BioSci 108 lecture 27 (Blumberg) page 1 © copyright Bruce Blumberg All rights reserved Bio /15/2000 Molecular Genetics of Pattern Formation II Contact information office hours W/F 3-4 phone (preferred contact mode) Lectures posted at Exam update –Dr. Cho and I will write the exam. –Questions will be approximately equally distributed between the sections each of us taught –The examination will not be identical to any given in previous years

BioSci 108 lecture 27 (Blumberg) page 2 © 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

BioSci 108 lecture 27 (Blumberg) page 3 © copyright Bruce Blumberg All rights reserved Segmentation genes subdivide the embryo The maternal genes have broadly divided the embryo into anterior, posterior, dorsal and ventral –this pattern of gene expression is interpreted by the next group of genes to act –the zygotically-expressed segmentation genes segmentation genes begin to be transcribed when zygotic transcription begins at the cellular blastoderm stage –segmentation genes control the number of segments and their polarity –segmentation genes do not influence the overall polarity of the embryo segmentation genes can be subdivided into three classes (Fig ) –criteria for classification phenotypes of the resulting embryos time that the genes act –gap genes - act first, interpret the maternal gradients and subdivide the embryos into broad domains begin to be transcribed in syncytial blastoderm mutations in a single gene eliminate one or more adjacent segments mutations in a different gap gene cause different but partially overlapping defects Krüppel mutations cause the loss of eight segments T1-A5

BioSci 108 lecture 27 (Blumberg) page 4 © copyright Bruce Blumberg All rights reserved Segmentation genes (contd) –Pair rule genes act next interpret the information specified by the gap genes and divide the embryos into 14 segments mutations cause deletion of alternating segments about eight genes have been identified all pair rule genes are expressed in two segment periodicity –but expression borders relative to segment boundaries differ among genes even-skipped (eve) mutants lack the whole of even-numbered parasegments fushi tarazu (ftz) mutants lack the whole of odd numbered parasegments hairy mutants lack periodic regions of similar width that are out of register with the parasegmental units –segment polarity genes are the last to act mutations produce larvae with a normal number of segments but part of each segment is deleted and replaced with a mirror image duplication of the region that is present in gooseberry mutants, the posterior half of each segment is replaced by a mirror image duplication of the anterior half segment

BioSci 108 lecture 27 (Blumberg) page 5 © copyright Bruce Blumberg All rights reserved Hierarchical regulation of segmentation genes Segmentation genes have all been cloned and the majority are transcription factors –can directly regulate the expression of other genes –one can compare how they act on one another and on other genes by comparing gene expression in normal and mutant embryos Molecular markers are a key concept in developmental biology –one can determine what has happened to a structure by observing the expression of genes that normally mark its presence –can actually deduce the regulatory circuits this way –example 1: even numbered parasegments are marked by eve expression. if eve expression is lost in a mutant, one can presume that even numbered parasegments are lost –example 2: in Xenopus, the organizer is marked by goosecoid expression. if gsc expression is lost, one can presume that the organizer has been lost –It is not possible to publish a paper today describing the morphological effects of a mutation - molecular markers are required

BioSci 108 lecture 27 (Blumberg) page 6 © copyright Bruce Blumberg All rights reserved Hierarchical regulation of segmentation genes (contd) phenotypes from mutations in gap genes do not precisely correspond to the regions where the mRNA for the gene is expressed –implies that the protein can diffuse beyond the boundaries of where the mRNA is expressed not possible after cellular blastoderm stage –example (Fig 21-61) both Kruppel and hunchback mRNAs show non- overlapping expression domains however, domains lost in mutants do overlap with each other significantly –implication gap genes regulate each other’s expression most are transcriptional repressors boundaries established between fields of gene expression are very important for subsequent patterning events expression of pair rule genes corresponds closely to the regions that are deleted in loss-of-function mutations (Fig 21-62). –products of some early acting genes can diffuse a bit from their site of synthesis –others (e.g. ftz) are expressed only in the structures that would be lost expression of segment polarity genes corresponds precisely with regions lost in loss-of-function mutations

BioSci 108 lecture 27 (Blumberg) page 7 © copyright Bruce Blumberg All rights reserved Hierarchical regulation of segmentation genes (contd) developing hierarchy –egg polarity genes provide global signals –these cause gap genes to be expressed in particular regions of the embryo –products of gap genes produce a second tier of positional signals that refine the pattern by regulating the pair rule genes –pair rule genes interact combinatorially to regulate the segment polarity genes ultimate aim is to precisely specify pattern while using a minimum number of components –this is accomplished by sequentially refining a broad pattern created by only a few genes –mechanism is not so different from intercalary regeneration that we discussed a few lectures back –two possible ways to interpret a gradient to generate pattern (Fig 21-63) cells can respond to morphogen gradient directly by adopting many states (analog model) –requires very sensitive discrimination cells can respond to morphogen gradient by adopting one state or another (digital model) –this sets up two new local signals (or positional confrontations) that can generate a new state in between –no sensitive discrimination required

BioSci 108 lecture 27 (Blumberg) page 8 © copyright Bruce Blumberg All rights reserved Hierarchical regulation of segmentation genes (contd) how does one map the hierarchy? –test expression of positional markers in mutant backgrount –experiment: test expression of ftz in Kruppel background observation –ftz expression is lost but only in the region where Kruppel is normally expressed conclusion –kruppel directly or indirectly regulates the expression of ftz in that part of the embryo –experiment: test expression of Kruppel in ftz background observation –no effect on Kruppel expression conclusion –ftz is downstream of kruppel –ftz product does not feed back to regulate kruppel combinatorial regulation of segmentation genes –interactions between genes in the same tier of the hierarchy are also important (e.g. gap genes) –Kruppel and hunchback are expressed with a sharp boundary between it turns out that these repress each other

BioSci 108 lecture 27 (Blumberg) page 9 © copyright Bruce Blumberg All rights reserved Hierarchical regulation of segmentation genes (contd) combinatorial regulation of segmentation genes –interactions between genes in the same tier of the hierarchy are also important (e.g. gap genes) –Kruppel and hunchback are expressed with a sharp boundary between it turns out that these repress each other also occurs with other gap genes, most of which are transcriptional repressors this mutual repression helps to establish the very sharp borders of expression and precisely position the expression of downstream genes –similar mechanisms aid in establishing the sharp expression boundaries of the pair rule genes (Fig ) these genes interpret information from gap genes to yield a reproducible expression pattern of mutual exclusions. combination of early expressed pair rule genes and gap genes helps to position the later pair rule and segment polarity genes each cell becomes distinguishable by a pattern of pair rule gene expression (~4 cells wide) (Figure 21-65) –combinations of pair rule genes can specify regions for segment polarity gene expression even down to the single cell level (e.g. engrailed) (other pix)

BioSci 108 lecture 27 (Blumberg) page 10 © copyright Bruce Blumberg All rights reserved Hierarchical regulation of segmentation genes (contd) entire process of regulating zygotic gene expression and the interactions among patterning genes occurs at the level of transcriptional regulation –promoters of the gap, pair rule and segment polarity genes contain multiple binding sites for other members of the families –many of these factors compete for the same binding sites allows precise control of spatial expression same sites can be used by different proteins that may bind to the same site but are expressed in different compartments –many homeodomain proteins bind to the same targets

BioSci 108 lecture 27 (Blumberg) page 11 © copyright Bruce Blumberg All rights reserved Combined action of maternal and segmentation genes Observation is that genes are first expressed in patterns that only approximate the final picture (Fig 21-65) –the pattern is later refined so that domains are precisely delineated –pattern is transient gastrulation movements disturb the spatial patterns of the gap and pair-rule genes but these have already imposed positional values onto the cells –these labels must be remembered for patterning process to continue expression patterns of segment polarity genes and homeotic selector genes are responsible for maintaining the cellular memory of early patterning events segment polarity genes mark the parts of each parasegment –expressed in repeated patterns –each parasegment is divided into three regions by the concerted action of the pair rule genes –this leads to specific localization of segment polarity genes –many of these genes are extracellular receptors and their ligands (e.g. wingless) –these rapidly set up autoregulatory loops to maintain expression but keep it localized

BioSci 108 lecture 27 (Blumberg) page 12 © copyright Bruce Blumberg All rights reserved Combined action of maternal and segmentation genes segment polarity genes mark (contd) –the chemical distinction created by segment polarity genes may persist into adulthood –e.g. engrailed is first expressed in on row of cells from each parasegment (Fig ) anterior border of each parasegment posterior border of segment it persists in the posterior part of each segment in the adult in addition to regulating the segment polarity genes, the product of the pair rule, gap and maternal genes act together to cause localized regulation of homeotic selector genes that we will talk about next lecture