Lecture 2 - Strategies for Studying Embryogenesis

Question 1

historically, what were the two early models of animal development? what did they generally entail?

Answer 1

mosaic model and regulative development

mosaic model - argued that differentiation was highly predetermined due to the distribution of copious cytoplasmic determinants (e.g. mRNA, proteins, signaling molecules, etc.); focus on autonomous cell diff.

regulative - cell fate is flexible and development occurs as an effect of cellular conditions (e.g. local signals, ECM transduction from nearby cells); argued that inheritance of determinants was either unimportant or nonexistent

Question 2

how does animal development occur realistically with respect to the two historical models of development?

Answer 2

no organisms develops solely “mosaically” or through “regulative development”. there will always be a mix of determinant inheritance and cellular localization that affects the development and diff. of blastomeres.

Question 3

what is controlled interference?

Answer 3

changing one parameter (e.g. genetic, cellular, enviro.) at a time + observing developmental changes. referred to as “controlled embryo mangling”.

Question 4

what was the process of ablation? what did it reveal and/or how was it argued in the context of models of animal dev.?

Answer 4

ablation - exp. done to test cell dev. and autonomy.

stabbing cell w/ hot needle (or a laser, as is used more often in modern times)

killing 1 cell in 2-cell stage of frog embryo produced half an embryo (one half normal development, one half undiff. cells)

argued as evidence for mosaic development; since the other half the embryo developed fine, it was argued that development was autonomous and not dependent on local conditions.

Question 5

what is a problem with the ablation experiment (i.e., what are possible complications that could affect the results of the experiment).

Answer 5

ablated remnants can still provide info (e.g. chemical, mechanical, etc.)

the ablated side was able to restrict the uninjured blastomere to make 1/2 an embryo

Question 6

what is the process of ligation? what did it reveal and/or how was it used as evidence in the context of animal dev.?

Answer 6

hair loop was used to split and completely separate two blastomeres.

at two cell stage, splitting of blastomeres led to full development of two viable embryos.

was argued as evidence for “regulative” development - rather than developing into “right” and “left” halves, each blastomere was able to recognize it was alone and develop into a viable embryo.

also revealed all embryo cells contain all inherited DNA.

Question 7

what did ligation prove about cytoplasmic determinants and how?

Answer 7

when the blastomeres were cleaved in a way such that one resulting cell did not get any of the “grey crescent” (responsible in forming polarity and will become Spemann’s organizer), that cell resulted in a ball of undifferentiated cells, whereas the cell w/ the crescent developed normally.

proved that cytoplasmic determinants were critical in development; proper inheritance was vital for gene expression and normal development.

Question 8

grey crescent - what is it, what does it do? how is it formed?

Answer 8

forms opposite of the site of fertilization (cortical cytoplasm rotates 30 deg. relative to internal cyto. when fertilization occurs). rotation exposes grey crescent below the cortical cyto.

marks future site of blastopore and dorsal structures.

determinants related to Wnt pathway thought to be involved in “inducers” of grey crescent.

Question 9

define cellular commitment. what are the two stages of commitment? what are their qualities?

Answer 9

commitment - when a cell’s fate (i.e. potency) becomes restriction (even if it is not pheno. diff. from neighbors).

two stages of commitment:

1) specified - if the cell (or organ) is isolated and put in a neutral enviro, it will develop into its original fate. if placed in a signaling enviro, it will adopt the fate of neighboring cells.
2) determined - when a cell’s fate is restricted such that it will differentiate into its fate regardless of environment (i.e. diff. part of embryo).

specification is reversible, determination is not

Question 10

what is indicated if a cell/tissue cannot carry out its fate in isolation?

Answer 10

its fate has yet to be restricted (it remains undifferentiated). not enough signaling has occurred for it to become committed.

Question 11

what could you infer by watching an embryo develop without a particular part of it?

Answer 11

it can be determined how committed that specific area. the removed piece in question could be determined to be an inducer if there is no differentiation of cells in that area without the piece vs. with (within the same timing, of course). it can also be determined how vital that piece is to the embryo overall with regard to signaling, development, etc.

Question 12

what are coculture experiments? what can be determined from them?

Answer 12

coculture experiments are isolating two tissues in a petri dish and seeing their effect on one another.

if one causes changes in differentiation or structure in the other, that tissue is called an “inducer”. important for determining which tissues affect or influence which.

sometimes, the tissues may be separated by a porous barrier. if influencing occurs (and to what extent it occurs), it may be determined the nature of the secreted signals, size, etc.

Question 13

what is ectopic transplantation? what can result?

Answer 13

(aka heterotopic transplantation) - embryonic part removed from donor + implanted into different place on comparable recipient. if the recipient is competent, abnormal structures may grow into these engrafted places (ex.: grafting of tissue can produce eyes in legs, or legs where antennae should be).

Question 14

what was the Spemann/Mangold famous experiment? what did it accomplish?

Answer 14

tissue from the dorsal lip (of blastopore) was grafted from one species of newt to another species (on the opposite side of its blastopore).

the grafted tissue induced new body axis formation w/ neural tubes and somites. determined that the dorsal lip is such a strong inducer that it was called an “organizer” - produced large scale differentiation and signaling in the embryo.

Question 15

what is heterochronic transplantation? what issue/question does it address?

Answer 15

heterochron. transplantation occurs when there is an age gap between the recipient and the donor.

address competence - when a cell/tissue is capable of responding to an inductive signal.

competent = capable of responding to such a signal

basically, competency is a result of receptor expression - how many receptors are on the cell membrane? are they there all the time? is the cell capable of transducing these signals?

Question 16

why test for heterospecificity?

Answer 16

testing for competency between species is important because it allows for the elucidation of shared signals/structures/tissues that are fundamental for different groups of species. results of these tests may have affects in phylogeny, evo-devo, and moreover species relationship.

Question 17

what is a genetic type of controlled interference? how are these denoted?

Answer 17

mutations in one or both alleles of a gene (by chemicals, radiation, insertions, etc.).

mutant versions are usually denoted with a minus and wildtype (“normal” type, the one that predominates in natural pop’s) are given a +. some will use uppercase for wildtype and lower case for mutants.

Question 18

how is the gene name specified from its protein name?

Answer 18

gene name often written in italics, protein product in plan text

or,

gene is in lowercase + protein is capitalized.

NOTE: GENES ARE OFTEN NAMED FOR MUTANT PHENOTYPE (E.G. WEE1)

Question 19

what are the 3 main types of mutations (regarding function), and what are their names?

Answer 19

null mutations - complete loss of function

loss-of-function - reduction in function

gain-of-function - causes gene to be expressed at an abnormal time or place (e.g. allele loses regulatory region that has it turned off until a specific condition arises)

gain-of-function mutations may result in proteins that are consititutively expressed (expressed all the time).

Question 20

do mutations need to be in zygotic genes to have an effect?

Answer 20

no; mutations in genes of the mother (called maternal effect genes) can have big impacts on dev.

ex: offspring of bicoid - mutants lack head + thorax, show partial duplication of abdomen at anterior end.

Question 21

how can deliberately mutating genes be beneficial? what does it reveal?

Answer 21

determination of epistasis - when one gene masks the expression of another.

Question 22

how to test for epistasis?

Answer 22

double mutant analysis - determines order of function of gene products in a dev. pathway:

1) single mutant strains are crossed to give rise w/ double mutants.
2) resultant phenotype is assessed
3) phenotype that matches single mutant phenotype is considered the “masker”. ex: recessive gene apterous in Drosophila produces wingless homozygotes. if flies are crossed w/ abnormal wing mutants (e.g. curled wings), that mutation will be masked by apterous mutation. thus, apterous is “upstream” of these wing mutations, or apterous is “epistatic” to wing mutations (“stands on”; apterous gene product acts before wing mutant products).

Question 23

what are homeotic mutations? outcome?

Answer 23

mutations in homeotic genes - genes involved in development of organs + anatomical structures. they result in misplaced organs, and as such, rarely do embryos w/ homeotic mutations survive.

Question 24

cell fate mapping - what is it, how is it performed?

Answer 24

mapping (or the attempt of) each blastomere to its end fate through the use of lipophilic dyes (bind to plasma mem.) or microinjection of high MW dyes (restricted to labeled cells + progeny).

visualization techniques like confocal microscopy can be used to track cells and determine where they move throughout the embryo and where they end up.

note: can also use DNA mutations if it results in color change.

Question 25

C. elegans has been entirely mapped (each blastomere’s fate is known). why is this not feasible for other organisms like birds or mammals?

Answer 25

C. elegans is very “mosaic” like - there is a relatively invariant pathway for development of the nematode (though conditional specification does exist). the same pathway is used for females as well, and the large amount of autonomous specification makes it easy to map their blastomeres.

other organisms have a lot of variation with how the embryo develops - blastomeres may act differently in different organisms within the same species, and there’s too much signaling/determinant determination happening for every blastomere to be mapped accurately (not including the ethics of such mapping). lots of conditional specification too, which changes the relative actions of blastomeres between organisms.