1. Vertebrate Limb Patterning Chris Campbell cc59@buffalo.edu 829-3462

2. *

3. Origin of the limb Intermediate Kidney, gonads Paraxial HeadSomite Cartilage, skeletal, muscle dermis Lateral Circulatory, Body lining, Limbs (except Muscle & nerve) Extraembryonic Notochord Dorsal view of early embryo showing Somites (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 *

4. Basic limb buds structures are conserved among vertebrates Vertebrate limbs originate as two pairs of limb buds that appear at specific levels of the embryonic flank. Forelimb buds appear first (~E9 in mouse) followed by hindlimb buds ~ 0.5 -1day later. Each limb bud consists of lateral plate mesoderm (LPM) covered by ectoderm. E10

5. The parts of the vertebrate limb stylopod zeugopod Duboc (2011) Dev Dyn 240, 1017

6. Bat wing snake

7. 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

8. What makes chick a good model system for studying limb development? Ease of physical manipulation

9. The developing limb bud is patterned along three axes : Proximal-Distal Anterior-Posterior Dorsal-Ventral

10. The apical ectodermal ridge (AER) is a specialized epithelium (derived from the ectoderm) that controls limb bud outgrowth. The AER is a transient structure that does not itself contribute cells to the mature limb. Proximal Distal Patterning E-cadherin

11. 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 required for M-E conversion and survival of MM MM specifies UB branching & differentiation UB can be replaced by 3T3Wnt-4 or L-cellWnt 9b

12. Cohn (1999 ) Nature 399, 474

13. What regulates limb bud outgrowth? Studies in the early 90s indicated that placing a bead soaked in one of several FGFs adjacent to 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 bead was 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 are normally 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 FGF and also is involved in AP patterning.

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

15. 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. Fgf4AER ck/o no effect Fgf8AER ck/o limb deformities Fgf9AER k/o no effect Fgf17AER k/o no effect Fgf10LPM k/o limb deformities FGFs and Their Mechanism of Action FGF10 in situ Sun (2000) Nat Gen 25, 83

16. 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 mesenchyme Tbx4 & Tbx5 are induced appropriately but expression is rapidly lost. Fgf8 E9.25 Shh E10.5 Fgf10 +/- Fgf10 -/- There is no morphological appearance of an AER H&E E10.5 Fgf10 -/- Fgf10 +/- Sekine (1999) Nat Gen 21, 139

17. 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 forelimb AER but not in hindlimb AER. Msx-Cre is expressed in both FL and HL but expression is turned on after Fgf8 in FL (see b-i). Fgf4 expressed for longer period of time in Fgf8 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.

18. 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

19. 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 -/-

20. Mercader (2000) Dev 127, 3961

21. Sun (2002) Nature 418, 501

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

23. 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-Distal Dorsal-Ventral Anterior-Posterior Dorsal proximal distal

24. BMPs and Dorsoventral Patterning normally expressed in ventral ectoderm En-1 Bmp4 & Bmp7 Normally expressed in dorsal ectoderm Wnt7-a & r-fng Normally expressed in dorsal mesenchyme Lmx-1 & Shh Normally expressed in AER Fgf8 Bmp4 Bmp7 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

25. BMPs signal by binding to BMP receptors. This binding can be inhibited by BMP antagonists such as Noggin or Gremlin Canalis Endocrine Reviews 24, 218 (2001)

26. Bmp4 Bmp7 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 Shh And 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

27. En-/-En+/+ En Wnt7a Fgf8 Bmp2 Engrailed knockout mouse Loomis (1996) Nature 382, 360

28. Li (2010) Dev 137, 1181

30. Patterning of the vertebrate limb Anterior-posterior patterning is controlled by signals from the mesenchymal cells of the ZPA (zone of polarizing activity) The developing limb bud is patterned along three axes : Proximal-Distal Dorsal-Ventral Anterior-Posterior proximal distal

31. The ZPA and Shh signaling ZPA ** notochord and floorplate organizing centers possess similar activity when grafted onto limb buds spinal cord will induce metanephric mesenchyme anterior posterior Duplication of posterior digits

32. Riddle et al. (1993) Cell 75, 1401 Tickle et al. (1982) demonstrated that applying RA can result in similar mirror-image duplications 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 evidence for 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 immediately adjacent. Based on the ability of drosophila hedgehog to act as a morphogen in flies, Riddle et al. sought to 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 activity had been mapped by assaying small blocks of limb tissue at different stages of development for 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.

33. Cohn (1999 ) Nature 399, 474

35. Shh -/- Mice Chiang et al. (2001) Dev. Biol. 236,421 Mutant mouse limbs contain a single digit Based on gene expression pattern and morphology it is digit 1. Therefore digit 1 doesn’t require Shh signaling. Shh -/- mice form truncated limbs with single digits and show inappropriate patterning of Hox genes (early) and gdf5 & Msx1 (markers expressed in digits). Unlike mesenchyme from wt mouse limb buds, Shh -/- mesenchyme has no polarizing activity when grafted onto chick limb buds. 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 of BMP 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.

36. Shh:GFPCre/+;R26R/+ Harfe (2004) Cell 118, 517 Shh:GFPCre/+;R26R/+ Shh:CreERT/+;R26R/+

37. Xu (2011) Dev Dyn 240, 1303

38. T-box genes and limb patterning FL or HL? Tbx2 Tbx3 Tbx4 Tbx5 Ancient T-box gene 2/34/5 Tbx2Tbx4Tbx3Tbx5 amphioxis

39. T-box genes & Limb specification Gibson Brown (1996) Mech. Dev.56, 93 reported that Tbx4 was expressed primarily in hindlimbs while Tbx5 is expressed primarily in forelimbs and proposed that these genes may specify limb identity. Tbx4 Tbx5 chick embryos zebrafish Tamura (1999) Mech Dev 87, 181

40. 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 genes by retroviral infection of chick limb buds EnTbx5 inhibits wing but not leg formation EnTbx5 in wing field results in loss of Wnt2b (EnTbx4 in leg field results in loss of Wnt8c)

41. CAGGS-Tbx5 EGFPCAGGS-Tbx4 EGFP

42. Tbx4 -/- knockout Tbx4 flox/flox Die by E10.5 (before hindlimb development has really started) due to defects in the allantois Limb bud cultures Early limb bud patterning relatively normal Naiche (2003) Devt. 190, 2681

43. Agarwal et al. (2003) Dev. 130, 623 Limb formation in the Tbx5 -/- mouse Rallis et al. (2003) Dev. 130, 2471

44. Limb field patterning is initiated in absence of Tbx5 as indicated by correct expression of Tbx5 itself & other early markers dHand, Hoxb8 & Pea3 Note Pea3 is expressed normally at E9 but is not upregulated in del/del embryos at E9.5 TBX5 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 growth In Tbx5fl/fl Prx1-cre mice. TBX4 DOES NOT SPECIFY HINDLIMB IDENTITY

45. Minguillon (2005) Dev Cell 8, 75

47. Neither Fgf8 nor Fgf10 is expressed in mutant embryos Since both Fgf10 & Tbx5 are expressed in mesenchymal cells and Tbx5 is a transcription factor … does Tbx5 regulate Fgf10 or LEF1 (involved in Wnt signaling) expression? What is the role of Tbx4 or Tbx5?

48. Tbx5 is a transcription factor - Are Lef/Tcf or Fgf genes direct targets of transcriptional regulation by Tbx5? Agarwal et al. (2003) Dev. 130, 623 Fgf10-luc

49. Naiche (2007) Dev 134, 93 Tbx4 cond/cond ; Rosa-ERcre T2 Tbx4 is not required to maintain hindlimb identity Deletion of Tbx4 by Tam injection at E9.5 –E10.5 does not lead to loss of hindlimb specific gene expression but does cause skeletal abnormalities. Tam injection at E7.5 – HL development failing by E11.5

50. Deletion of Tbx5 after initiation of FL bud outgrowth does not perturb normal patterning and results in only minor skeletal abnormalities Hasson (2007) Dev 134, 85 lacZ reporter

52. Delete Tbx4 or Tbx5 early in devt = no limb Delete slightly later = skeletal abnormalities Delete later yet = muscle & tendon abnormalities Muscle abnormalities are non cell autonomous (can be shown by using a Cre that is expressed in muscle progenitors prior to migration into limb Hasson (2010) Dev Cell 18, 148

53. Naiche (2011) Dev Dyn 240, 2290

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