1!

Karina S. Cramer!10/1/13!

Developmental Neurobiology!N152!

Review Notes!•  Focus of course: How does the nervous system develop?!

•  Strategies for studying neural development!

•  Selection of model organisms!•  Experimental tools!•  Experimental design: observation and perturbation; gain of function and loss of function!

•  Diverse species share some molecular mechanisms during development.!

2!

Figure 22-7 Molecular Biology of the Cell (© Garland Science 2008)

Tissue transplants to test cell fate determination!

Figure 22-8 Molecular Biology of the Cell (© Garland Science 2008)

The thigh tissue has already been programmed to become leg-associated tissue, but has not yet been specified to become thigh vs. toes.!

Determination of structure is progressive!

3!

Cre Recombinase – Lox P Transgenics!

(Imayoshi et al., Nature Neuroscience, 2008)!

Allows for genetic control of limited populations of cells, determined by the promoter that drives expression of cre.!

Cre-Lox technique used to ablate new-born neurons!

Nestin: Expressed in neural progenitors!!CreER: Modified Cre recombinase that requires activation of estrogen receptor through administration of tamoxifen; allows temporal control!!NSE: Neural specific enolase, a gene expressed after differentiation!!DT-A: Encodes for diphtheria toxin receptor fragment A. Cells that express it are killed after administration of diphtheria toxin.!

4!

Only new-born neurons are killed in vivo!

(Imayoshi et al., Nature Neuroscience, 2008)!

Neural induction!

•  Overview of early embryogenesis!

•  Neural induction; similarity across animals!

•  Cellular and molecular interactions induce neural tissue!

•  Role of BMP signaling and “default” pathways!

•  Control of neuroblast maturation !

•  Lateral inhibition and Notch/Delta signaling!

!

5!

Early development in Xenopus !

Similar early embryonic events across phyla !

•  After fertilization, early cleavage divisions lead to blastula.!

•  Animal and vegetal poles reflect initial asymmetry in the oocyte.!

•  The dorsoventral axis forms early after fertilization.!

•  Gastrulation leads to formation of three germ layers.!

6!

Figure 22-70 Molecular Biology of the Cell (© Garland Science 2008)

Blastomeres!Cleavage divisions produce many smaller cells from the egg without change in total mass!

Figure 22-71 Molecular Biology of the Cell (© Garland Science 2008)

Blastula: formation of an internal cavity !

Formation of an epithelial sheet to isolate the interior of the embryo--faster division in animal pole!

7!

Figure 22-88 Molecular Biology of the Cell (© Garland Science 2008)

Mouse Blastocyst!

Figure 22-89 Molecular Biology of the Cell (© Garland Science 2008)

Blastocyst!Morula!4-cell stage!2-cell stage!

Scanning EM of Mouse Blastocyst!

8!

Gastrulation!

“It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life.” - Lewis Wolpert

Gastrulation!

Formation of 3 germ layers that give rise to all cells and tissues.!

9!

The Spemann organizer!

Transplantation of dorsal mesoderm (organizer) induces a secondary axis, where only the notochord (mesodermal tissue) is derived from the transplanted tissue!

The neural inducer – a secreted protein expressed by the organizer that induces ectoderm to form neural tissue!

The Organizer sends “dorsalizing” signals!

1.  Induces neural tissue on the overlying ectoderm!

2. Makes mesoderm more dorsal (somites)!

3.  Induces secondary gut (endodermally derived)!

10!

Nervous system and ectoderm share lineage in nematodes (c. elegans)!

Progeny of AB blastomere give rise to most neurons and the epidermis.!

Cells in the neural plate thicken, roll up into a tube, and then pinch off from the rest of the ectoderm creating the neural tube and later, the central nervous system.!

Neurulation!

11!

Early Development-genesis of neural tissue in Drosophila!

Nervous system derived from ventral region.!!Mesoderm involutes at ventral surface.!!Gives rise to three germ layers.!

Early Development-genesis of neurons in Drosophila!

Neuroblasts scattered in neurogenic region enlarge and are squeezed out of epithelium (delamination).!!Once they leave the ectoderm, they divide and give rise to the neuronal lineages, and make neurons and glia.!

12!

Development of chick embryo!

Development of chick embryo - Gastrulation!

Figure 47.13!

Epiblast!

Future!ectoderm!

Migrating!cells!(mesoderm)!

Endoderm!

Hypoblast!

YOLK!

Primitive!streak!

Hensen’s Node!

13!

Development of chick embryo!

Hensen’s node is the organizer, analogous to Spemann’s organizer in amphibians.!

Animal cap assays!•  Tissue culture of fragments reveals potential fates of cells within the tissue.!

•  Animal caps from pre-gastrula develop as epidermis.!

•  Animal caps from gastrula develop into neural tissue.!

•  Shows that neural lineage arises during gastrulation.!

•  Provides useful assay for studying molecular components from the organizer.!

14!

Mesoderm induction: interactions between animal and vegetal cells.!

Mesoderm induction: interactions between animal and vegetal cells.!

15!

Mesoderm induction: interactions between animal and vegetal cells.!

Indirect vs. direct neural induction!

Mesoderm is induced, then in turn induces neural tissue, turning on neural genes!

Mesoderm is not induced. Candidate turns on neural specific genes but not mesodermal genes.!

16!

Noggin is a direct neural inducer!

Strategy:!•  UV treatment inhibits dorsal structures!

•  Lithium is hyperdorsalized!

•  mRNA from hyperdorsalized embryos rescues UV embryos!

•  cDNA from organizer can also rescue UV embryos!

•  Use animal cap assay to test cDNA’s. Noggin, a protein secreted from the organizer, was identified using this approach.!

Chordin and follistatin are also direct neural inducers!

Both are also expressed in the organizer region at the time of neural induction.!

17!

Neural tissue is the “default” state!

Activin, a member of TGF-β family, can induce mesoderm.!!Isolated animal caps normally form epidermis.!!Truncated activin receptor blocks signaling through activin. In treated embryos, animal caps form neural tissue. !!Neural tissue is actively inhibited in the ectoderm; organizer blocks this inhibition in the overlying ectoderm.!

Neural tissue is the “default” state!

Neural fate is actively suppressed by cellular associations in the ectoderm.!!BMP4 blocks this suppression, so that cells can obtain epidermal fate.!

18!

Neural induction in Xenopus !

Wilson and Edmond, Nat. Neurosci, 2001!

At gastrulation, the organizer forms and begins to release inhibitors of BMP!

How are these molecules related?!

BMP s bind to BMP receptor to prevent neural fates.!!The organizer releases proteins (including noggin, chordin, follistatin) that bind BMP and prevent it from activating its receptor.!!

19!

How are these molecules related?!

Evidence:!BMP4 inhibits neural differentiation in animal caps treated with chordin, noggin, follistatin.!!BMP4 inhibits neural differentiation in dissociated animal caps.!!Animal caps treated with antisense BMP4 mRNA show neural differentiation without any of the inducers. !

Vertebrates and invertebrates use similar molecules to pattern the D-V axis!

BMP is related to decapentaplegic (dpp). Both inhibit neural differentiation in the ectoderm.!!Short gastrula (sog) is related to chordin.!

20!

Mutations cause defects in brain development!

SoxD expression also induces neural differentiation !

SoxD is upregulated in neural ectoderm after induction, and encodes a transcription factor.!!Overexpression leads to formation of ectopic neurons.!!!

21!

Neural fate is determined by activation of proneuronal bHLH transcription factors!

•  These genes are expressed in neuronal precursors!

•  Proneural bHLH gene expression initiates when the inhibition of neural fate is removed with BMP antagonists!

•  Bind to DNA at “E Box” and initiate expression of neural-specific genes, starting a transcription factor cascade!!

Neural induction leads to expression of proneuronal bHLH genes!

BMP signaling leads to inhibition of Sox transcription.!!Inductive signals inhibit BMP and permit transcription of Sox, which activates proneural genes.!!Other signaling pathways interact with these genes: Wnt, FGF !

22!

Generation of neurons!In nervous system development, the right number of neurons must be made. The number of progenitors is controlled by intercellular interactions. In Drosophila, clusters of cells that could become neurons each give rise to a single neuroblast. These cells are distributed across the neural ectoderm.!

Achaete-scute (a complex of bHLH genes) expression is seen in all proneuronal cells at first, then becomes restricted to the neuroblasts.!

Genes that regulate neurogenesis!

e.g., Achaete-scute: mutants have fewer neuroblasts.!

e.g., Notch, Delta: Mutants have too many neuroblasts.! From Molecular Biology of the Cell, 4th ed.!

Each cluster of proneural cells yields a single neuroblast!

23!

Lateral inhibition!

Laser ablation of delaminating neuroblasts causes neighboring ectodermal cell to adopt neural fate. Provides evidence that neuroblasts normally inhibit their neighbors from becoming neuroblasts.!!

Lateral inhibition!

Cells in the proneural cluster express achaete and scute, and all have the potential to become a neuroblast.!

One of the cells starts to express more proneural genes, and sends signals to neighboring cells that reduce their expression of proneural genes.!

This cell is eventually the only one to express proneural genes, and it delaminates from the ectoderm to become a neuroblast.!

24!

Lateral inhibition is mediated by the neurogenic genes Notch and Delta!

When notch is inactive, ASC genes activate their own transcription and also Delta, a ligand for Notch. This cell would become a neuroblast.!

When Notch is activated by Delta expressed on a neighboring cell, Notch is cleaved. The intracellular domain binds to SuH (CSL) then the complex enters the nucleus and activate expression of HES, which represses both ASC and Delta expression.!

Lateral inhibition!

While the proneural cells all start out with the same levels of Notch and Delta, this process amplifies any small differences, until one of the cells is the clear winner.!!This pathway regulates the number of neural progenitors, and ultimately, the number of neurons and glia in the brain.!

25!

Notch and Delta regulate neurogenesis in vertebrates- studies in frog embryos !

Dominant negative Delta blocks Notch signaling!

Notch and Delta regulate neurogenesis in vertebrates !

Notch ICD was injected in left blastomere. No neurons developed on this side.!

Control! NICD!

26!

Notch and Delta in the mammalian nervous sytem!

During brain development: !•  Several homologs found for Notch, Delta and other Notch ligands!•  Control of neural progenitors and their proliferation and survival!•  Relative numbers of neurons and glia!

In the adult brain:!•  Neural plasticity - LTP and neurite outgrowth!•  Learning and memory!!Neurological disease:!•  Response to ischemic injury!•  Adult proliferation of neurons after injury!•  Alzheimer’s disease - presinilin-1 cleaves both APP and Notch-1!•  Other degenerative diseases!

Summary!

•  The embryonic origins of neural tissue have common features across phyla.!

•  Tissue interactions lead to neural induction through BMP signaling. In the default state, BMP signaling inhibits neuronal fates. At induction, BMP signaling is blocked, permitting neuronal fate.!

•  Neuronal fate results from activation of proneural genes of the bHLH transcription factor family.!

•  Lateral inhibition uses antagonistic Notch and Delta signaling to regulate activation of these proneural genes.!