Developmental Mechanisms Of Morphological Change Flashcards
Developmental mechanisms of morphological change
Finding the pathways that change
When evolution changes development
Can we find the pathways/networks involved?
Two main strategies
1) candidate pathway approach
2) hypothesis-free approach
Transposition
- “sliding” of homologous gene segments along a segmented body plan
- e.g. no of ribs changes in different segments in vertebrate skeletons
- change segment specification? Add segment?
Is transposition underpinned by
Hox genes?
Hox gene expression has changed between species
- to modify the end-product of development
- e.g. Hoxc6 in mice, chick and goose by in situ localisation
Modification of the Hox pathway correlated with
Development change
Crustacea
- transposition
- head appendages = feeding
- thorax appendages = locomotion
Artemia
- brine shrimp
- Ubx-Ab stains thorax and posterior (swimming limbs /=/ feeding)
Ubx
A hox protein
Triops
- Ubx-Ab stains thorax and posterior (swimming limbs /=/ feeding)
Mysidium
- T1 modified for feeding, not locomotion
- maxilliped
- Ubx not expressed; slid
- faint in T2
- expressed in T3
Lobster (Homanes)
- T1 & 2 modified for feeding
- Ubx not rxot eeed
Crustacean evidence
- correlational
- we need interventional
Parhyale
- amphipod
- T1: Ubx not expressed (maxilliped)
- T2: Ubx expressed (gnathopod)
- RNAi @ embryo stage (siRNA injection) induces partial transformation: T2->T1-like
- hatchling SEM
Oligodactyly
- mice = ancestral mammal
- cow/pig/camel digits: stands on 2, 2 highly reduced (more symmetrical)
Oligodactyly H
- Shh
- expressed in limb bud posterior
- manipulating concs changes digits no
- e.g. in chicle
Shh
It is not where the RNA, but the protein is, that matters
Compare Shh protein patterns
- travels further
- downstream target genes more symmetrical
- cellular cascade of initiation
patched (ptc) and smoothened; Gli
- sequester Shh
Less ptc in Oligodactyly
- Shh diffuses further back
Oligodactyly mutation
- ptc receptor has insertion mutations in cis-regulatory region of limb regulatory module in intrinsic regions A (2.4kb) and B (1.4kb)
- suppressed expression and altered spatial distribution
Observing Shh regulation
- GFP-lacZ transgenics
- in mice: expression lower and shifter distribution
- mutation expands Shh influence
- not necessarily causative; could be many mutations in other genes
Tunicates
- Molgula oculata
- tadpole larva
- oral/atrial siphon
- brachial basket
- tunic
- digestive system
- tail-less form is derived; lost
Hypothesis
In tail-less tunicates, notochord cells don’t stack and extend; was it changes to structural genes?
M. oculata
- 40 notochord cells that intercalate
M. occulta
20 notochord cells in that do not intercalate
Tunicate hybrid
20 notochord cells that intercalate
Notochord
- secrete collagen and stack as discs; causes tail growth
Methodology :
1) extract RNA from different species @ right developmental stage
2) RNA-Seq
3) map
4) DEG analysis
- examine known notochord genes
Tunicates results
- 32 known notochord genes ^ in tailed but not tailless
- not pseudogenes, so must be regulatory change
- 2 collagen, 2 laminin, 2 collagen-processing enzymes
Does changing collagen expression affect the tail?
- intervention yet
- Ciona robusta: CRISPR collagen KO
- tail formation affected
- mild: wonky
- severe: lost
What else to do re Tunicates?
- Sequence before and after tail emergence ; what has changed?
- are collagens or laminins represented
Darwin’s finches
- adaptive radiation
- one of the first studies adopting a hypothesis-free approach
Short, stumpy beaks
- medium ground finch (G. fortis)
- large ground finch (G. magnirostris)
Long, pointy beaks
- cactus finch (G. scadens)
- large cactus finch (G. conirostris)
Beak shape
- shape of mandible (upper jaw) via skull
- crucial
- RNA-Seq
How to study gene regulation in embryonic mandible
1) RNA-Seq
2) microarray
RNA Seq
- newer
- more expensive
- more sensitive
- need a large amount of tissue
- Darwin’s finches are protected
Microarray
- traditional
- extract RNA from different species and compare expression to outgroup/ reference species
Microarray process
1) extract from maxilla (embryo beak region)
2) different species different label (Cy3/Cy5)
3) hybridise
4) convert to cDNA
5) spot cDNA into glass slide; each spot represents an RNA
6) scan
7) set results: look for genes affecting beak length
Microarray results
- c100 show consistent difference correlating w beak shape but not overall size
- e.g. calmodulin (Ca2+-binding protein) affects calcium envrt; signalling
Test: is calmodulin capable of changing beak length? If
- chickens: experimentally tractable
- expression in frontonasal mandible
- use RCAS delivery to constitutive express downstream CaMKII
- beak length increases 10%
- sufficient
RCAS
Retroviral
CaMKII
CaM effector
Evolution has tweaked a calcium signalling pathway during radiation of Darwin’s finches
- one developmental perspective
- probably other pathways too
Other potential factors
- IGFBP (insulin)
- β-catenin (Wnt pathway)
- Kruppel Factor TF
- incomplete understanding!
