Tomáš Bárta tbarta@med.muni.cz Regulation of cell and organ size in development and examples of their necessity Contents •Introduction Embryo size Signaling pathways in individual/organ size regulation Aspects of regulation Growth rate/duration Examples – Drosophila, Human Regulation of organ size Size control - food for thought •The Basic Question of Developmental Biology We still don't know the clear answer Suncus etruscus vs ? Balaenoptera musculus The same plan of the body (propositions must be preserved), the size of tissues and organs largely differs Size control - food for thought •Improper regulation leads to dwarfism, gigantism, and/or hypo- or hyperplasia in organs. •Size is the most basic phenotype It affects many aspects of animal biology: anatomy, physiology, behavior... Therefore, control of body/organ size is a key developmental process that ensures that an animal grows to a size that is typical of its species. Size control - food for thought Cell number regulation Cell size control How is the size of embryo regulated? Lewis and Rossant 1982 „Size regulation appeared to be brought about by alteration in cell cycle length. There was no obvious increase in cell death in the double embryos nor an increase in the non-dividing cell population.„ If we aggregate two embryos together - a normal individual is formed •How does an embryo know that it should have the right number of cells? How and when does it adjust the number of cells? By lengthening the cell cycle, the number of cells is adjusted. This happens after implantation, but before gastrulation If there are too many cells: How is embryo size regulated? If we aggregate two embryos together - a normal individual is formed In a double embryo, apoptosis is induced only by epiblast cells (no others) that are not in contact with the basement membrane and only in the early embryonal stage. Orietti, 2020 Demonstrated on an isolated epiblast in vitro - without trophectoderm contribution Orietti, 2020 If there are too many cells: How is embryo size regulated? Demonstrated using na Embryonic Stem Cells Inhibition of apoptosis Inhibition of apoptosis did not lead to the formation of an epithelium. Orietti, 2020 If there are too many cells: How is embryo size regulated? Apoptosis, however, does not regulate the size of the embryo! Embryos with inhibited apoptosis were able to adjust their size Orietti, 2020 If there are too many cells: How is embryo size regulated? If there are too many cells: How is embryo size regulated? Summary: •The embryo compensates for the increased number of cells by lengthening the cell cycle, not by apoptosis. However, apoptosis is necessary for the proper formation of the epiblast epithelium. Two different mechanisms: apoptosis x slowing of proliferation Apoptosis is necessary to arrange the epiblast epithelium in "double" embryos Slowing the cell cycle If there are too many cells: How is embryo size regulated? So, is there a checkpoint in development where the embryo "checks" its size? ► Half-embryos have the same timing of development as normal embryos, but their developmental potential is reduced. ► In order to maintain development, the presence of at least 4 pluripotent cells is required. ► Fgf/Wnt modulation leads to correction of the development of half embryo Morris, 2012 If there are too few cells: How is embryo size regulated? Morris, 2012 If there are too few cells: How is embryo size regulated? Summary: •Half embryos complete their development (not all) No 1/4 embryo completes its development - it lacks cells. Can be compensated by modulation of FGF, Wnt signaling. If there are too few cells: How is embryo size regulated? What is involved in size control? Insulin/IGF-, RAS/RAF/MAPK-, TOR, Hippo, and JNK pathways •Crosstalk between them Growth rate Length of growth Size (organ tissues) Growth coordination Include: •Genes Signal pathways Hormones Together they provide a properly proportioned and reasonably sized body Signaling pathways that are involved in size control Size – Signalling pathways – Insulin/IGF1 Insulin/Insulin-like growth factor (IGF) Signaling (IIS) pathway Amount of IGF depending on nutrition/nutrients Size – Signalling pathways – Insulin/IGF1 Insulin/Insulin-like growth factor (IGF) Signaling (IIS) pathway Amount of IGF depending on nutrition/nutrients Velikost – signální dráhy – Insulin/IGF1 Size - signalling pathways - Ras/Raf/MAPK IGF activated – see previous slides receptor tyrosine kinases EGF/FGF/VEGF Size - signalling pathways - TOR •Similar to IGF, it also regulates growth based on the presence of nutrients, energy, oxygen Conserved from yeast to man IGF and TOR are the major players involved in the transmission of information on the presence of nutrients. TOR = target of rapamycin Size - signalling pathways - TOR Size - signalling pathways - Hippo Drosophila v. Mammals Hippo = Mst1/2 Salvador = Sav1 Warts = Lats1/2 Mats = Mob1A/B Size – signalling pathways - JNK •Stress pathway, regulates cell death, tissue regeneration, wound healing Size - signalling pathways - Other •Signal pathways that control pattering: • •They control the shape and arrangement of the organ - changes in cell growth and proliferation Hh, Wnt, TGF-β Size – Signalling pathways – Conclusion and Questions •Know the basic signal pathways that regulate size. Aspects of Size Control – Drosophila Imaginal discs – model system The size of the disc determines the size of the organ Aspects of Size Control – Drosophila Engrailed GAL4 GFP UAS GAL4/UAS system: a geneticist's Swiss army knife Aspects of Size Control – Drosophila GAL4/UAS system: a geneticist's Swiss army knife Aspects of Size Control •The above-mentioned (in intro) signaling pathways regulate growth and proliferation, but this does not explain the very nature of size control. The size of the tissue/organ/individual determines the growth rate and the duration of growth. Growth rate Controlled by signaling pathways that regulate cell proliferation and growth Growth duration It is regulated by systemic hormonal signals that coordinate the cessation of growth throughout the body, as well as organ-autonomic processes that ensure that organs stop growing once they reach their final size. Growth rate Controlled by signaling pathways that regulate cell proliferation and growth. Aspects of Size Control Aspects of Size Control – Growth Rate •Regulated by cell growth and proliferation => more larger cells/time = larger organ/individual Growth and proliferation are regulated by different pathways. Cell growth: 75% of the cell is made up of water, ions and small molecules 18% is made up of proteins => growth depends on protein synthesis. •S6K (S6 kinase) is a ribosomal 40S protein => controls ribosomal protein synthesis S6K deficiency (I-1/I-1) – delayed development, smaller individual but the same number of cells Aspects of Size Control – Growth Rate S6K is a ribosomal 40S protein => controls protein synthesis S6K deficiency (I-1/I-1) – delayed development, smaller individual, smaller organs, but the same number of cells Montagne et al., 1999 Aspects of Size Control – Growth Rate S6K is a ribosomal 40S protein => controls ribosomal protein synthesis dS6K (overexpression) – larger cells Montagne et al., 1999 Aspects of Size Control – Growth Rate Aspects of size control - Insulin/Insulin-like growth factor (IGF) Puig et al, 2003 •FOXO regulates organ size by controlling the number of cells Higher FOXO expression, fewer cells Aspects of size control - Insulin/Insulin-like growth factor (IGF) Puig et al, 2003 •FOXO regulates organ size by controlling the number of cells Higher FOXO expression, fewer cells Rescue phenotype through overexpression of dAkt (growth inhibitor inhibition) Aspects of size control – Growth rate – cell cycle Aspects of size control - Growth rate - cell cycle - MYC •Oncoprotein Induces the expression of many growth factors It positively regulates the biogenesis of ribosomes and thus also protein synthesis Aspects of size control - Growth rate - cell cycle - MYC Cells in close contact with the dMyc clone have a growth disadvantage. Claire de la Cova, Cell, 2004 Myc overexpression Is a difference in growth rates between populations of cells sufficient to induce competition? Aspects of size control - Growth rate - cell cycle - MYC Myc overexpression Aspects of size control - Growth rate - cell cycle - MYC Johnston et al., 1999 Myc deficiency Wnt controls the size Summary of MYC: Myc overexpression = more larger cells, faster proliferation, larger individual, loss of size control Myc deficiency = smaller cells, smaller individual The size of the wing is controlled by Wnt (Wg) Aspects of size control - Growth rate - cell cycle - MYC Aspects of Size Control – Growth Duration Growth duration It is regulated by systemic hormonal signals that coordinate the cessation of growth throughout the body, as well as organ-autonomic processes that ensure that organs stop growing once they reach their final size. •Drosophila – 3 larval stages The size of the individual is determined by: rigid exoskeleton – there is no further growth, the size of the larva at the stage when it stops receiving food, before the stage of pupa (larval wandering). However, the decision to pupate is at a much earlier stage (the beginning of the 3rd instar) and is associated with a certain size (critical size) Aspects of Size Control – Growth Duration •Regulation of the growth rate and proliferation is not enough to fully control growth. Differences between individuals (and also between species) are due to differences in cell size and number of cells -> also regulated by growth duration Many signaling pathways and molecular mechanisms that regulate growth rates are also involved in regulating growth duration. Aspects of Size Control – Growth Duration - IGF •However, the decision to pupate is at a much earlier stage (the beginning of the 3rd instar) and is associated with the acquisition of a certain size (critical size), which is accompanied by the synthesis of the hormone ecdysone Ecdyson hormone is synthesized in increasing pulses, and each pulse is associated with a specific event in development (metamorphosis). The only period for determining growth rate and duration TGP = Terminal Growth Period •Critical size is absent in humans. Aspects of Size Control – Growth Duration - IGF IGF regulates growth rate during TGP Shingleton et al., 2005 Because the rate of cell proliferation is slowed, the imaginal discs are smaller when they begin to differentiate, reducing final organ size. When ecdysteroid levels rise above a maximum threshold, the discs cease cell proliferation and undergo differentiation, fixing their final size Aspects of Size Control – Growth Duration Lin et al., 2011 Prolonged growth time leads to the "rescue" of the phenotype of the cycE hypomorphic mutation Aspects of size control – Growth duration – protein synthesis Lin et al., 2011 Aspects of Size Control – Growth Duration The exact mechanism by which the larvae monitor their critical size is not clear... Here we show that this size-sensing mechanism depends on the limited ability of a fixed tracheal system to sustain the oxygen supply to a growing individual. As body mass increases, the demand for oxygen also increases, but the fixed tracheal system does not allow a corresponding increase in oxygen supply. We show that interinstar molting has the same size-related oxygendependent mechanism of regulation as metamorphosis. We show that low oxygen tension induces molting at smaller body size, consistent with the hypothesis that under normal growth conditions, body size is regulated by a mechanism that senses oxygen limitation. however… Aspects of Size Control – Growth Duration The exact mechanism by which the larvae monitor their critical size is not clear, but signaling pathways regulating the synthesis of Ecdysone and thus responding to critical size are known IIS, TOR, RAS/RAF/MAPK Aspects of Size Control - Growth Duration - Human What about human? Aspects of Size Control - Growth Duration - Human •Growth arrest is associated with the end of puberty -> timing puberty is an important factor in size regulation Hormonal changes in puberty are known, but the mechanisms that control when these hormonal changes are initiated are much less understood. What about human? •Higher BMI, earlier puberty This suggests that the timing of puberty and growth stop in humans, as well as in Drosophila, are regulated by the state (amount) of nutrition and body size of adolescents. „For example, children displaying precocious puberty are typically tall for their age because of an advanced adolescent growth spurt, but generally become shorter adults because they enter maturation and, therefore, adulthood earlier. Conversely, delayed puberty commonly leads to individuals with high stature.“ Aspects of Size Control - Growth Duration - Human study for age at menarche in 4,714 women independent replication studies in 16,373 women „This allele was also associated with earlier breast development in girls; earlier voice breaking and more advanced pubic hair development in boys; a faster tempo of height growth in girls and boys; and shorter adult height in women and men in keeping with earlier growth cessation.“ LIN28B the first genetic determinant regulating the timing of human pubertal growth Aspects of Size Control – Growth Duration – LIN28 •Important in microRNA processing (inhibits Let-7). Controls growth and metabolism. Aspects of Size Control – Growth Duration – LIN28 •Back to the flies Lin-28 deficiency leads to earlier pupation, smaller and lighter individuals Aspects of Size Control – Growth Duration – LIN28 •Back to the flies Overexpression of lin-28 leads to larger individuals and problems during hatching (problems during metamorphosis) Aspects of Size Control – Growth Duration – LIN28 Let-7 deficiency Caygill et al., 2008 Larval wandering pupation Cells are not able to "get out" of the cell cycle. Aspects of Size Control - Growth Duration - LIN28B Let-7 overexpression leads to cessation of development in L1, L2 and high lethality => overexpression of miRNA in vestigial (vg) – wing-specific Caygill et al., 2008 Negative growth controls * Apoptosis White et al., 1994 Aspects of size control - Size of organs The organs "know" what size they should be. And they also "know" when to stop growing => autonomy in terms of organ size. The concept was introduced back in the 70s. Silber, 1976 Aspects of size control - Size of organs The same goes for the fruit fly. Imaginary wing disc The same disc, cultured in vivo in an adult Bryant, Levinson, 1985 Aspects of size control - Size of organs •Mammals have a similar body plan, but organ sizes vary dramatically The size is not random, but the result of thorough regulation The size of the organ must also be adapted to physiological needs (when a kidney is removed, the other one hypertrophies) Coordination of proliferation and cell death is crucial for the correct size of the organ. Size was determined by integration of a limb-intrinsic “potential,” which was greater in tigrinum, and a systemic “regulator” more active in punctatum •Each organ has autonomous size control (but is also partially subject to system control) Growth duration/timing seems to be the key Aspects of size control - Size of organs Aspects of size control - Size of organs •Each organ has an autonomous size control Link to the first lecture Aspects of size control - Size of organs Coordination of proliferation and cell death is crucial for the correct size of the organ. Hippo Aspects of size control - Organ size - Hippo YkiS168A Dong et al., 2007 Aspects of size control - Organ size - Hippo And in mammals? Dong et al., 2007 Aspects of size control - Organ size - Hippo Hippo and cell death BrdU TUNEL YAP Overexpression Aspects of size control - Organ size - Hippo BONUS: Hippo and cancer Aspects of sizing control — Organ size — Hippo — Conclusion BONUS:Back to the lecture on size control •ARHGAP11B gene is present is only in humans It was formed about 5 million years ago by partial duplication of the ARHGAP11A gene (Rho-GAP in the nucleus) However, ARHGAP11B has lost this activity due to the point mutation and is localized in the mitochondria, where it participates in glutamine metabolism Increased number of radial glia, neural precursors of the neocortex Ethics? Did this gene really separate human • from primates? “The cells amplified upon ARHGAP11B expression in fetal marmoset neocortex exhibited a marker signature consistent with the identity of basal radial glia.” 77 Conclusion and questions 78 •Control of growth/size: Growth rate vs Growth duration + examples Organ growth/size control (Hippo) Know that there is also negative regulation. Thank you for your attention 79 Tomas Barta tbarta@med.muni.cz Rate of growth Duration of growth Cell count regulation Cell size control X X