Dorso-ventral patterning of the mouse limb: Lmx1b

1. Dorso-ventral patterning of the mouse limb: Lmx1b Carlos G. Arques and Cristina Clavería

2. LIM A LIM B HOMEODOMAIN The LIM homeodomain (LIM-HD) protein family • Mammalian genomes, such as those of mice and humans, contain at least 12 LIM homeodomain (LIM-HD) genes that encode key regulators of developmental pathways. • LIM homeodomain (LIM-HD) proteins are transcription factors that contain: • - Two LIMprotein/protein interaction domains, eachof them binding two zinc ions toform a finger-like structure. • A DNA binding homeodomain. DNA PROTEIN PROTEIN

3. Lmx1b protein Lmx1b is a member of the LIM-HD family of transcription factors that plays a variety of roles during development to determine body pattern in vertebrates and invertebrates. Dorso-ventral limb patterning in vertebrates is thought to be controlled by the Lmx1b protein, which is expressed in a spatially and temporally restricted manner along the dorso-ventral limb axis.   Schweizer et al., 2004 Lmx1b is expressed in multiple murinetissues, including the developing limbs and eyes,the kidneys, the brain, and in cranial mesenchyme.

4. Lmx1b null mouse displaysdorsal-to-ventral conversion of the limbs and alack of the patella and nails. Chen et al., 1998 Lmx1b null mouse model mimics the humandisease associated with Lmx1b mutations, nail-patella syndrome (NPS).

5. Nail-patella syndrome

6. Nail-patella syndrome Nail-patella syndrome (NPS) is an autosomal dominant disorder caused by heterozygous mutations in Lmx1b. It is characterised by developmental defects of dorsal limb structures, the kidney, and the eye, manifested by nail dysplasia, patellar abnormalities, elbow dysplasia, iliac horns, nephropathy and glaucoma. Missense mutations are concentrated within the homeodomain and the LIM domains, with frameshift and nonsense mutations more widely distributed throughout the coding region.

7. LIM A LIM B HOMEODOMAIN Lmx1b protein conservation

8. Pathologic mutations * * ** * * * * * * * * * * * * ** * * * * 149 * * * * * * LIM A LIM B 149 Both LIM domains are highly conserved, although they have diverged one from each other. Pathologic mutations affect the most conserved residues between LIM A and B domains.

9. LIM A Pathologic mutations Disruption of Zn-finger LIM B

10. 201 Pathologic mutations * * * ** * * * * HOMEODOMAIN 253 Homeodomain is highly conserved along evolution. Pathologic mutations affect conserved residues.

11. HOMEODOMAIN Pathologic mutations Disruption of DNA binding

12. Conclusions • As nail-patella syndrome (NPS) is an autosomal dominant disorder, Lmx1b mutants might be acting as (partial) dominant-negative proteins. • Pathologic mutations in LIM domains affect the most conserved residues between A and B domains, and all of them seem to disrupt the zinc finger structure. Interestingly, no pathologic mutations have been described in these domains not directly affecting the zinc finger structure. • Pathologic mutations in homeodomain affect the most conserved residues, and all of them seem to affect DNA binding. • Mutations in other regions do not seem to be pathologic, at least in heterozygosity.

13. How does Lmx1b domain get its shape in the mouse limb?

14. Current model

15. LOGO plus multi-agent capabilities plus a expanded graphical interface Kid-oriented… or not Free (as in “free beer”)

19. Exploring alternative models of rostral-caudal paatterning in the zebrafishh neurectoderm with computer4 simulations Chitnis and Itoh Current Opinion in Genetics & Development 2004

20. Wnt7a drives Lmx1b expression in the dorsal mesenchime Wnt7a is expressed in dorsal ectoderm Is diffusion enough?

22. Wnt7a gradient shape is not the observed Lmx1b domain shape

23. Lmx1b expression does respond to wnt7a Lmx1b promoter activation is all or nothing Frontier between lmx1b and not lmx1b cells is a straight line

24. Lmx1b expression does respond to wnt7a Lmx1b promoter activation seems to be all or nothing wnt7a exposure changes cell state? Frontier between Lmx1b and not lmx1b cells is a straight line different affinities between lmx1b and not Lmx1b cells?

25. Lmx1b expression does respond to wnt7a Lmx1b promoter activation is all or nothing wnt7a exposure changes cell state? Frontier between lmx1b and not lmx1b cells is a straight line different affinities between lmx1b and not Lmx1b cells? MODEL:Exposure to high concentrations of wn7a changes cells to lmx1b expressing state Lmx1b on state is maintained in the absence of wnt7a Lmx1b on state conffers special affinity for other lmx1b expressing cells

27. Affinity-driven reshaping of Lmx1b domain allows other cells to enter the high wnt7a region… and become new Lmx1b-expressing cells

28. Finally, the system stops itself… achieving the expected domain shape

30. Conclusions This model could explain what is observed It predicts something (is falsable) Would these be compartments? (scholastic) Netlogo can be an useful tool