Vertebrate Limb Patterning

Chris Campbellcc59@buffalo.edu 829-3462

*

Origin of the limb

Intermediate

Kidney, gonads

Paraxial

Head Somite

Cartilage,skeletal,muscle dermis

Lateral

Circulatory,Body lining,

Limbs (exceptMuscle &

nerve)Extraembryonic

Notochord

Dorsal view of early embryo showingSomites (arrows) and neural folds *. Somites form starting near the head region and progressing towards the tail. Counting somites indicates the developmental stage of an embryo

*

Basic limb buds structures are conserved among vertebrates

Vertebrate limbs originate as twopairs of limb buds that appear atspecific levels of the embryonic flank. Forelimbbuds appear first (~E9 in mouse) followed byhindlimb buds ~ 0.5 -1day later. Each limb bud consists of lateral platemesoderm (LPM) covered by ectoderm.

E10

The parts of the vertebrate limb

stylopod zeugopod

Duboc (2011) Dev Dyn 240, 1017

Bat wing

snake

Techniques to Study the Regulation of Differentiation

During Development• Observation of normal development in animals of different species

• Reporter genes or other markers (such as fluoresceinated lectins or antibodies) to delineate particular cell types

• Ablation and transplantation experiments

• Use classical genetics to identify genes in which mutations affect limb development. (phenotype to genotype)

• Loss or gain of function mutations (knockouts, transgenics, injection of sense, antisense or small inhibitory RNAs) (genotype> phenotype)

• In vitro organ and cell culture

What makes chick a good model system for studying limb development?

Ease of physical manipulation

The developing limb bud is patterned along three axes : Proximal-Distal Anterior-PosteriorDorsal-Ventral

The apical ectodermal ridge (AER) isa specialized epithelium (derived from theectoderm) that controls limb bud outgrowth.The AER is a transient structure that does notitself contribute cells to the mature limb.

Proximal Distal Patterning

E-cadherin

What regulates limb bud outgrowth?

AER is required for

limb bud outgrowth

Limb mesenchyme is required for and specifies type of limb.

AER can be replaced

by FGF bead. **

Inductive interactions between AER and limb mesenchyme similar to the inductive interactions that take place

between metanephric mesenchyme and ureteric bud.

UB is requiredfor M-E conversionand survival of MM

MM specifies UBbranching & differentiation

UB can be replaced by3T3Wnt-4 or L-cellWnt9b

Cohn (1999 ) Nature 399, 474

What regulates limb bud outgrowth?Studies in the early 90s indicated that placing a bead soaked in one of several FGFs adjacentto chick flank between stage 13 and stage 17 of development could induce ectopic limbs.

These limbs were frequently quite well developed and tended to be more wing like if the beadwas placed anteriorly (i.e. near where wing normally forms) and more leg like if placed posteriorly.

Digit patterns were almost always reversed (indicating a defect in AP patterning) as were Shh (signaling molecule) and hoxd13 (transcription factor) expression patterns. These genes arenormally expressed in the posterior part of the bud but were expressed anteriorly in the ectopic buds.

Cohn et al. (1995)Cell 80, 739

Shh

AER functions by providing an FGFand also is involved in AP patterning.

Proximal elements are patterned prior to distal elements and patterning is dependent on signals(FGFs) from the AER

Fgfs (fibroblast growth factors) are soluble secreted molecules that bind to their receptors and activate kinase signaling pathways leading to the modulation of transcription of specific genes. Fgf4 AER ck/o no effectFgf8 AER ck/o limb deformitiesFgf9 AER k/o no effectFgf17 AER k/o no effect

Fgf10 LPM k/o limb deformities

FGFs and Their Mechanism of Action

FGF10in situ

Sun (2000) Nat Gen 25, 83

Fgf10 and limb bud

Fgf10 is expressed in the mesenchyme. In its absence:

There is no Fgf8 expressed in the forelimb bud AER(although Fgf8 is expressed elsewhere in the embryo)There is no Shh in the FL or HL mesenchymeTbx4 & Tbx5 are induced appropriately but expressionis rapidly lost.

Fgf8E9.25

ShhE10.5

Fgf10+/- Fgf10-/-

There is no morphological appearance of an AER

H&EE10.5

Fgf10-/-Fgf10+/-

Sekine (1999) Nat Gen 21, 139

Msx-Cre;Fgf8-/flox

Msx-Cre;Fgf8-/flox

Lewandoski (2000) NatureGenetics 26, 460

Fgf8

Fgf8-/- is an early embryonic lethal and therefore two different conditional knockouts were made

Moon (2000) Nat Genetics 26, 455

*RAR-cre;Fgf8-/flox

*RARCre is expressed in forelimbAER but not in hindlimb AER.

Msx-Cre is expressed in both FL and HL butexpression is turned on after Fgf8 in FL (see b-i).

Fgf4expressedfor longerperiod oftime inFgf8 ko

Contrary to the predictions of the progress zone model, it is the zeugopod that is most affected in RAR-cre;Fgf8 mutants, while the stylopod and more distal autopod is relatively normal.

Fgf4/Fgf8 conditional double knockouts have a more severe phenotype than Fgf8 single knockouts

Msx2-Cre;Fgf8-/flox

Msx2-Cre;Fgf4-/flox;Fgf8-/flox

Sun (2002) Nature 418, 501

Dt = deltoid tuberosity

Dt = deltoid tuberosity

AER forms in mutant hindlimb buds (unlike what happens in Fgf10-/- mice) but at ~E10.5 expression of many mesenchyme markers is perturbed.By ~E11.25 the AER will begin to degenerate.

Knocking out Fgf4 & Fgf8 in the AER causes altered gene expression in the mesenchyme including expansion of a normally proximally expressed gene.

Fgf4-/-Fgf8-/-

Mercader (2000) Dev 127, 3961

Sun (2002) Nature 418, 501

Zeller (2010) Curr Op Gen & Dev 20, 384

Patterning of the vertebrate limb

Signaling by the non AER ectoderm controls dorsal ventral patterning

The developing limb bud is patterned along three axes : Proximal-DistalDorsal-Ventral

Anterior-Posterior

Dorsal

proximaldistal

BMPs and Dorsoventral Patterningnormally expressed in ventral ectoderm

En-1Bmp4 & Bmp7

Normally expressed in dorsal ectodermWnt7-a & r-fng

Normally expressed in dorsal mesenchymeLmx-1 & Shh

Normally expressed in AERFgf8

Bmp4Bmp7

Shh

Fgf8

dorsal

ventral

In situ for Shh (molecular marker for ZPA) & Fgf8(molecular marker for the AER)

Pizette et al. (2001) Dev.128,4463

BMPs signal by binding to BMPreceptors. This binding can beinhibited by BMP antagonistssuch as Noggin or Gremlin

Canalis Endocrine Reviews 24, 218 (2001)

Bmp4Bmp7

Fgf8

Downregulate BMP(v) signaling by misexpression of Noggin (BMP antagonist) by retroviral injections to the ectoderm leads to loss of en1(ve) and expansion of Wnt-7a (de), Lmx1 (dm) & Shh (dm) into ventral region of bud (i.e. Dorsalization of bud). Also leads to misexpression of ShhAnd Fgf8

Upregulate BMP(v) signaling by misexpression of a constitutively active Bmp receptor leads to expansion of en1, and loss of

Wnt-7a & Lmx1(d) (i.e.Ventralization of bud). Also leads to loss of AER

Pizette et al. (2001) Dev.128,4463

Shh

En-/-En+/+

En Wnt7a Fgf8 Bmp2

Engrailed knockout mouse

Loomis (1996) Nature 382, 360

Li (2010) Dev 137, 1181

Patterning of the vertebrate limb

Anterior-posterior patterning iscontrolled by signals from the mesenchymal cells of the ZPA(zone of polarizing activity)

The developing limb bud is patterned along three axes : Proximal-DistalDorsal-Ventral

Anterior-Posteriorproximal

distal

The ZPA and Shh signaling

ZPA

** notochord andfloorplate organizingcenters possess similaractivity when graftedonto limb buds

spinal cord will inducemetanephric mesenchyme

anterior

posterior

Duplication of posterior digits

Riddle et al. (1993) Cell 75, 1401

Tickle et al. (1982) demonstrated that applying RA can result in similar mirror-imageduplications to what is seen with ZPA. However, a series of experiments suggested that RA does not act as a morphogen in this system but acts by inducing an ectopic AER. The evidencefor this was 1) required more RA to induce limb bud duplications than is normally seen in the ZPA.2) it is the tissue DISTAL to an RA implant that acquires ZPA activity not the area immediatelyadjacent.

Based on the ability of drosophila hedgehog to act as a morphogen in flies, Riddle et al. soughtto clone a hedgehog related gene(s) expressed in chicken limb buds in the ZPA. Found Shh.

Evidence that Shh is the morphogen that mediates ZPA polarizing activity

Expressed in correct temporal and spatial pattern during limb bud development. (ZPA activityhad been mapped by assaying small blocks of limb tissue at different stages of developmentfor their ability to induce digit duplication.)

Shh is also expressed in other tissues that can induce digit duplications when grafted onto anterior chick limb buds (i.e. notochord).

RA induces Shh.

Shh induces digit duplications and Hox gene expression repatterning similar to ZPA grafts.

Cohn (1999 ) Nature 399, 474

Shh-/- Mice

Chiang et al. (2001) Dev. Biol. 236,421

Mutant mouse limbscontain a single digitBased on gene expressionpattern and morphology itis digit 1. Thereforedigit 1 doesn’t requireShh signaling.

Shh-/- mice form truncated limbs with single digits and show inappropriate patterning of Hoxgenes (early) and gdf5 & Msx1 (markers expressed in digits). Unlike mesenchyme from wt mouselimb buds, Shh-/- mesenchyme has no polarizing activity when grafted onto chick limbbuds. Several AER markers were expressed early but later disappeared (eg Fgf8). Bmp2&4 are expressed at low levels, Gremlin(known target of Shh signaling and inhibitor ofBMP signaling) is turned on but rapidly downregulated & Gli3 expression (negatively regulated by Shh) is extended.

In the absence of SHH, AER formation is initiated but doesn’t persist as long as in wt.

Shh:GFPCre/+;R26R/+

Harfe (2004) Cell 118, 517

Shh:GFPCre/+;R26R/+

Shh:CreERT/+;R26R/+

Xu (2011) Dev Dyn 240, 1303

T-box genes and limb patterning FL or HL?

Tbx2Tbx3Tbx4Tbx5

Ancient T-box gene

2/3 4/5

Tbx2 Tbx4 Tbx3 Tbx5

amphioxis

T-box genes & Limb specification

Gibson Brown (1996) Mech. Dev.56, 93 reported that Tbx4 was expressed primarilyin hindlimbs while Tbx5 is expressed primarily in forelimbs and proposed that these genesmay specify limb identity.

Tbx4 Tbx5

chick embryos

zebrafishTamura (1999) Mech Dev 87, 181

RCAS-EnTbx5

RCAS-EnTbx4

Tbx4 and Tbx5 Specify Legs and Wings Respectively in Chicks

Takeuchi (2003) Dev. 130, 2729

Express dominant negative or wt Tbx genesby retroviral infection of chick limb buds

EnTbx5 inhibits wing butnot leg formation

EnTbx5 in wing field results in loss of Wnt2b(EnTbx4 in leg field results in loss of Wnt8c)

CAGGS-Tbx5 EGFP CAGGS-Tbx4 EGFP

Tbx4-/- knockout

Tbx4flox/flox Die by E10.5 (before hindlimb development has really started)due to defects in the allantois

Limb bud cultures

Early limb bud patterning relativelynormal

Naiche (2003) Devt. 190, 2681

Agarwal et al. (2003) Dev.130, 623

Limb formation in the Tbx5-/- mouse

Rallis et al. (2003) Dev. 130, 2471

Limb field patterning is initiated in absence of Tbx5as indicated by correct expression of Tbx5 itself & other early markers dHand, Hoxb8 & Pea3Note Pea3 is expressed normally at E9 but is not upregulated indel/del embryos at E9.5TBX5 DOES NOT SPECIFY FORELIMB IDENTITY

Agarwal et al. (2003) Dev.130, 623

Tbx4 doesn’t specify leg identity in mice

Minguillon (2005) Dev Cell 8, 75

Tbx4 but not Pitx1 can rescue forelimb growthIn Tbx5fl/fl Prx1-cre mice.TBX4 DOES NOT SPECIFY HINDLIMBIDENTITY

Minguillon (2005) Dev Cell 8, 75

Minguillon (2005) Dev Cell 8, 75

Neither Fgf8 nor Fgf10 is expressed in mutantembryosSince both Fgf10 & Tbx5 are expressed inmesenchymal cells and Tbx5 is a transcriptionfactor … does Tbx5 regulate Fgf10 or LEF1(involved in Wnt signaling) expression?

What is the role of Tbx4 or Tbx5?

Tbx5 is a transcription factor - Are Lef/Tcf or Fgf genesdirect targets of transcriptional regulation by Tbx5?

Agarwal et al. (2003) Dev.130, 623

Fgf10-luc

Naiche (2007) Dev 134, 93

Tbx4cond/cond; Rosa-ERcreT2

Tbx4 is not required to maintain hindlimb identity

Deletion of Tbx4 by Tam injectionat E9.5 –E10.5 doesnot lead to loss of hindlimb specificgene expression but does causeskeletal abnormalities.

Tam injectionat E7.5 – HLdevelopmentfailing by E11.5

Deletion of Tbx5 after initiation ofFL bud outgrowth does not perturbnormal patterning and results in onlyminor skeletal abnormalities

Hasson (2007) Dev 134, 85

lacZ reporter

Delete Tbx4 or Tbx5 early in devt = no limbDelete slightly later = skeletal abnormalitiesDelete later yet = muscle & tendon abnormalities

Muscle abnormalities are non cell autonomous(can be shown by using a Cre that is expressed inmuscle progenitors prior to migration into limb

Hasson (2010) Dev Cell 18, 148

Naiche (2011) Dev Dyn 240, 2290

Questions will be handed out at class on Tuesday (Nov 15)