1. BioSci 108 lecture 28 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved Bio 108 - 3/17/2000 Molecular Genetics of Pattern Formation III Contact information office hours W/F 3-4 phone 824-8573 blumberg@uci.edu (preferred contact mode) Lectures posted at http://blumberg-serv.bio.uci.edu/bio108-w2000/index.htm http://blumberg.bio.uci.edu/labtemp/bio108-w2000/index.htm 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

2. BioSci 108 lecture 28 (Blumberg) page 2 © copyright Bruce Blumberg 2000. All rights reserved Homeotic selector genes specify segment identity Hierarchy –maternal egg polarity genes divide the embryo into broad regions anterior/posterior, dorsal/ventral –gap genes respond to this information and divide the embryo into smaller domains along the A/P axis –pair-rule genes respond to the gap genes by being expressed in 7 stripes along the A/P axis –segment-polarity genes respond to the pair-rule genes by subdividing the embryo into 14 parasegments, each of which contains 3 compartments. –Homeotic selector genes interpret the pattern created by the actions and interactions of the egg polarity, gap, pair-rule and segment-polarity genes to create unique anteroposterior positional codes for each compartment of the embryo Drosophila has two clusters of homeotic selector genes –antennapedia and bithorax complexes –most other invertebrates and insects have a single complex that contains both antennapedia and bithorax complexes can think of antp and bx complexes as the two halves of HOM-C –The invertebrate complex is collectively referred to as HOM-C. The correxponding complexes in vertebrates are called the HOX-C

3. BioSci 108 lecture 28 (Blumberg) page 3 © copyright Bruce Blumberg 2000. All rights reserved HOM-C (contd) Homeotic selector genes have precise domains of action –like the pair-rule genes, the domain of action is the parasegment (Fig 21-50) –homeotic genes have precise expression boundaries, but these are typically only at either the anterior or posterior –mutations are dramatic -> antp (Fig 21-67) homeotic selector genes are recessive lethal –embryos typically do not survive beyond hatching of the egg into a first instar larva –practically speaking, this means that one must evaluate such mutations at ~24 hours or so (just when the larva would ordinarily hatch –clear view of what is happening (Fig 21-68) –larvae deficient in the entire bithorax complex are particularly interesting. All of the parasegments posterior to P5 take on the character of P5 P5 is T2/3 these flies consist of a head,T1 and then 11 segments which look more or less like T2 T2 has a pair of wings and a pair of legs –ancestral “ground state” –essential role of homeotic selector genes is to define the differences among parasegments loss of gene function = loss of differences

4. BioSci 108 lecture 28 (Blumberg) page 4 © copyright Bruce Blumberg 2000. All rights reserved HOM-C (contd) Homeotic selector genes are molecular address labels –first activated at cellular blastoderm –all of HOM-C genes are cloned and expression patterns have been analyzed –to a first approximation, the anterior border of each gene coincides with the regions that develop abnormally in loss-of-function mutations –this implies that these genes encode molecular address labels for each cell if the address labels are changed, the cells behave as if they are somewhere else –expression patterns of HOM-C genes coincides with parasegmental boundaries established by segmentation genes combination of HOM-C and segmentation gene products defines a unique address for cells in one subdivision of one segment Homeotic selector genes are the “keepers” of positional information –all homeotic selector genes are transcription factors –all contain a highly conserved DNA sequence called the “homeobox” –the homeobox encodes a segment of the protein called the “homeodomain” homeoboxes were first identified in homeotic selector genes, hence the name

5. BioSci 108 lecture 28 (Blumberg) page 5 © copyright Bruce Blumberg 2000. All rights reserved HOM-C (contd) Homeotic selector genes and positional information (contd) –together the antennapedia and bithorax complexes contain 8 genes (the HOM-C) these are very large transcription units (more than 300 kilobases each) these large transcription units contain a large number of regulatory elements that control the temporal and spatial expression of the genes these regulatory elements contain binding sites for the products of egg polarity and segmentation genes –this is the molecular mechanism underlying the combinatorial regulation elicited by these genes –these binding sites are the interpreters of positional information

6. BioSci 108 lecture 28 (Blumberg) page 6 © copyright Bruce Blumberg 2000. All rights reserved HOM-C (contd) Position of genes within the complex corresponds to the regions of expression along the A/P axis (Fig 21-69) –order of genes is invariant across species –all genes are transcribed in the same direction –most proximal genes are expressed in the anterior, more distal genes are expressed in the posterior –appears that the genes are serially activated by an unknown process that spreads along the chromosome –most of the genes have unique 5’ borders but the expression domains can overlap posteriorly it turns out that the proteins are expresses in a more restricted pattern than the mRNAs –overlapping expression patterns means more genes are expressed in the posterior –this leads to predictions about the effects of mutations loss-of-function -> anterior transformations gain-of-function -> posterior transformations –questions remaining: why is direction of transcription the same? why is order conserved? –accident or design? why are inversions and rearrangements not tolerated?

7. BioSci 108 lecture 28 (Blumberg) page 7 © copyright Bruce Blumberg 2000. All rights reserved HOM-C (contd) There are not enough HOM-C genes for all parasegments –only 8 HOM-C genes but 14 parasegments –bithorax complex contains only 3 genes but is responsible for the differences among 10 parasegments –many of Bx-C mutations affect only a single segment or part of a segment map along the chromosome in the order of expression most map to noncoding regions of DNA, putative regulatory sequences –suggests that differences between body regions are defined not only by the presence of homeotic gene products but by persistent differences in states of the control regions implies that control regions are not simple on-off switches but integrate the signals received in a complex way

8. BioSci 108 lecture 28 (Blumberg) page 8 © copyright Bruce Blumberg 2000. All rights reserved HOM-C (contd) What is the mechanism that retains positional memory? –one good mechanism is positive feedback once a gene product is expressed, it stimulates its own expression many HOM-C genes have autoregulatory binding sites in their promoters –but positive feedback, in itself, is insufficient to maintain memory without other factors –another group of genes, the Polycomb group, are required to repress homeotic selector genes that should not be expressed in a particular region loss-of-function mutations result in indiscriminate upregulation of HOM-C genes all over the embryo polycomb is bound to the chromatin of the genes it controls –genes related to polycomb appear to be involved in the control of chromatin structure this suggests that persistent modifications in chromatin structure can also have an important role in positional memory

9. BioSci 108 lecture 28 (Blumberg) page 9 © copyright Bruce Blumberg 2000. All rights reserved HOX-C Vertebrates have homologous developmental control genes to invertebrates –homologous means derived from a common ancestor –when Drosophila homeobox genes were identified, researchers screened for homologs in vertebrates –an important point to remember is that although not all developmental mechanisms are conserved, the genes employed to control development are the same across species. e.g. bicoid and goosecoid dpp and BMP-4 –the vertebrate homologs of the HOM-C are called HOX-C Genes homologous to HOM-C are found in all organisms that have been examined –C. elegans –Hydra –they are invariably grouped into complexes that maintain the order and direction of transcription HOX-C and HOM-C complexes contain very similar genes (Fig 21-80) –similar (paralogous) genes occupy the same position in each complex! –this suggests that the HOM-C(HOX-C) complex was invented very early in evolution retained retained and used with only few changes

10. BioSci 108 lecture 28 (Blumberg) page 10 © copyright Bruce Blumberg 2000. All rights reserved HOX-C (contd) Expression of HOX-C genes is highly ordered along the A/P axis (Fig 21-81) –most clearly seen in the developing neural tube –A/P position mirrors position in the complex Gene expression domains draws parallels between the body parts of insects and vertebrates (fig 21-82) –stop and think about this for a minute - the genes that specify head in Drosophila are the same genes that pattern your head e.g. labial and HOX-A,B,D-1 genes –as in Drosophila compartments, the regions specified by HOX-C genes do not cross compartment boundaries –clearest example is in the vertebrate hindbrain segments are called rhombomeres lateral to rhombomeres are the branchial arches –one branchial arch/two rhombomeres Vertebrates have four HOX-C complexes –two genome duplications in the vertebrate lineage four complexes are organized like HOM-C –some genes may be lost in one complex –additional internal duplications have occurred »paralog 3 genes and dfd »paralog 10-13 and AbdB

11. BioSci 108 lecture 28 (Blumberg) page 11 © copyright Bruce Blumberg 2000. All rights reserved HOX-C (contd) HOX-C genes specify a discrete combinatorial code that uniquely identified each cell in the body –called the HOX code –alterations in the HOX code cause predictable changes in patterning loss-of-function cause anterior transformations gain-of-function cause posterior transformations –since there are multiple copies of each HOM-C gene, it is difficult to obtain perfect homeotic transformations important concept: functional redundancy this means that the if one gene is knocked out, the remaining paralogous genes can partially compensate –HOX-C and HOM-C genes bind to the same target DNA sequence suggests that differences in affinity may be an important factor in regulation also suggests that the genes may be functionally redundant vertebrate HOX genes can rescue Drosophila homeotic mutations –best proof of conserved function –this is also a good justification for studying simpler model organisms that only have a single gene at each position in the complex

12. BioSci 108 lecture 28 (Blumberg) page 12 © copyright Bruce Blumberg 2000. All rights reserved Final comments You may have noticed that prices at the UCI bookstore are not especially good. –in fact, the bookstore marks items up by a fixed amount (26%) over cost –the very frequent result is that books cost MORE than the publisher’s list price –what can you do? buy your books direct from the publisher online buy your books from an online discount bookstore but be careful about edition numbers –a good resource is http://www.bestbookbuys.com/ this site has a search engine that compares prices and availability of a book among many booksellers. some students are not happy with the selection of upper division satellite courses required –one solution is to complain to the Dean, Susan Bryant –another possibility is to take graduate courses. Most of these are open to undergraduates who have completed the core and a few other satellites. feel free to ask the instructor