BioSci 145A lecture 18 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved...

BioSci 145A lecture 18 (Blumberg) page 1 ©copyright Bruce Blumberg 2000. All rights reserved

BioSci 145A Lectures 18 - Gradients, cascades, and signaling pathways regulate development I

• last lecture we talked about how gene regulation influences cancer

• this week we will try and put together the types of techniques and experiments we have been talking about to solve problems of transcriptional regulation in development

– three interesting problems in transcription

• development (includes cancer)

• metabolism

• homeostasis (includes hormonal signaling)

– some of the material overlaps with Bio108 and Larry Marsh’s genetics course but we will emphasize the role of transcriptional regulation in the patterning process


Fundamentals of patterning

• How to pattern an embryo

– begin with a small asymmetry

– create cell diversity with short range inductive events between cells

– send positional signals between cells and continue inductive events

– differential cellular responses to these signals will lead to the elaboration of pattern

• Multiple axes need to be considered

– anterior/posterior

– dorsal/ventral

– proximal/distal (limbs)

– left/right (vertebrates)

• As we will see, the signaling mechanisms along these axes are largely conserved across evolution

• Central assumption about the molecular basis of development - each cell type may be characterized by its pattern of gene expression

– since gene expression is controlled primarily at the level of transcription it follows that transcription factors will be important developmental regulators

– these will include activators and repressors that act in various ways to modulate transcription

• Important principle - activation and repression are equally important developmental mechanisms


Transcription factor cascades in development

• groups of transcription factors form regulatory circuits

– interactions are responsible for patterning

– known to be true for all multicellular organisms

– currently, details are only well worked out in Drosophila and C. elegans

• Since gene expression regulates development, we will explore in some detail

– interaction between transcription factors

– interaction between and among signaling pathways

– widespread conservation of these pathways

• many of the genes that control development are also involved in the development of cancer

– in many ways cancer is a disease of development

• tumor cells share properties of embryonic cells

– capacity for division

– ability to migrate throughout the body

– secretion of bioactive substances

– understanding regulatory circuits that pattern the embryo will aid in understanding how cancers develop

• What do we want to understand?

– begin with an initial asymmetry

– asymmetry triggers positional differences

– these are translated into differential gene expression

– regions of the embryo acquire specific properties


Transcription factor cascades in development (contd)

• How is asymmetry translated into gene expression?

– mechanisms differ among the various systems BUT

• the types are conserved among all animals

– may involve localization of

• mRNAs

• proteins

• regulatory factors (RNA binding proteins, inactive dimerization partners)

– end result is always the same

• localized control of gene expression

• After asymmetrical beginning, the body begins to be subdivided

– regional specification - areas are specified (determined) -> particular tissues or structures

– genes that regulate this process were identified by mutations that cause body parts to be absent, duplicated or formed in inappropriate places

• famous Drosophila screen by Christianne Nusslein-Volhardt and Eric Wieschaus (Nature, 1980 287, 795-801)

• led to the identification of the major classes of patterning genes

– opened up the field, Nobel prize

– combined with molecular techniques, these led to the elucidation of regulatory pathways



• subdivision (contd)

– these genes are strong candidates to function as genetic switches

• typically act on themselves and other regulatory genes to create a hierarchical cascade of gene expression

• but they must also act on downstream target genes that actually form the structures involved

– can’t only have regulatory proteins, must also have structural proteins and enzymes to build tissues, organs and body parts

– one should not lose sight of the importance of these “effector genes” that actually do the work of development.

– most of these switch proteins act by binding to DNA sequences in the promoters of target genes

• can be studied using all of the techniques we have been talking about for the last few weeks

• a small number are RNA binding proteins that modulate the localization or stability of mRNAs

• a few are proteases that modulate the activity of other factors

• a very few regulate the transport of proteins into the nucleus in particular parts of the embryo



• Developmental pathways are predetermined in the egg

– the program is usually sequential and interactive

• cells can be moved around

• some plasticity in outcome

– program may be invariant in time and space (C. eleg)

– despite widely different appearances, the regulatory mechanisms that control development are very highly conserved in vertebrates and invertebrates

• this justifies the use of weird model organisms such as Drosophila and C. elegans

• we use these because they have particular advantages that allow questions to be asked and answered precisely

• many mutations in developmental regulatory genes are lethal early in development

– single mutations that cause gross changes in development are particularly interesting

– three classes in Drosophila -> act successively

• maternal genes - these broadly specify regions in the embryo -> loss causes absence of large regions

• segmentation genes - lead to changes in segment number, size or polarity

• homeotic selector genes - control the identity of particular segments but not the number or size


Drosophila and gradients

• early embryo is patterned by molecular gradients along major body axes

– anteroposterior (A/P)

• anterior ->head

• posterior -> tail

– dorsoventral (D/V)

• dorsal -> top (except for Xenopus)

• ventral -> bottom (except for Xenopus)

• A/P axis is already determined in the Drosophila egg

– determined after fertilization in amphibians and fishes

• D/V axis is determined during zygotic development in Drosophila

– concurrently with A/P in amphibians and fishes


Drosophila and gradients (contd)

• identification

– Drosophila embryo is composed of segments (parasegments)

• all invertebrates are overtly segmented

– segment boundaries are respected

• vertebrates have some vestiges of segmentation

– boundaries may not be respected

– Drosophila has very clear markers for each segment - shape and number of denticles (hairs) varies between segments and between dorsal and ventral side

• absolutely key for identifying the functions of genes

• frequently, mutations are evaluated at an early larval stage rather than in the adult fly

• although larval segments do not contribute to adult structures, they reflect what will happen in the adult

– adult fly has head, thorax and abdomen

• 3 thoracic

• 8 abdominal segments

• fly is a very specialized insect and the details of its development are not characteristic of insects or invertebrates as a whole

– but the molecular mechanisms are virtually identical



• Drosophila has unique early development

– early embryo develops as a syncytium (no cell membranes)

– first asymmetry is pole plasm vs cytoplasm

– after 13 nuclear divisions, the nuclei are positioned at the periphery

– cell membranes then form - cellular blastoderm

– at about this stage, the first determination events occur

– nuclei do not move in predetermined patterns

– behave according to position in A/P and D/V gradients



• What are the gradients that specify position in the early embryo?

– many components are put into the oocyte by the nurse cells during oogenesis, (proteins, RNAs)

– mutations in maternal genes important for early development -> female sterile mutations (no effect on mother)

• females have no progeny

• embryos may have cuticular defects

• Critical components of four developmental pathways are laid down in the oocyte by follicle cells

– anterior

– posterior

– terminal (both ends)

– dorsoventral



• Each pathway has a morphogen

– morphogens are diffusible substances that form concentration gradients that pattern the embryo - many definitions, loosely applied

• Gradient model was popularized by Child, Huxley and De Beer

– activity gradients -> formation of specialized regions in the embryo

• in its strictest definition, a morphogen patterns tissues directly depending on its concentration

– Classic paper in the field is Turing, A.M. (1952) The chemical basis of morphogenesis. Phil. Trans. R. Soc. Lond. B 237, 37-72.

– Alan Turing was a British mathematician whose claim to fame was the solving of the German wartime code called “enigma”

» enabled the allied to eavesdrop on military transmissions and win the war

• Louis Wolpert -> many theoretical treatments of morphogenesis

– Wolpert, L. (1989). Positional information revisited. Development 107s, 3-12.

• Francis Crick provided legitimacy to the model with a quantitative treatment of the activity of diffusible substances - worked over small distances 0.1-1mm

– Crick, F. (1970). Diffusion in embryogenesis. Nature 225, 420-422.



• morphogens (contd)

– most common definition of morphogen is a diffusible substance responsible for patterning the embryo

• important components of each pathway are localized in the oocyte before fertilization

– anterior system patterns the head and thorax. Products in the maternal germline are required to localize the bicoid mRNA. bicoid protein is the morphogen

– posterior system patterns the abdominal segments. many components act to cause the localization of the nanos gene.

– terminal system is responsible for the specialized structures in unsegmented termini of the head and tail. morphogen acts through the transmembrane receptor torso

– dorsoventral system is responsible for patterning the d/v aspect of the embryo. Patterning occurs through a gradient of the dorsal protein in the nucleus

• key feature is that these pathways all use somewhat different mechanisms to generate pattern.

– many of these mechanisms are widely used in other organisms and in transcriptional regulation in general


Gradients in the oocyte

• summary of the four systems

– multiple levels at which the pathways can be affected

– ~30 maternal genes may be grouped by location, and activity

– with the exception of dpp, all of the zygotic targets of the patterning systems are transcription factors

• many are repressors

– anterior and posterior systems interact, d/v and terminal act independently of others


Gradients in the oocyte (contd)

• How does one go about testing whether gene products are required?

– inject cytoplasm or purified components from wt embryos into mutants and assay for rescue

– can use this method to map where mutants are in the pathway (but very tedious)

– only the morphogen itself can elicit localized, concentration-dependent rescue

– rescue is sufficient to conclude that the mutation causes a deficiency of some material in the embryo.


A/P patterning uses localized regulators• Bicoid is an anterior morphogen in the strictest sense

– bicoid protein or wt cytoplasm rescues bicoid embryos in a concentration-dependent manner with a high point at the site of injection

• this implies that the targets are ubiquitous– bicoid mRNA is localized to the very anterior of the

embryo– bicoid protein diffuses after translation forming a

concentration gradient


A/P patterning uses localized regulators (contd)

– bicoid is a homeodomain protein that directly specifies anterior -> instructive

• bicoid is a positive regulator of hunchback -> band of hb expression where bcd is expressed

• Posterior development involves many genes

– females mutant for any of these genes produce progeny that lack abdominal structures

– apparently, each of these gene products is required for localizing the next one in the pathway -> all required to localize the morphogen nanos (can rescue all)

– reason for all of these components may be the requirement for transport the entire length of the egg



• Posterior development (contd)

– posterior gene cascade illustrates many levels of gene regulatory control

– cappuccino, and spire are required to localize staufen protein at the pole

– staufen protein localizes oskar mRNA

– oskar/staufen localize vasa

• target of vasa is not known

– vasa is a RNA binding protein similar to translation initiation factors (RNA helicase)

• vasa protein found in germ cells of most species

– valois has not been cloned

– tudor appears to be a RNA binding protein that is similar to a EBV coactivator protein

• required both for abdominal development and germ cell formation

– unknown how vasa, valois and tudor affect nanos but they do

– actually, it is fairly remarkable that the functions of these genes is not known after so many years of intensive study.



• anterior vs posterior

– both have a localized mRNA that produces a diffusible protein that acts as a morphogen by injection experiments

– bicoid and nanos both function by regulating the expression of hunchback

BUT

– nanos is required to repress hunchback

• it is permissive rather than instructive

– localization of the posterior components is much more complicated than anterior

• bicoid is required to transcribe hunchback mRNA

– hunchback protein is a repressor

– nanos represses translation of hunchback protein in the posterior

• it represses a repressor - common in development

– net effect is to have hunchback protein in the anterior half of the embryo only


Dorsoventral patterning

• D/V patterning differs from A/P patterning

– occurs after cellularization has occurred

– therefore, it uses localized receptor/ligand interactions

– much more typical developmental pathways since most organisms never have syncytial state as does Drosophila

• complex interaction between oocyte and follicle cells

– process begins again with the localization of a mRNA - gurken

– multistep process - details are not critical here.

– cap and spire are also required for gurken localization


Dorsoventral patterning (contd)

• dorsalizing pathway looks like this

– gurken encodes a protein resembling TGF• TGF is a growth factor related to EGF

– torpedo is downstream and encodes the Drosophila EGF-receptor

• probably gurken receptor

– activation of torpedo (receptor tyrosine kinase) leads to the activation of a typical MAP kinase signaling pathway

– ultimate result is to prevent activation of the ventral pathway on the dorsal side of the embryo

– a very similar pathway is used in cell differentiation in the eye

– illustrates common modus operandi - activates its own dorsal specifying genes (dpp) while repressing the activity of genes of the opposite (ventral) pathway



• Ventral pathway (dorsal group mutants) is required for the formation of a variety of structures

– dorsal midline

– dorsal ectoderm

– neural ectoderm

– mesdoderm

– ventral midline

• gene products produce ventral phenotype

– mutants produce dorsalized embryos (hence dorsal pathway)

– mutations in any of the dorsal group gene prevent the formation of ventral and intermediate phenotypes

– ventral determinant can rescue



• pathway in summary

– initial signal is not known

– signal leads to a protease cascade

snake -> easter -> spatzle

– spatzle is the ligand for toll - a critical step in the cascade

• toll - embryos have no ventral structures

• toll injection rescues ventral structures

• paradoxically, any cytoplasm from a wt embryo will rescue a toll mutant

– toll activity is found in all parts of a wild type embryo

– toll is ubiquitously expressed but only activated in the ventral part of the embryo

• but the ligand is in the perivitelline space and can presumably diffuse

• spatzle either can not diffuse far or cleavage does not occur unless toll is in proximity

– toll is similar to the interleukin 1 receptor and the entire pathway from spatzle -> dorsal is completely homologous

• IL-1 pathway is required for activation of helper T lymphocytes and antibody-mediated immunity

• binding of spatzle (IL-1) is sufficient to trigger the whole ventral-determining pathway



• comparison of IL-1 and spatzle -> dorsal signaling pathways

– similarity is actually rather remarkable

– nothing is new in evolution!

– also remarkable that other genes in the pathway have not been identified yet

– in lymphoid cells, activation of the pathway results in the release of NF-B from I- B and its transport to the nucleus

– cactus appears to have the same function as I- B

– remember that NF- B is also a viral oncogene called v-rel

– this should reinforce the link in your minds between development and cancer



• dorsal (NF- B) is uniformly distributed throughout the embryo

– it is specifically transported to nuclei in ventral cells where the spatzle->toll pathway is activated

– amount of dorsal protein in the nucleus is associated with the degree of ventralization

– dorsal is a transcriptional activator that turns on twist and snail

• these are required for ventral structures

– it also represses decapentaplegic and zerknült that are required for dorsal structures



• D/V patterning requires three localized systems to work properly

– gurken->torpedo-->represses spatzle dorsally

– snake->easter->spatzle-->dorsal induces ventral cells

• represses dorsal pathway locally

– dpp mediates dorsal development via several members of the TGF-/BMB signaling pathway that we will talk about next time.


Terminal system

• terminal system is not dissimilar to the D/V system but the downstream genes are quite different

– torso is the key player and it is another receptor tyrosine kinase

– its activation ultimately leads to the production of two transcription factors

• tailless - a nuclear receptor that probably functions as a constitutive repressor

• huckebein - a zinc finger protein that can both activate and repress transcription

– tailless is required for the formation of terminal structures

• mutations lead to loss of specialized terminal structures

– huckebein is required for mesoderm and endoderm patterning

• it represses the formation of ventral mesoderm in the ends of the embryo

• it activates genes that cause invagination of gut primordia at both ends of the embryo

• also regulates wingless and engrailed expression in the head


Axis determination in summary

• maternal patterning genes act to broadly partition the embryo into large, exclusive domains

– anterior system leads to hunchback anterior

– posterior system represses hunchback and promotes knirps and giant

– dorsal/ventral system leads to decapentaplegic and zerknült dorsally, twist and snail ventrally

• the presence of these proteins, and the boundaries that they establish are interpreted by the next set of genes to refine patterning along the axes.


Next lecture

• patterning of the embryo by zygotically-expressed genes

• final comments

BioSci 145A lecture 18 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved...

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Transcript of BioSci 145A lecture 18 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved...