide click chemistry (CuAAC) with an azide containing fluorphore (B). http://www.jenabioscience.com/cms/en/1/catalog/2205_apoptosis_tunel_assay.html

Nurse cell death and transfer of material blocked in dcp-1-deficient mutants. (A, B) Control egg chambers, at stage 10A and 14, showing the death of the nurse cells and the transfer of their cytoplasmic contents (here visualized by the staining of b-galactosidase) into the oocyte. (C, D) Similar stages of a dcp-1-deficient mutant showing neither cell death nor the transfer of material into the oocyte. (From McCall and Steller, 1998. Reprinted with permission from Science and the American Association for the Advancement of Science.) McCall, K. and Steller, H. 1998. Requirement for DCP-1 caspase during Drosophila oogenesis. Science 279: 230-234

Surface dwelling (eyed) and cave-dwelling (eyeless) forms of Astyanax mexicanus. Adjacent to them are sections of the embryonic eyes, stained with a reagent (TUNEL) that binds to and stains DNA fragments produced by apoptosis. A1. Surface form with eyes and pigmentation. A2. 25 hour embryo of surface form, showing very little apoptosis in the lens. B1. Cavefish from La Cueva Chica with reduced eyes and reduced pigmentation. B2. 25 hour embryo of the Chica cave-dwelling form, showing severe apoptosis in the lens vesicle (arrowhead). C1. Cavefish from Cueva de El Pachón showing neither eye nor pigmentation. C2. Apoptosis in lens and corneal epithelium in the 25 hour embryo of the Cueva de El Pachón cavefish. (After Jeffery and Martasian, 1998.)

Notch signaling Mechanism of Notch activity. Model for the activation of Notch. 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 activate a transcription factor of the CSL family (such as Suppressor of hairless or CBF1). The activated CSL can then transcribe its target genes. Immuno reactivity to Notch Intra-Cellular Domain (NICD) is used as a readout of Notch signaling

Lateral Specification and lateral inhibition

The Molecular Basis of Neuronal Generation Is Similar Throughout Phylogeny Clues to the mechanisms controlling the differentiation of a dividing progenitor cell into a postmitotic neuron have emerged from molecular genetic studies of neurogenesis in Drosophila melanogaster. The selection of a single neuron from a large and initially uniform population of ectodermal cells in Drosophila involves a program of cell interactions that gradually restrict the fate of a cell. The initial step in this program is the recruitment of a small cluster of ectodermal cells that acquire the potential to give rise to neuronal precursors This region of the ectoderm is known as the proneural region.

Neuronal fate is decided by a process of signaling between adjacent cells in the proneural region. This process depends on the interactions between two cell-surface proteins encoded by the neurogenic genes delta and notch. Both proteins span the cell membrane: delta functions as a ligand, and notch is its receptor. Notch signaling between cells is initially similar in the proneural region of the ectoderm that express the achaete-scute class basic helix-loop-helix genes (brown). The activation of notch by delta initiates a local feedback signal between adjacent cells whose purpose is to ensure that even a minor difference in the initial level of notch activity is amplified rapidly to generate a large discrepancy in the state of notch activation and consequently in cell fate. Cells in which notch is activated to high levels are inhibited from acquiring a neuronal fate, whereas cells with a relatively low level of notch signaling become neurons.

The activation of notch by delta initiates a local feedback signal between adjacent cells whose purpose is to ensure that even a minor difference in the initial level of notch activity is amplified rapidly to generate a large discrepancy in the state of notch activation and consequently in cell fate. Cells in which notch is activated to high levels are inhibited from acquiring a neuronal fate, whereas cells with a relatively low level of notch signaling become neurons.

Activation of notch in one cell (cell A) initiates an intracellular signaling cascade that involves proteolytic cleavage of the cytoplasmic domain of notch, the translocation of this domain into the nucleus, and the activation of a transcription factor, called suppressor of hairless. Suppressor of hairless is one of a large group of transcription factors of the basic helix-loop-helix class that are encoded by proneural genes and play critical roles in neurogenesis. Suppressor of hairless activates the expression of inhibitory basic helix-loop-helix proteins of the enhancer of split class, which represses expression of still other helix-loop-helix genes, achaete-scute genes. Finally the activation of achaete-scute proteins leads to a decrease in expression of delta on the cell surface.