BioSci 108 lecture 26 (Blumberg) page 1 © copyright Bruce Blumberg All rights reserved Bio /13/2000 Molecular Genetics of Pattern Formation I Contact information office hours W/F 3-4 phone (preferred contact mode) Lectures posted at Many students are concerned with who will write the test and what will be covered –both Dr. Cho and I will contribute to the exam very likely that the questions will be similar to those given in previous years –everything in the assigned chapters is fair game –anything lectured about is fair game –You need to learn everything. ultimately, you will need to know this stuff for MCAT and GRE may as well learn it now while we are here to explain the parts that may not be completely clear

BioSci 108 lecture 26 (Blumberg) page 2 © copyright Bruce Blumberg All rights reserved Genesis of the body plan As we have been discussing, the structure of an organism is controlled by the action of its genes. –mechanisms by which genes controlled development remained a mystery until the early 1980s Drosophila is the key system that allowed progress –genes that regulate development 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, , ) led to the identification of the major classes of patterning genes and categorization into a coherent framework –opened up the field -> Nobel prize Why Drosophila? –T.H. Morgan abandoned amphibians in the 1930s because he presciently realized genetics were required to answer major embryological questions –small, easy to keep, short generation time –genetics allowed identification of genetic loci long before it was possible to clone genes field was ripe for picking when recombinant DNA tools became available in late 1970s understanding of tools since 1980s have made it possible for anyone to do these experiments

BioSci 108 lecture 26 (Blumberg) page 3 © copyright Bruce Blumberg All rights reserved Genesis of the body plan (contd) Why Drosophila? (contd) –fancy genetic techniques allow difficult questions to be answered mitotic recombination and compartment boundaries –in situ hybridization was first perfected and widely applied in Drosophila no easier organism to do in situs in than Drosophila –polytene chromosomes allowed relatively precise mapping of mutations later advances allowed chromosomes to be microdissected and genes in a region to be cloned Reward: genes identified in Drosophila turn out to have close counterparts in virtually all animals, including humans Anatomy of the fly (Fig 21-47) –adult fly has characteristic structures –important to note that each part has characteristic patterns of hairs and pigmentation allows identification of subtle differences in mutants (much like cockroach leg bristles)

BioSci 108 lecture 26 (Blumberg) page 4 © copyright Bruce Blumberg All rights reserved Segmentation Stages of development (Fig 21-48) –egg -> adult takes 9 days very large differences between larval stages and adult -> metamorphosis is important other insects have more direct development - primary differences are in size, e.g.. grasshopper –larval stages are called instars –in the pupa, larval structures are recycled and the adult form (imago) appears Regions of the early embryo map to adult structures (Fig 21-49) –but they do not actually give rise to them except indirectly via imaginal disks –fly larva does not show overt segmentation until gastrulation –at the completion of major morphogenetic movements (~10 hrs) all of the future segments are visible ancestral insects had numerous identical segments (like a millipede) –evolution has led to the modification of certain segments to produce specialized structures –the segments are different from each other but built according to a similar plan

BioSci 108 lecture 26 (Blumberg) page 5 © copyright Bruce Blumberg All rights reserved Segmentation (contd) adult fly has –head with various appendages head appendages are homologous to legs –three thoracic segments (T1-T3) all 3 have legs T2 has wing T3 has haltere (wing in dragonfly) –8-9 abdominal segments (A1-A9) no legs or wings –terminal structures nonsegmented (acron and telson) these segments can be mapped to the larva (Fig 21-50) –fly can be discussed in terms of segments or parasegments segments correspond to physical units parasegments correspond to patterns of gene expression. –1/2 segment out of register with physical segments »implies that each segment is derived from two parasegments –invented to explain why gene expression did not correspond with segments –convenient to think of embryo in terms of parasegments when discussing patterns of gene expression that elaborate the embryo

BioSci 108 lecture 26 (Blumberg) page 6 © copyright Bruce Blumberg All rights reserved Drosophila has no cells early in development Fundamental weirdness of Drosophila (and other insects) is that first 13 mitotic divisions occur without cell division (Fig 21-51) –end up with ~6000 nuclei in a single cell, the egg –this means that regulatory molecules are free to diffuse around the embryo fundamentally different from other animals which always have cells patterning mechanisms are therefore a simplified version of what goes on in other animals –first nine nuclear divisions occur very rapidly and synchronously (as with Xenopus) at this point the nuclei are randomly distributed throughout the egg cytoplasm migrate to the surface - syncytial blastoderm –four more divisions occur -> cell membranes form cellular blastoderm - after cellularization, DNA replication slows and transcription begins pole cells (at the posterior end) separate a few divisions earlier -> primordial germ cells –as in the amphibian embryo, the rapid rate of DNA replication and nuclear division prevents transcription from occurring early development runs on mRNAs and proteins deposited during oogenesis patterning is determined by maternal information

BioSci 108 lecture 26 (Blumberg) page 7 © copyright Bruce Blumberg All rights reserved Drosophila has no cells early in development (contd) Drosophila cellular blastoderm corresponds to the hollow blastula of the amphibian embryo –gastrulation movements are quite different from amphibians and mice but end result is the same: germ layers are formed endodermal cells invaginate to form the gut mesoderm surrounds the gut rudiment and comes to lie between endoderm and epidermis –cells are determined at the cellular blastoderm fate map using marking techniques (Fig 21-52) future segmented areas are outlined positions of most tissues are maintained, however gut forms by invaginating at both ends

BioSci 108 lecture 26 (Blumberg) page 8 © copyright Bruce Blumberg All rights reserved Developmental hierarchy Developmental genes in Drosophila act according to a strict developmental hierarchy –early acting genes divide the embryo in broad strokes –the later acting genes sequentially refine these subdivisions as development proceeds MATERNAL GENES (egg polarity genes) anterior group (e.g. bicoid) posterior group (e.g. nanos) terminal group (e.g. torso) dorsoventral group (e.g. dorsal) ZYGOTIC GENES gap genes (e.g. hunchback) pair rule genes (e.g. even-skipped) segment polarity genes (e.g. engrailed) Homeotic selector genes (e.g. antennapedia)

BioSci 108 lecture 26 (Blumberg) page 9 © copyright Bruce Blumberg All rights reserved Maternal genes pattern the early embryo Two coordinate systems are required to specify the position of any cell in the embryo (Fig 21-52) –four groups of egg polarity genes were identified in searches for mutations that affect polarity 12 genes were identified that affect dorsoventral (D/V) polarity –loss-of-function in any one of 11 genes gave dorsalized embryos (no ventral structures) –other one gave ventralized embryos (no dorsal structures) –these 12 genes are part of a pathway that is required to set up a D/V morphogen gradient in the early embryo anteroposterior genes (A/P) can be divided into three subsystems (Fig 21-53) –anterior group (4 genes) - head and thoracic segments lost –posterior group (11 genes) - abdominal segments lost –terminal group (6 genes) - non-segmented structures at both ends of the embryo lost –each of these three subsystems sets up its own morphogen gradient –these four primary spatial signals organize the early embryo by setting up broad patterns interpreted by later acting genes that refine the pattern sequentially

BioSci 108 lecture 26 (Blumberg) page 10 © copyright Bruce Blumberg All rights reserved Patterning genes are laid down in the egg egg polarity genes are transcribed in the cells surrounding the developing oocyte and transported there as maturation proceeds –transcribed from the maternal genome during oogenesis –products act before, or soon after fertilization –phenotype of embryo is determined by genotype of mother, not the embryo –almost all recessive mutations in these genes lead to effects two generations downstream (Fig 21-54) –maternal effect mutations identified by screening for mutant embryos from apparently normal mothers usual mutations are dependent 50% on genotype of father maternal effect mutations depend solely on genotype of mother –phenotype of offspring is dependent on genotype of mother, not embryo homozygous offspring appear normal but are sterile when mated with wt fathers alternative figure shows comparison of std and maternal effect mutations bottom line - components placed into the developing oocyte by follicle cells are responsible for setting up the initial pattern (Fig 21-55)

BioSci 108 lecture 26 (Blumberg) page 11 © copyright Bruce Blumberg All rights reserved Dorsovental patterning Dorsoventral patterning involves an extracellular signaling cascade not dissimilar to vulva induction in C. elegans. –actually two pathways a dorsally acting pathway acts to inhibit the action of ventral gene products a ventrally acting pathway acts to specify ventral and also inhibit the action of zygotic dorsalizing factors that we will talk about next time –all of so-called maternal dorsal group genes are actually genes responsible for forming ventral structures mutations in these dorsalize the embryo ventralizing pathway (dorsal gene product) (supp figure) –seven genes are required to localize the ventral morphogen spatzle –spatzle is the ligand for toll which is the key player –toll is a transmembrane tyrosine kinase receptor homologous to the IL-1 receptor IL-1 is required for antibody-mediated immunity in vertebrates! –after several steps, cactus protein is cleaved which releases dorsal protein from the cytoplasm into the nucleus –dorsal is the ventral morphogen

BioSci 108 lecture 26 (Blumberg) page 12 © copyright Bruce Blumberg All rights reserved Dorsovental patterning (contd) Dorsal protein acts on zygotic target genes in two ways (Fig 21-56) –activates expression of twist and snail which are required for mesoderm and other ventral structures –represses expression and action of decapentaplegic which is a dorsal morphogen in regions where dorsal is high enough to repress dpp but too low to activate twist, neurogenic ectoderm is formed –this is very similar to how neural tissue is induced in vertebrate embryos! –the book is behind the times in its description of how neural tissue is induced in vertebrates so wait for Developmental Biology to worry about this

BioSci 108 lecture 26 (Blumberg) page 13 © copyright Bruce Blumberg All rights reserved Terminal patterning terminal system is patterned through the action of a transmembrane receptor –torso is the key player and it is another receptor tyrosine kinase –torso ctivation 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 –huckebein is required for mesoderm and endoderm patterning important point to get from the D/V and terminal systems is that these use transmembrane signaling cascades to specify pattern –very much like the situation in vertebrate embryos –very unlike the A/P patterning system

BioSci 108 lecture 26 (Blumberg) page 14 © copyright Bruce Blumberg All rights reserved Anteroposterior patterning A/P patterning is performed by opposing morphogen gradients that work on the same target gene –active before the cellular blastoderm stage –morphogens regulate gene expression directly by diffusing along the embryonic A/P axis Existence of an anterior and posterior morphogens was suspected from experiments in which the embryo was punctured and cytoplasm allowed to leak out (Fig 21-58) –injection of anterior cytoplasm from wt embryos was able to rescue anterior structures –injection of anterior cytoplasm into the posterior of wt embryos could cause duplicated anteriors –injection of posterior cytoplasm into anterior led to duplicated posteriors taken together, these experiments demonstrated that the factors which determined anterior and posterior were located at the ends of the embryos and were diffusible –elegant confirmation of the morphogen gradient model

BioSci 108 lecture 26 (Blumberg) page 15 © copyright Bruce Blumberg All rights reserved Anteroposterior patterning (contd) Posterior system involves many genes -most involved in localizing the next most downstream gene –mutations cause loss of: abdominal structures (all) germ cells (all but two most downstream) –posterior and germ cell pathways branch at tudor –apparently, each of these gene products is required for localizing the next one in the pathway –book mentions oskar - about in the middle of the pathway one of the targets of oskar is vasa vasa protein found in germ cells of most species –end result of the posterior gene cascade is the localization of the nanos mRNA –nanos protein diffuses toward the anterior and forms a morphogen gradient specifies degree of posterior can replace posterior cytoplasm in injection experiments or rescue experiments

BioSci 108 lecture 26 (Blumberg) page 16 © copyright Bruce Blumberg All rights reserved Anteroposterior patterning (contd) anterior system involves only a small number of genes –most of these are responsible for localizing bicoid mRNA –mutations in the bicoid gene cause the loss of anterior structures –increase in the number of copies of the bicoid gene cause increases in the extent of the anterior bicoid protein is the active anterior morphogen (fig ) –concentration of bicoid protein, or the number of bicoid genes directly influences patterning –bicoid is a homeodomain protein that functions by directly activating the expression of its target gene hunchback –wherever bicoid is injected into an embryo, head structures form (supp figure) this proves that it is a true morphogen that acts directly to pattern the anterior –a vertebrate relative of bicoid exists, called goosecoid goosecoid is involved in patterning the anterior mesodermal tissues in the vertebrate embryo works by repressing transcription of target genes, rather than activating –can’t diffuse either since vertebrate embryos always have cells