1 Molecular mechanisms of Development Dr. Robert Moss Human body contains 50 TRILLION CELLS! 1. Pattern formation/Morphogenesis 2. Differentiation 1.Determination

2 Determinatin is a step-wise process, involving: Communications between cells; Activation of genetic developmental programs

l At 8 cell stage of human embryo, cells begin to read positional cues, either by contact, or morphogens. l Cues influence expression of genes. l In a chicken embryo, if take tissue at base of leg bud [normally gives rise to thigh], transplant to tip of identical-looking wing bud, tissue develops into a TOE rather than THIGH!! Tissue already determined to be leg, but took position information from new position to decide on tissue. –Cells determine fate both by reading their own gene expression, and by reading environmental cues. –Position info in leg and wing must be the same! –Nature often re-uses systems, and genes!

4 Developmental mechanisms: morphogen gradients

5 Regulative development mechanisms: INDUCTION l If separate animal and vegetal pole cells, neither develops mesoderm! But if moved together, some of the ectoderm becomes mesoderm! What does that tell you? l Induction seen in many places in development; see that cells communicate with eachother to decide what to become.

6 Experiments show that the notochord "induces" any ectoderm above it to become neural tube, which will become brain and spinal cord. How can I demonstrate this? Neural tube later induces formation of vertebrae.

7 EXPERIMENTAL DEVELOPMENTAL BIOLOGY l DROSOPHILA MELANOGASTER: –Life cycle –Body plan

Homeotic Genes l Homeotic genes: –“Master Control Genes”: Activate an entire developmental program for a particular structure. –determine identity of each segment/organ. –Activates genetic program appropriate to organ. –Overexpress “eyeless” throughout the body, get eyes developing on wings, antenna, legs, etc. l Lepidopteran, with addition of two new genes, transforms posterior pair of wings into “halteres”.

Network of cross- regulation determines position l Gradients of ‘morphogens’ activate particular genes in particular regions. l Example: To express eve in the leftmost two even bands, nuclei must first express hb, and bicoid. These bind enhancers near eve. l Balance between eve activators, such as hb, and repressors, such as Kr.

10 Bithorax & Antennapedia complexes l Order on chromosome matches expression pattern!

11 l Homeo Box: 180 base pairs –60 a.a. DNA binding domain; regulators of transcription. l Found in all multicellular organisms! l Homeo box protein gradient in frog limbs, could convey information about position along limb. l Same homeo box gene involved in limb development in mammals. l Must have evolved very early! l Mouse homeobox gene can substitute for a flies!!

l Just as in flies, in mice order of genes reflects order in body plan!! l Must be some significance to order!! l “Major changes between major animal phyla are correlated with duplications in Hox genes or an increase in the number of Hox genes”

13 Limb/digit formation l Limb development stimulated by expression of FGF8 l Internal mesodermal core stimulates formation of a thickened ectoderm, “Apical Ectodermal Ridege” [AER] l AER stimulates proliferation and development of underlying mesoderm to construct the proximal/distal axis of the limb l Grafting an additional AER creates additional limb. »Photos:

14 l Anterior/posterior gradient and digit formation: –Zone of Polarizing Activity [ZPA] »Sonic Hedge Hog, a homeotic gene first identified in flies! »Photos: l Primitive snakes, evolved from legged lizardes, and some retain hindlimb vestiges. l Limbless lizards: AER regresses, and limbs do not appear. Only requires changes in a few genes! l In mouse, eliminate FGF4 and 8, no limb development at all! l Thalidomide: Inhibits expression of Sonic hedge hog, which prevents limb extension.

15 Along trunk, co-expression of Hoxc6 and Hoxc8 seems to INHIBIT limb development. Change in expression of these two genes can create a dramatic change in body plan!

16 Mammalian Pattern Formation l Cilia of blastomeres beat asymmetrically, due to their structure. Probably determines axis. l At about 8 cell stage, homeobox transcription factors first show distinction between “inner cell mass” and trophectoderm. l First ‘bilateral’ indicator: Nodal, expressed on left side only. –If absent, randomization of orientation occurs. l Same hox genes used for A/P position of CNS in mammals as in flies!

17 Epigenomics l Remember, every cell in the body has the same set of genes! l So it’s the EXPRESSION of different genes in different cells that determines pattern, differentiation, organ formation, cell behavior. l How do we get different expression in different cells? l Developmental signals/morphogens initiate changes, that are then “heritable” as the cell divides. l “Epigenomics”.