BioSci 145A lecture 19 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved...

BioSci 145A lecture 19 (Blumberg) page 1 ©copyright Bruce Blumberg 2000. All rights reserved

BioSci 145A Lectures 19 - Gradients, cascades, and signaling pathways regulate development II

• Office hours

– tu/th 2-3

– [email protected]

– 824-8573

• review session Thursday bring questions

– we will also go over answers to last year’s final examination

• evaluations will be done on Thursday as well so please come and give us your candid assessment of the course

• today

– patterning by zygotic genes

– cross-regulatory interactions among segmentation genes

– cell-cell communication

– homeotic genes


Zygotic D/V patterning

• D/V patterning is the same in all organisms!

– subject of a famous debate in 1830 between Georges Cuvier and his mentor Etienne Geoffroy St.Hilaire

• Geoffroy - function follows form - one body plan

• Cuvier - form follows function - no evolutionary connection between four distinct forms

• Cuvier considered to have won debate until 1995

– simple observation suggests that most invertebrates are upside down relative to vertebrates

• vertebrates - dorsal nerve cord and ventral mesenchyme

• invertebrates have a ventral nerve cord and dorsal mesenchyme

– the same signaling pathway is initiated on the dorsal side of flies and ventral side of vertebrates (Geoffroy)

– ventral (vertebrate) or dorsal (invertebrate) structures are induced by members of the TGF/BMP family

• both are small, dimeric polypeptides that act through cell-surface receptors by activating transcription factors called SMADS

• TGFs - transforming growth factors were first identified as proteins that caused cancers when loss-of-function mutations occurred.

• BMBs - bone morphogenetic proteins identified as factors from cartilage that could induce the growth of bone when injected into animals


Zygotic D/V patterning (contd)



• TGF/BMP family members act through specific receptors

– high-affinity receptors are heterodimers of type I and type II receptors

– first receptor in the family to be identified was the activin receptor

• identified by expression cloning using a radiolabeled ligand (activin) that was purified biochemically

– Wylie Vale’s laboratory at the Salk Institute

• Xenopus versions followed soon thereafter and it was thought for a time that these were the key players in early development

– the author of your textbook found that the model was sexy (but the data were weak)

• Dorsal determination in Drosophila

– Drosophila dorsal morphogen is decapentaplegic (dpp)

– receptors are encoded by three genes in Drosophila

• two type I receptors

– saxophone

– thick veins (tkv)

• one type II receptor

– punt

– phenotype of tkv and pun is the same as dpp suggesting that these two genes encode the dpp receptor

– receptors phosphorylate smads that activate transcription

• mad = mothers against decapentaplegic



• mechanism and components are conserved in evolution

– extracellular components are ligand, antagonist and protease

– tolloid/xolloid=BMP1

– intracellular components are smads and receptor kinase domains



• conservation of mechanism should give you pause

– remember in Drosophila that both dorsal and ventral have active morphogens that antagonize each other

– Drosophila

• dorsal pathway has

– dpp that actively specifies dorsal

– gurken->torpedo pathway antagonizes ventral

• ventral pathway has

– spatzle->dorsal pathway that ACTIVELY promotes ventral (twi, sna)

– same pathway represses dorsal by repressing dpp and zen

– Xenopus

• ventral pathway has

– BMP4 that actively specifies ventral

• dorsal pathway has

– noggin,chordin, follistatin and cerberus that all antagonize BMP4 and/or wnt activity

– Isn’t there something missing ???

• Homologous pathways predicts a ventrally specified antagonist that acts against dorsal

AND

• a positively acting dorsal factor

– Is anyone looking?



• D/V patterning illustrates several important concepts in developmental gene regulation

– morphogens may work both by being present and being absent

• presence of BMP-4 -> ventral mesoderm

• absence of BMP-4 -> neural tissue

– antagonists are frequently combined with agonists to tune response of tissues

• dpp and sog

• BMP-4, noggin, and chordin

– multiple gene products may be required to produce the same result

• noggin, chordin, cerberus, and follistatin all act by inhibiting BMP-4

• single KO (knockout) of chordin or noggin are not informative

• double KO produces expected result (no head)

– extracellular proteases may be required both to activate morphogens and degrade antagonists

• active cleavage of BMP-4 required for function

• degradative cleavage of antagonist complexes by tolloid/xolloid/BMP-1 required for function

– multiple and varied responses can be obtained by altering the location or concentration of players in the pathway


Segmentation genes

• a fate map of the embryo may be drawn at the cellular blastoderm stage

– segments are overt physical separations formed in the larva

– parasegments mirror gene expression -> contribute to adjacent segments

– embryonic regions directly correspond to adult structures, even though they do not contribute to adult structures at all

• these compartments are formed by the successive and combinatorial interaction of a group of segmentation genes

– mutations cause specific, predictable changes in larval structures


Segmentation genes (contd)

• three broad groups of segmentation genes

– defined by the type of defect that mutations in each causes

– gap genes - lof mutations cause loss of large, adjacent chunks of the pattern

– pair-rule genes - lof mutations cause pattern deletion in alternate segments

– segment polarity genes - lof mutations cause mirror image duplications

• these genes are successively expressed in the early embryo

• a fundamental principle is that the boundaries established between areas of gene expression are very important for establishing the expression of subsequent genes

– Hans Meinhardt is the major theoretician

– The Algorithmic Beauty of Seashells is a “tour de force” in understanding patterning mechanisms (beautiful too)


Hans Meinhardt


Hans Meinhardt (contd)



• Temporal and spatial hierarchy

– maternal genes establish gradients that activate or repress gap genes

– gap genes, and boundaries between them combine to set up the pattern of 7 stripes for the pair rule genes

– pair rule genes borders set up segment polarity genes

– gap and pair rule genes are expressed before cellularization

– segment polarity genes at time of cellularization

• most of maternal, gap and pair-rule genes are transcription factors

– most types represented

• zn finger (hb, kr)

• nuclear receptor (tll, kni)

• homeodomain (eve, ftz)

• bHLH (hairy



• Principle: combinatorial interactions among a small number of genes specifies each region of the embryo

• gap genes are regulated in two ways– respond directly to bicoid– regulate each other– four bands are created thusly

• bcd -> hb• hb -> kr• hb -| kni, gt **figure 29-22 is

mislabeled**

• nos -| hb allowing kni and gt posteriorly

• transition to 7 stripes

– high hb -| kr

– but some hb is required for kr hence the decline when hb is lost

– kni requires absence of hb

– gets complicated

• general principle is that combinatorial interactions among proteins control where they are expressed.



• gap genes directly regulate transcription of pair rule genes

– each pair rule gene is expressed in a pattern of 7 stripes that can be directly traced to combinatorial interactions among gap genes (example coming)

– always expressed in pairs

• one in even

• other in odd parasegments

• primary pair rule genes are eve and hairy -

– first ones expressed

– they affect expression of other pair rule genes

– expressed in stripes that are 3-4 nuclei wide

– stripes in which a gene are expressed correspond to the regions that are missing in loss-of-function mutations

– compartments are fundamental units of morphological development in Drosophila

• boundaries are respected

• these are set by segmentation gene expression



• example of how combinatorial regulation of pair-rule gene expression occurs

– eve promoter has various binding sites for gap genes in 480 bp

• bicoid

• hunchback

• giant

• Kruppel

– these were identified by the usual types of experiments

• footprinting

• transfection

• biochemistry

– note competition on four sites

– general rule is that broadly distributed proteins are needed for activation (e.g. bicoid, hunchback)

– localized proteins set borders by repression (kr, gt)



• Generalized theoretical model for pattern formation uses a combination of long acting and local factors

– Meinhardt shows that all complex patterns can be created by combining the activity of a long acting repressor and local activator (or vice versa)

– http://www.eb.tuebingen.mpg.de/abt.4/meinhardt/primary.html

– provides a computer program that you can use to alter the stability and diffusibility of these morphogens and observe results on patterning

• pair rule genes are first expressed in broad domains that touch anteriorly and have a gap posteriorly

– pattern is later refined by repression

– the repression is mediated by engrailed protein that is expressed as a single cell wide stripe



• Segment polarity genes

– segment polarity genes are expressed at or after the cellular blastoderm stage

– engrailed is expressed as a single cell wide stripe at the anterior border of both eve and ftz expression

– how can it be that en is exclusively transcribed in a single cell width?

• expression is important because en sets boundaries of actual compartments

• engrailed is a homeodomain protein with a strong repression domain -> dominant repressor

– two models proposed

• combinatorial model - as with gap gene control of pair-rule genes, the overlapping expression of pair-rule genes may regulate segment polarity genes

• boundary model - interactions involving cell-cell communications at boundaries are important for causing subdivisions to arise within a compartment (like positional confrontations)

– boundary model seems more probable

• en is initially expressed in response to ftz and eve expression at boundaries

• establishment is followed by a completely different maintenance phase



• maintenance of expression at boundaries

– en and wingless are expressed in adjacent cells

– after en expression starts an autoregulatory loop is established that makes expression of en and wg required for each other’s expression

– wg product is a secreted protein that induces cells to express en

• en expression allows cells to secrete hedgehog

• hh induces wg expression in adjacent cells

– mechanism keeps the boundary sharp

• reciprocal short range interactions that stabilize cell fate are very important in development

– e.g. in fly eyes, legs and wings

– developing or regenerating vertebrate limb


Cell-cell communication

• many segment polarity genes are secreted, transmembrane, kinases, cytoskeletal components, suggesting that cell-cell communication is now important

• wingless pathway is very interesting for a variety of reasons (Larry Marsh, Ken Cho)

– like many others, it begins with the expression of an extracellular ligand that leads to the expression of a transcription factor

– unique feature:

• transcriptional mediator - armadillo is a component of the cytoskeleton

• interacts with cadherins to promote adhesion


Cell-cell communication (contd)

• wingless (contd)

– arm is inactive when phosphorylated by zeste-white3

– activation of wg signaling pathway blocks phosphorylation of arm and it now translocates to the nucleus and heterodimerizes with pangolin to activate transcription

• a nearly exact copy of this pathway is operational in vertebrate cells

– only the names have been changed

– remember the colon cancer example from a few lectures ago?

– APC tumor suppressor gene normally binds to -catenin (arm)

– loss of APC results in inappropriate activity of target genes and increases cell growth


Homeotic genes

• Homeotic genes control the identity of the particular segments in which they are expressed

– homeotic mutations “transform” one compartment into another

– practically speaking, this means that they transform one body part into another

– virtually all are transcription factors that regulate each others expression as well as other target genes

• most are homeodomain proteins

– homeotic genes interpret the information laid down by the segmentation genes.

• expression coincides with the peak expression of the segment polarity genes

– phenotype of mutation in one homeotic locus depends not only on the gain or loss of function in that gene but also how other homeotic genes alter their expression to in response to the loss.

• genetic loci are large and complex with multiple promoters and enhancers operative in particular temporal and spatial patterns.

– two major loci in Drosophila (HOM-C)

• bithorax - BX-C

• Antennaedia - Ant-C

– these are together in one large complex in most insects and other animals including vertebrates (HOX-C)


Homeotic genes (contd)



• Homeotic genes lead to a sequential elaboration of pattern

– complex is transcribed in one direction

– each gene in the complex acts on a successively more posterior region of the animal

– formation of a particular compartment (structure) depends on the expression of all preceding genes and a new activity encoded by the next gene in the cluster

• loss-of-function mutations transform the segment toward the next more anterior

• gain-of-function transform segments toward the next more posterior structure

• Ant-C patterns head through T2

– many weird mutations all put legs where other structures belong

• antennapedia

• proboscipedia

• nasobemia

– antp is an example of a gene that is required to suppress the formation of head structures

• lof alleles transform T2-> T1

• gof alleles transform antenna->leg



• BX-C patterns T2 though the abdomen

– only 3 proteins are encoded by BX-C

• how can three proteins specify the identify of 10 segments?

• two RNAs that do not encode proteins are also produced

– mutations in these RNAs cause phenotypes!

– some recent data from other systems suggesting that RNAs can act as transcriptional coactivators

– numerous cis-acting mutations throughout the complex

• many genetic loci are really regulatory sequences in one of the three coding units

– BX-C loss of function is lethal

• segments all look like T2 (not unlike ancestral insect)

• like Ant-C, BX-C acts to refine structure in terms of a combinatorial code

• mutations in cis acting sequences cause inappropriate gain or loss of function in particular compartments



• Homeodomain is highly conserved

– vertebrates have Hox complexes (HOX-C)

– typical vertebrate has four

• four genome duplications in vertebrate lineage

• most fish have had an additional duplication and may have 7-8 Hox complexes.

– genes at the same position in different clusters are called paralogs (e.g. Hox-A1, B1, D1)



• Hox genes are clustered

– organized and transcribed in the same direction along the chromosome

– like HOM-C genes, the downstream genes in the cluster specify more posterior compartments

– all homeodomains in HOM-C and HOX-C have the same in vitro DNA-binding specificity

• corresponding vertebrate gene can rescue Drosophila mutations

• specificity must be conferred by flanking sequence and context of proteins expressed in the cell

• depending on set of proteins expressed, various Hox genes can be transcriptional activators or repressors

– these genes are considered to act together to specify a Hox code that determines position along the A/P axis

• gain- and loss-of-function experiments completely support this model

• some functional redundancy exists between paralogs

– knockouts do not completely ablate structures expressing the gene



• Hox-C and HOM-C genes are expressed at similar positions in the developing embryo

– implies that the Hox code is an ancient and successful mechanism to specify position

• still an open question why the organization of the clusters has not changed

– it was once thought that there were a few “master promoters” that transcribed the entire complex at one time

– promoter bashing has identified control elements for each gene but it remains unclear why no rearrangements are tolerated


Summary

• genetic control of patterning

– combinatorial interactions among transcription factors is the primary mechanism for determining position and establishing pattern in the body

– genes and mechanisms are highly conserved among all animals

– pattern is sequentially elaborated

• early acting genes divide the embryo in broad strokes

• later genes refine this pattern

– repression is an important means of patterning

• it wasn’t so long ago that many researchers believed that repression was not a viable patterning mechanism

– reciprocal interactions at boundaries of gene expression are a primary means of refining and subdividing patterns

– patterning genes regulate the expression of other patterning genes as well as structural genes

– many patterning pathways are aberrantly regulated in various cancers

BioSci 145A lecture 19 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved...

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Transcript of BioSci 145A lecture 19 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved...