Dorso-ventral patterningof the mouse limb:

Lmx1b

Carlos G. Arques and Cristina Clavería

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 LIM protein/protein interaction domains, each of them binding two zinc ions to form a finger-like structure.- A DNA binding homeodomain.

The LIM homeodomain (LIM-HD) protein family

LIM A LIM B HOMEODOMAIN

PROTEIN PROTEIN DNA

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.  

Lmx1b protein

Schweizer et al., 2004

Lmx1b is expressed in multiple murine tissues, including the developing limbs and eyes, the kidneys, the brain, and in cranial mesenchyme.

Lmx1b null mouse displays dorsal-to-ventral conversion of the limbs and a lack of the patella and nails.

Lmx1b null mouse model mimics the human disease associated with Lmx1b mutations, nail-patella syndrome (NPS).

Chen et al., 1998

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.

Nail-patella syndrome

hs_lmx1b 1 -----------------------------------------------------------------------------------------------MLDGI mm_lmx1b 1 -----------------------------------------------------------------------------------------------MLDGI xl_lmx1b 1 ------------------------------------------------------------------------MDIATGPESLDRCFTRGPSDCAKMLDTI dr_mx1b.1 1 -----------------------------------------------------------------------------------------------MLDGI dm_6CG4328-PA 1 -----MDIKCQQQQLGAGQLPQTGGLNQLLLLNGNERMLTTPTTAATSVVVAAGRSASTKAGVAHNDKMASVSRTVNGSCSNHNSSNSSSSSSTNSSSNM ce_ldfp6 1 ---------------------------------------------------------------------------------------------------- 2dm_apterous_A 1 MGVCTEERPVMHWQQSARFLGPGAREKSPTPPVAHQGSNQCGSAAGANNNHPLFRACSSSSCPDICDHSTKPFGNAYGTESFRSYETADRATFEDSAAKF hs_lmx1b 6 KMEEHALRPGPAT------------------------------------------------------------LGVLLG--SDCPHPAVCEGCQRPISDR mm_lmx1b 6 KMEEHALRPGPAT------------------------------------------------------------LGVLLG--SDCPHPAVCEGCQRPISDR xl_lmx1b 29 KMEDHPLRTGTAT------------------------------------------------------------LGVLLG--SECQHQAVCEGCQRPISDR dr_mx1b.1 6 KIEDHPLRSGQAT------------------------------------------------------------LGVMLG--TECHHQAVCEGCQRPISDR dm_6CG4328-PA 96 AINKQPMGMGPGTGTGMGTGTGHGPGPPNHTHCNRITLGECSLNGMDGFATPAAPPSASNTPQAPLGMASNSGMGMELGLAMASPQLSQCAHCCQPICDR ce_ldfp6 1 ---MSLLLISATT--------------------------------------------------------------------SSTTEDKLCSGCGCLIKDR 2dm_apterous_A 101 SISRSRTDCTEVSDETTSG------------------------------------------ISFKTEPFGPPSSPESTSDSKITRNLDDCSGCGRQIQDR hs_lmx1b 44 FLMRVNESSWHEECLQCAACQQALT--TSCYFRDRKLYCKQDYQQLFAA-KCSGCMEKIAPTEFVMRALECVYHLGCFCCCVCERQLRKGDEFVLKEGQL mm_lmx1b 44 FLMRVNESSWHEECLQCAACQQALT--TSCYFRDRKLYCKQDYQQLFAA-KCSGCMEKIAPTEFVMRALECVYHLGCFCCCVCERQLRKGDEFVLKEGQL xl_lmx1b 67 FLMRVNEASWHEECLQCTVCQQPLT--TSCYFRDRKLFCKQDYQQLFAA-KCSGCMEKIAPTEFVMRALECVYHLSCFCCCVCERQLRKGDEFVLKEGQL dr_mx1b.1 44 FLMRVNESSWHEECLQCAVCQQPLT--TSCYFRERKLYCKYDYQQLFAT-KCSGCLEKIAPTEFVMRALECVYHLNCFCCCVCDRQLRKGDEFVLKDGQL dm_6CG4328-PA 196 YIMRVVENSFHEGCLKCTACSLHLV--HSCYAREGKLYCRVDYERLYIRNHCLGCGLKIAADELVMRCHENVFHLKCFACVVCGALLKKGEQYVVKQGQL ce_ldfp6 30 YIYRVMEDSYHESCLRCSCCQLSLSSFKKCFSRHGNIYCEHDHQMLYGK-RCRRCMTLLLPTDIVHRVHFMYYHAQCFSCCSCQRPFNLGDEYHVFDGEV 2dm_apterous_A 159 FYLSAVEKRWHASCLQCYACRQPLERESSCYSRDGNIYCKNDYYSFFGTRRCSRCLASISSNELVMRARNLVFHVNCFCCTVCHTPLTKGDQYGIIDALI hs_lmx1b 141 LCKGDYEKEKDLLSSVSPDESDSVKSEDEDGDMKPAKGQGSQSKGSGDDGKDPRRPKRPRTILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRV mm_lmx1b 141 LCKGDYEKEKDLLSSVSPDESDSVKSEDEDGDMKPAKGQGSQSKGSGDDGKDPRRPKRPRTILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRV xl_lmx1b 164 LCKSDYEKEKDLLSSGSPDDSDSVKSDDEEGDVKPGKGRVNQGKGS-DDGKDPRRPKRPRTILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRV dr_mx1b.1 141 LCKSDYEREKDLLGSVSPDDSDSEKSEDEELDIKPEKG--SGGTGKGDDGKDPRRPKRPRTILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRV dm_6CG4328-PA 294 FCRFDYEKEVEMLQG----------YDFYGDELFPPKL----------DGR--RGPKRPRTILNTQQRRAFKASFEVSPKPCR-KVRENLAKDTGLSLRI ce_ldfp6 129 FCRNDYQSICNFQTISNP--------DPLMEEVVRSEIH-------------RKTPKRPRTILNAQQRRQFKTAFERSSKPSR-KVREQLANETGLSVRV 2dm_apterous_A 259 YCRTHYSIAREGDTASSSMSATYPYSAQFGSPHNDSSS---------------PHSDPSRSIVPTGIFVPASHVINGLPQPARQKGRPRKRKPKDIEAFT hs_lmx1b 240 VQVWFQNQRAKMKKLARRHQQQQEQQ---NSQRLGQEVLSSRMEGMMASYTPLAPPQQQIVAMEQSPYGSSDPFQQGLTPPQMP-------GNDSIFHDI mm_lmx1b 240 VQVWFQNQRAKMKKLARRHQQQQEQQ---NSQRLGQEVLSSRMEGMMASYTALAPPQQQIVAMEQSPYGSSDPFQQGLTPPQMP-------GNDSIFHDI xl_lmx1b 262 VQVWFQNQRAKIKKLARRHQQQQEQQ---NSQRLGQEVMSSRMEGMMTSYAPLAPSQQQIVTMDQNSY-STDPFQQGLTPPQMPGDHMNPYGNDTIFHDI dr_mx1b.1 238 VQVWFQNQRAKMKKLARRQQQQQEQQ---NSQRLGQEVMSNRMEGMMNSYTPLAPAQQQMVALE-NGY-STDPFQQGLTPPQMPGDHMNPYGNDSIFHDI dm_6CG4328-PA 371 VQVWFQNQRAKVKKIQKKAKQEPPSKGASDSQDSQESLDSSLATKIKDEAHSDSESQLESPYSTTSDGLTRMRCTIKDEQEQVPFNCMETNKENCNKNSE ce_ldfp6 207 VQVWFQNQRAKIKKLNKK---DSDSG---DTFKHGPGSEGRSTEDIRSSDD----------------------------------------EEESVISKL 2dm_apterous_A 344 ANIDLNTEYVDFGRGSHLSSSSRTKR---MRTSFKHHQLRTMKSYFAINHNPDAKDLKQLSQKTGLPKRVLQVWFQNARAKWRR-----MMMKQDGSGLL hs_lmx1b 330 DSDTSLTSLSDCFLGSSDVGSLQARVGNPIDRLYSMQSSYFAS----------------- mm_lmx1b 330 DSDTSLTSLSDCFLGSSDVGSLQARVGNPIDRLYSMQSSYFAS----------------- xl_lmx1b 358 DSDTSLTSLSDCFLASSEVTSMQARVGNPIDRLYSMQSSYFAS----------------- dr_mx1b.1 333 DSDTSLTSLSDCFMASSDAGSMQARVGNPIDRLYSMQSSYFAS----------------- dm_6CG4328-PA 471 PILNTILGLSYATFQQLMGPFAQTPMINPIDRLYSMQSSYFRPEELQSYGECGVKDSMDH ce_ldfp6 261 KRIG--IDIGELWLYKLKSVFQKVRKFVP------------------------------- 2dm_apterous_A 436 EKGEGALDLDSISVHSPTSFILGGPNSTPPLNLD--------------------------

LIM A LIM B HOMEODOMAIN

Lmx1b protein conservation

hs_lmx1b 92 CSGCMEKIAPTEFVMRALECVYHLGCFCCCVCERQLRKGDEFVLKEGQLLCKGDYEKE 151 mm_lmx1b CSGCMEKIAPTEFVMRALECVYHLGCFCCCVCERQLRKGDEFVLKEGQLLCKGDYEKE xl_lmx1b CSGCMEKIAPTEFVMRALECVYHLSCFCCCVCERQLRKGDEFVLKEGQLLCKSDYEKE dr_mx1b.1 CSGCLEKIAPTEFVMRALECVYHLNCFCCCVCDRQLRKGDEFVLKDGQLLCKSDYERE dm_6CG4328-PA CLGCGLKIAADELVMRCHENVFHLKCFACVVCGALLKKGEQYVVKQGQLFCRFDYEKE ce_ldfp6 CRRCMTLLLPTDIVHRVHFMYYHAQCFSCCSCQRPFNLGDEYHVFDGEVFCRNDYQSI 2dm_apterous_A CSRCLASISSNELVMRARNLVFHVNCFCCTVCHTPLTKGDQYGIIDALIYCRTHYSIA

Both LIM domains are highly conserved, although they have diverged one from each other.

LIM_A 33 CEGCQRPIS-DRFLMRVNESSWHEECLQCAACQQALTTS--CYFRDRKLYCKQDYQQLFA 89 LIM_B 92 CSGCMEKIAPTEFVMRALECVYHLGCFCCCVCERQLRKGDEFVLKEGQLLCKGDYEKE-- 151

149

hs_lmx1b 33 CEGCQRPISDRFLMRVNESSWHEECLQCAACQQALT--TSCYFRDRKLYCKQDYQQLFA 89 mm_lmx1b CEGCQRPISDRFLMRVNESSWHEECLQCAACQQALT--TSCYFRDRKLYCKQDYQQLFA xl_lmx1b CEGCQRPISDRFLMRVNEASWHEECLQCTVCQQPLT--TSCYFRDRKLFCKQDYQQLFA dr_mx1b.1 CEGCQRPISDRFLMRVNESSWHEECLQCAVCQQPLT--TSCYFRERKLYCKYDYQQLFA dm_6CG4328-PA CAHCCQPICDRYIMRVVENSFHEGCLKCTACSLHLV--HSCYAREGKLYCRVDYERLYI ce_ldfp6 CSGCGCLIKDRYIYRVMEDSYHESCLRCSCCQLSLSSFKKCFSRHGNIYCEHDHQMLYG 2dm_apterous_A CSGCGRQIQDRFYLSAVEKRWHASCLQCYACRQPLERESSCYSRDGNIYCKNDYYSFFG

149

LIM A

LIM B

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Pathologic mutations

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Pathologic mutations affect the most conserved residues between LIM A and B domains.

LIM A

LIM B

Pathologicmutations

Disruption of Zn-finger

hs_lmx1b 197 TILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRVVQVWFQNQRAKMKK 255 mm_lmx1b TILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRVVQVWFQNQRAKMKK xl_lmx1b TILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRVVQVWFQNQRAKIKK dr_mx1b.1 TILTTQQRRAFKASFEVSSKPCR-KVRETLAAETGLSVRVVQVWFQNQRAKMKK dm_6CG4328-PA TILNTQQRRAFKASFEVSPKPCR-KVRENLAKDTGLSLRIVQVWFQNQRAKVKK ce_ldfp6 TILNAQQRRQFKTAFERSSKPSR-KVREQLANETGLSVRVVQVWFQNQRAKIKK 2dm_apterous_A SIVPTGIFVPASHVINGLPQPARQKGRPRKRKPKDIEAFTANIDLNTEYVDFGR

201 253

HOMEODOMAIN * * * ** * * * *

Pathologic mutations

Homeodomain is highly conserved along evolution.

Pathologic mutations affect conserved residues.

HOMEODOMAIN Pathologicmutations

Disruption of DNA binding

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.

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

Current model

LOGO plus multi-agent capabilities plus a expanded graphical interface

Kid-oriented… or not

Free (as in “free beer”)

Exploring alternative models of rostral-caudal paatterning in the zebrafishh neurectoderm with computer4 simulations

Chitnis and Itoh

Current Opinion in Genetics & Development 2004

Wnt7a drives Lmx1b expression in the dorsal mesenchime

Wnt7a is expressed in dorsal ectoderm

Is diffusion enough?

Wnt7a gradient shape is not the observed Lmx1b domain shape

Lmx1b expression does respond to wnt7a

Lmx1b promoter activation is all or nothing

Frontier between lmx1b and not lmx1b cells is a straight line

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?

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

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

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

ConclusionsThis model could explain what is observed

It predicts something (is falsable)

Would these be compartments? (scholastic)

Netlogo can be an useful tool