1	Introduction to Molecular Embryology Dr Luc Laurier OLIGNY Pediatric pathologist CHU Sainte-Justine Université de Montréal luc_oligny@ssss.gouv.qc.ca
2	Course objectives Exposure to major actors in molecular embryogenesis Understanding the interaction of these actors in organogenesis Elaboration of a molecular approach to malformations Links between embryology & oncology
3	Course objectives Quick overview of molecular embryology More details provided in IAP’s web-based handout
4	Overview All our cells have identical DNA Epigenetic mechanisms control cell-specific gene expression, by modifying chromatin Differentiation is achieved through the control of transcription (epigenetic regulation)
5	Overview Master switch genes control differentiation, by activating & inhibiting downstream cascades
6	Overview Embryonic development: - cell proliferation & death - segmentation ( HOX, PAX, etc) - cell adhesion / migration (CAMs, integrins, chemotaxis)
7	Course objectives Review of the interactions between these key players, with formation of the Central Nervous System
8	Course objectives Development of a molecular diagnostic approach to malformations
9	Zinc-finger transcription factors
10
11
12
13
14
15
16
17
18
19	From zygote (1 cell) to newborn (10¹⁴ cells)
20
21	Cellular differentiation For a cell to differentiate into a hepatocyte, it must: activate all the genes necessary for achieving a hepatocyte’s structure and function and inactivate all the other genes
22	Cellular differentiation Differentiation is thus achieved through the control of the transcription of a cell’s genes
23	Chromatin Vs DNA DNA + Protein backbone, including: - transcription factors - histones
24	Epigenetics - Definition Alterations in chromatin that do not change the inherent DNA sequence but which affect gene transcription
25	Epigenetics - Definition Epigenetic marks are transmitted from one cell to all its daughter cells: - Marks are erased in late morula, partly through a global de-methylation - Marking re-initiated during late blastocyst stage (64 - 128 cell-stage, 4 days pc)
26	Differentiation is normally irreversible In cancer: - normally silenced genes can be reactivated - normally active genes can be silenced
27	Epigenetic control of transcription Methylation of cytosines Acetylation of histones Methylation, ubiquitination and phosphorylation of histones Polycomb and Trithorax
28
29
30	Methylation of cytosine by DNA methyltransferase
31
32
33	DNA methylation Methylation of cytosine in CG decreases binding of transcription factors: - methylation of gene promoters inhibits transcription
34	DNA methylation Initial gene inhibition accomplished through transcription factors DNA methylation plays a major role in the long-term maintenance of gene inhibition
35	DNA methylation and cell replication
36
37
38
39
40	Histones and acetylation Acetylation favors transcription: - acetylation of histones alters the 2^ry and 3^ry structure of DNA - promotes interaction of DNA with transcription factors
41
42	Histone modifications (histone code) Acetylation Phosphorylation Ubiquitination Methylation Effect on transcription depends on which histone and which amino acid is modified, and on the number of methyl groups added Very complex and poorly understood
43	Histones and acetylation Acetylation of histones modulates: - nucleosome formation - compaction of DNA into chromatin - interaction with transcription factors, Pc and TTX
44
45	Histones and acetylation Through poorly understood mechanisms, acetylation is passed on from one differentiated cell to all its descendants
46	Trithorax and Polycomb proteins Large families of proteins TTX generally activates transcription Pc generally inhibits transcription - Histone-code controls Pc-binding to chromatin
47
48
49	Cell proliferation Proto-oncogenes ~ growth factors Anti-oncogenes ~ tumor suppressor genes
50	Cell proliferation Proto-oncogenes and anti-oncogenes: - Proto-oncogenes ~ growth factors - oncogenes are mutated proto-oncogenes - the mutation leads to an unregulated over- expression
51	Cell proliferation Proto-oncogenes and anti-oncogenes: - Anti-oncogenes ~ tumor suppressors In embryos, it is the physiologic interaction between proto-oncogenes and anti-oncogenes which regulates the rate of cell proliferation
52	Apoptosis During normal embryogenesis, a large number of cells and tissues are eventually resorbed: Apoptotic stimulation Autophagic enzymes activated Cellular suicide
53
54	Limb bud / hand development
55	Diabetes causes apoptosis
56	Master Switch Genes Master switch genes = Selector genes Master switch genes code for Transcription Factors
57	Master Switch genes - They are activated at different moments during embryogenesis - It is the cumulative effect of many selector genes’ proteins (TF’s) at the level of promoters, enhancers and inhibitors which determines the level of expression of their subordinate genes
58	Control of the transcription of the gene even-skipped
59	Master Switch Genes Activation of a single master switch gene Synchronous regulation of a large battery of subordinate genes necessary for the differentiation of a cell or tissue
60
61	MYO-D: an example of a master switch gene MYO-D: myoblast differentiation gene Any primitive mesenchymal cell expressing MYO-D becomes a skeletal myocyte
62	MYO-D - A master switch gene MYO-D is a transcription factor: - activates genes involved in skeletal muscle differentiation - inhibits non-muscular differentiation
63	MYO-D The MYO-D protein also binds and activates its own promoter to ensure epigenetic transmission: - positive feedback-loop activation of its promoter: once a cell expresses MYO-D, it cannot stop its production - auto-activation causes all the descendents of this cell to express MYO-D - cell line committed to a muscular differentiation
64
65	MYO-D, MYOGENIN and double insurance MYOGENIN is a gene distinct from MYO-D: - both code for proteins with very similar functions - both are normally expressed - each can substitute for the other in the event of a mutation Such a “double insurance” mechanism is found in many developmentally critical molecular cascades
66	Myo-D / Myogenin Double insurance (redundance)
67	Master switch genes - conclusions The genes which control the fundamental aspects of development do so by controlling a whole battery of subordinate genes
68	Homeotic genes By definition, a mutation of a homeotic gene causes one body segment to differentiate into the structures of another segment: - the segment loses its “positional identity” - e.g., a mutation of antennapedia results in the replacement of antennas by legs
69	Homeotic genes Each homeoprotein acts on multiple promoters, enhancers and inhibitors, to regulate the expression of its subordinate genes It is the cumulative effect of the multiple activating and inhibitory transcription factors at a given gene which will determine the extent of expression of that gene, i.e., from nil to extreme
70	Homeotic complexes and HOX
71
72
73
74	Myotomes originate from the HOX segmentation
75	Vertebrate segmentation - Synopsis There is a unique combination of homeoproteins at the level of each segment These different combinations of homeoproteins control the expression of specific morpho-proteins whose role is to control the differentiation of the cells expressing them
76
77	PAX transcription factors
78	PAX - control of cellular interactions
79
80	PAX6 Sine qua non of ocular development: - ectopic PAX6 → formation of a normal eye in Drosophila (without an optic nerve) - PAX6 +/- : → microphthalmia - PAX6 -/- : → anophthalmia
81	Vertebrate segmentation - Synopsis Each cell of the embryo has a molecular address, which corresponds to the transcription factors (master switch genes) expressed by that cell. It is this address which determines the differentiation path which that cell follows
82	Cell Adhesion Molecules (CAMs) Divided in large super-families: - Immunoglobulin-like family of CAMs (each of which contains many members): - Cadherin families (E- , N- and PCadherin) - N-CAM, Ng-CAM, L-CAM, A-CAM and I-CAM families - Integrin family (> 20 members)
83
84	Cell Adhesion Molecules - CAMs are localized on cell surfaces - Different cell types express different CAMs - CAMs mediate the adhesion of cells with their neighbors
85	Cell Adhesion Molecules A CAM is chemically attracted by its identical type, and may attract or repulse other types of CAMs: - When cells express CAMs which are attracted to one another, these cells are essentially attached or glued together: - CAMs cause a dissociated frog embryo to reassemble into the 3 germ layers
86
87
88
89	Cell Adhesion Molecules Integrins anchor cells onto the extra-cellular matrix
90	Cell Adhesion Molecules CAMs and integrins are part of the cell migration machinery: - normal embryogenesis - tumor invasion - metastasis
91
92	Basic neural development Rapid proliferation of neuroblasts throughout neural tube Differentiated neurons can no longer divide: each has a “date of birth” regulating its differentiation birth of neurons of same type generally occurs during a very limited period
93	Basic neural development Radial migration of neurocytes, following glial cells role of CAMs The birth place of a neuron is as important as its birth date: - controls the expression of HOX and multiple other morphogens
94
95
96	Basic neural development Once radial migration completed, neurons send neurites guided by chemotaxis and CAMs to make connections with other neurons Neurites of a similar group share the same CAMs and thus migrate together, forming “tracts” Tracts migrate independently from one another
97	NGF stimulates: - growth of neurites - chemotactic attraction of neurites
98	Basic neural development Targets secrete trophic substances (e.g., NGF): - neurites reaching target early survive - non-specific connections are formed - later neurites undergo apoptosis Ten-fold reduction of neurites and neurons
99	Basic neural development Non-specific synapses eliminated by retrograde depolarization Ten-fold reduction of neurons
100	Elimination of non-specific connections
101	Epilogue Morphogenesis can be reduced to a few molecular concepts which provide a molecular approach to teratology
102	DiGeorge syndrome Abnormal development of 3^rd and 4^th branchial arches 22q12 +/-: In familial cases i.e., with same deletion: - variable expressivity - reduced penetrance
103	DiGeorge syndrome Haploinsufficiency: - both 22q11.2 alleles required for normal phenotype (locus of TBX1 gene) - 22q11.2 +/-: insufficient production of TBX1: - phenotype depends on amount of TBX1 - phenotype depends on sensitivity of TBX1 promoters and of its downstream effectors Need to think in terms of cascade
104
105	Polymorphisms in upstream cascades (acting on promoter)
106	Polymorphisms in downstream cascades
107	Limb malformations - a molecular approach Diagrams from Manouvrier-Hanu, in Potter’s Pathology of the Fetus, Infant and Child, 2^nd ed, Courtesy of Elsevier Publishers
108
109
110
111
112
113
114	Need to think of molecular embryology in terms of cascades !
115
116	“Classical cancer genetics”
117	“Classical cancer genetics” Point mutations Frameshift mutations Numerical anomalies
118	Methylation and cancer Early: 9 DNA Methyl Transferase (DNMT) - non-specific gene activation - de-differentiation Reactivation of : - oncogenes - telomerases - angiogenic factors - metastasis-promoting CAMs
119	Methylation and cancer Late: 8 DNMT - non-specific gene inactivation Inactivation of : - anti-oncogenes - apoptotic cascade - angiogenesis inhibitors - metastasis-promoting CAMs
120	Methylation and cancer Early: 9 DNA Methyl Transferase (DNMT) - non-specific gene activation Late: 8 DNMT - non-specific gene inactivation Fertile grounds for a “Darwinian” clonal evolution
121	Normal cell replication . . . . . . . . . C^metG . . . . . . . . . . . . . . . . . . . GC^met . . . . . . . . . . DNMT (~ 100 % ) . . . . . . . . . C^metG . . . . . . . . . . . . . . . . . . . GC^met . . . . . . . . . .
122	Cell replication in cancer . . . . . . . . . C^metG . . . . . . . . . . . . . . CG . . . . . . . . . . . . . . . . . GC^met . . . . . . . . . . . . . . . GC . . . . . . . . Most Rare Most Rare ... C^metG ... ... CG ... ... CG … ... C^metG ... ... GC^met … ... GC … ... GC ... ... GC^met ...
123	Cell replication in cancer . . . . . . . . . C^metG . . . . . . . . . . . . . . CG . . . . . . . . . . . . . . . . . GC^met . . . . . . . . . . . . . . . GC . . . . . . . . Most Rare Most Rare ... C^metG ... ... CG ... ... CG … ... C^metG ... ... GC^met … ... GC … ... GC ... ... GC^met ... Loss of DNMT specificity
124	Similarities - Embryo & Cancer - Proliferation - Invasion - Migration - Angiogenesis - Immortality Genes reactivated in cancer cells
125	Epigenetic changes in cancer Inhibition of H19 6 Re-expression of oncogenes (e.g., IGF2) Inhibition of other tumor suppressor genes e.g., p16^INK4A, APC Inhibition of genes involved in DNA repair e.g., MLH1, BRCA1
126	Epigenetic changes in cancer Alterations of extracellular matrix and cell adhesion molecules: - re-expression of mitogenic integrins - changes in CAMs to promote metastasis Reactivation of telomerase
127
128	Epigenetic changes in cancer Inhibition of angiogenesis inhibitors (e.g., TSP-1, TIMP-3) 6 neovascularization Inhibition of protease inhibitors (e.g., TIMP-3) 6 digestion of extracellular matrix
129	Epigenetic changes in cancer Modifications of Master Switch Genes Allow abnormal inactivation of some genes And abnormal activation of other developmental genes Leading to de-differentiation (e.g., presence of muscle cells, cartilage, etc. in Wilms tumors)
130	Diagnostic implications Hypermethylation is an early event in oncogenesis: - very early marker of cancer - lung cancer cells in sputum - colorectal adenomas in feces Methylation-sensitive PCR
131	Knudson’s “two hit” theory Knudson’s two hit theory must include epigenetic hits in addition to conventional mutations
132	Therapeutic implications Clinical trials: DNMT-inhibitor: - e.g., 5-azad-C (5-aza-2’-deoxycytidine) HDAC-inhibitor: - e.g., TSA (trichostatin-A)
133	Therapeutic implications DNMT-inhibitors and HDAC-inhibitors: Reactivation of TSGs (anti-oncogenes)
134	Therapeutic implications DNMT-inhibitors in clinical trials - best reported response rates: Adenocarcinomas Breast: 63% Ovary: 25% Colorectal: 30% Lung: 20% Prostate: 16%
135	Therapeutic implications DNMT-inhibitors in clinical trials - best reported response rates - others: Melanoma: 40% Mesothelioma: 17% AML: 89%
136
137	References Wilson GN, Oligny LL. Mechanisms of development and growth: molecular genetics. Potters pathology of the fetus, infant and child, 2nd ed. Edited by E. Gilbert-Barness, RP Kapur, LL Oligny and JR Seibert. Elsevier publishers, in press (Dec 2006). Bird A: DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16:6-21. Petronis A: Human morbid genetics revisited: relevance of epigenetics. Trends Genet 2001; 17:142-146.
138	"Goll MG," Goll MG, Bestor, TH: Histone modification and replacement in chromatin activation. Genes Dev 2002; 16:1739-1742. Li, E : Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 2002; 3: 662-673. Jenuwein T, Allis CD: Translating the histone code. Science 2001; 293:1074-1080. Mohd-Sarip A, Verrijzer P. A higher order of (chromatin) silence. Science 2004; 306:1484-1485.
139	"Gilbert SF:" Gilbert SF: Developmental Biology, 8^th ed. Sunderland MA: Sinauer 2006. Mahmoudi T, Verrijzer, CP: Chromatin silencing and activation by Polycomb and Trithorax group proteins. Oncogene 2001; 20:3055-3066. Yelon D, Stainier DYR: Pattern formation: swimming in retinoic acid. Curr Biol 2002; 12:R707-R709. Shintani T, Klionsky DJ. Autophagy in health and disease: A double-edged sword. Science 2004; 306:990-995.
140	"Lohmann I," Lohmann I, McGinnis W. Hox Genes: It's All a Matter of Context. Curr Biol 2002; 12:R514-R516. Akin ZN, Nazarali AJ. Hox genes and their candidate downstream targets in the developing central nervous system. Cell Mol Neurobiol. 2005; 25:697-741. Odenwald WF. Changing fates on the road to neuronal diversity. Dev Cell 2004; 8:133-134. Wen Z, Zheng JQ. Directional guidance of nerve growth cones. Curr Opin Neurobiol. 2006; 16:52-58.
141	"Grier DG," Grier DG, Thompson A, Kwasniewska A et al. The pathophysiology of HOX genes and their role in cancer. J Pathol 2005; 205:154-171. Yuspa SH, Epstein EH Jr. An anchor for tumor cell invasion. Science 2005; 307:1727-1728. Baldini A. Dissecting contiguous gene defects: TBX1. Curr Opin Genet Dev 2005; 15:279-84. Swales AK, Spears N. Genomic imprinting and reproduction. Reproduction. 2005; 130:389-99.
142	"Thompson JR," Thompson JR, Williams CJ. Genomic imprinting and assisted reproductive technology: connections and potential risks. Semin Reprod Med 2005; 23:285-95. Baylin SB, Ohm JE. Epigenetic gene silencing in cancer – a mechanism for early oncogenic pathway addiction? Nat Rev Cancer 2006; 6:1107-116. Yoo CB, Jones PA. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 2006; 5:37-50.