Tbx5 Buffers Inherent Left/Right Asymmetry Ensuring Symmetric Forelimb Formation

Abstract

The forelimbs and hindlimbs of vertebrates are bilaterally symmetric. The mechanisms that ensure symmetric limb formation are unknown but they can be disrupted in disease. In Holt-Oram Syndrome (HOS), caused by mutations in TBX5, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor Tbx5 in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of Tbx5 produces asymmetric forelimb defects that are consistently more severe in the left limb than the right, phenocopying the left-biased limb defects seen in HOS patients. In Tbx hypomorphic mutants maintained on an INV mutant background, with situs inversus, the laterality of defects is reversed. Our data demonstrate an early, inherent asymmetry in the left and right limb-forming regions and that threshold levels of Tbx5 are required to overcome this asymmetry to ensure symmetric forelimb formation.

Fig 1. A mouse Tbx hypomorph mutant produces left-biased asymmetric forelimb defects.

A-B, E17.5 control (A) and Tbx5^lox/lox;Prx1Cre;Prx1-Tbx mutant (B). C, Tbx5^lox/lox;Prx1Cre;Prx1-Tbx mutant forelimb skeletal preparations shown in descending order of severity (top to bottom) and E17.5 control forelimbs. The defects are consistently more severe in the left forelimb than the right (n = 11). Absent digit 1 (thumb) (arrows), triphalangeal digit 1 (arrowhead). D, A transverse section at the level of the forelimb bud of a E9.5 Z/AP/+;Prx1Cre embryo. E, qPCR analysis of left and right forelimb buds of Prx1Cre embryos. F, qPCR analysis of the left and right forelimb buds of Prx1-Tbx transgenic embryos. G-J. X-ray radiographs of a HOS patient. The left forelimb is more affected than the right one. The thumb is absent on the left hand (G, arrow) while it is present on the right (H). The radius is missing on the left side (I, arrow), while it is formed on the right (J). K. Numbers of patients showing left-biased, right-biased and symmetrical forelimb defects.

Fig 2. Marker gene expression in left and right forelimb buds of Tbx hypomorphs.

WISH analysis of E10.5 embryos. A, Fgf10 expression in a control embryo B, Left forelimb bud is not formed and Fgf10 expression is lost on the left side (arrowhead) in a severely affected embryo C, Fgf10 is weaker in left forelimb of a mildly affected embryo (arrowhead). D, Fgf8 expression in control E, Fgf8 is absent in left AER of a severely affected embryo (arrowhead). F, Fgf8 is disrupted in left AER of a mildly affected embryo (arrowhead). G, Sall4 expression in control. H, Sall4 is absent in left LPM of a severely affected embryo (arrowhead). I, Sall4 is expressed at similar levels bilaterally in a mildly affected embryo. A minimum of 4 mutants for each phenotype were analysed with each probe.

Fig 3. Comparison of Cre activity in Prx1Cre and Prx1Cre(98) transgenic deleter lines.

A-F, Lateral views of right side of embryos are shown. LacZ staining of Rosa26RLacZ/+;Prx1Cre embryos (A, C and E) and Rosa26RLacZ/+;Prx1Cre(98) embryos (B, D, and F). In the Prx1Cre line, Cre is active throughout the forelimb-forming region (marked by black asterisks in the adjacent somites) by 16 somites stage (A) while cre activity is not detected in Rosa26RLacZ/+;Prx1Cre(98) at this stage (B). At 22 somites stage, staining is seen throughout the nascent forelimb bud as well as the LPM rostral and caudal to the forelimb-forming region in Rosa26RLacZ/+;Prx1Cre embryos (C). At this stage cre is active in the nascent forelimb bud of Prx1Cre(98) embryo in a ‘salt and pepper’ mosaic manner (D). At E10.5 strong staining is observed throughout the forelimb buds of Rosa26RLacZ/+; Prx1Cre (E, arrowhead) and in a mosaic manner in the forelimb buds of Rosa26RLacZ/+;Prx1Cre(98) embryo (F, arrowhead). G, qPCR analysis of left and right forelimb buds of Prx1Cre(98) embryos. Cre mRNA is expressed at a similar level on both sides. H, All the elements of the forelimb have failed to form in E17.5 Tbx5^lox/lox;Prx1Cre embryo (arrow). I, Abnormal forelimbs are formed in E17.5 ^Tbx5lox/lox;Prx1Cre(98) embryo (arrow).

Fig 4. The mosaic and delayed deletion of Tbx5 results in the formation of left-biased forelimb defects.

A, a selection of 5 pairs of the left and right forelimbs of E17.5 Tbx5^lox/lox;Prx1Cre(98) mutants and control embryos illustrating the range in forelimb defect severity. Mutant forelimbs are shown in descending order of severity from top to bottom. B, Tabulation of forelimb defects observed in 18 mutant embryos. Defects are observed more frequently in the left than the right forelimbs. C-H, WISH analysis of E10.5 embryos. Left forelimbs are smaller than right forelimbs (n = 12) (arrowheads). Fgf8 expression is disrupted in left AER (F) while an obvious reduction of Fgf10 (D) and Sall4 (H) is not observed.

Fig 5. Tbx5^lox/lox;Prx1Cre;Prx1-Tbx;INV/INV mutants with situs inversus have right biased asymmetric forelimb defects.

A, Tbx5^lox/lox;Prx1Cre;Prx-Tbx mutant embryo at E14.5. The heart is on left (asterisk). B, Skeletal preparation. Right forelimb has 4 digits, while left forelimb has 3 digits (red arrow). C, Tbx5^lox/lox;Prx1Cre;Prx1-Tbx;INV/INV mutant embryo at E14.5. The heart is on the right (asterisk), indicating situs inversus. The right forelimb is more severely affected than the left (black arrow). D, Skeletal preparation. The left forelimb has 4 digits. In contrast, the right forelimb is more severely affected having 3 digits with the most anterior bifurcated (red arrow).

Fig 6. Fgf10 expression at an optimal level in the forelimb LPM cannot rescue asymmetric defects of Tbx5^lox/lox; Prx1Cre; Prx1-Tbx mutants.

A-B, Ventral views of mutant embryos at E17.5. The left forelimb is severely truncated compared to the right in Tbx5^lox/lox;Prx1Cre;Prx1-Tbx mutant (arrow) (A). Tbx5^lox/lox;Prx1Cre;Prx1-Tbx;Z/EGFgf10 mutant lacks digit one in the left forelimb (arrow) (B). C-E, Skeletal preparation of Tbx5^lox/lox;Prx1Cre;Prx1-Tbx (C), Tbx5^lox/lox;Prx1Cre;Prx1-Tbx;Z/EGFgf10 (D) and control (E) forelimbs. Left forelimbs are more severely affected than right in both examples. Absent digit 1 (thumb) (arrow), bifurcated digit (arrowheads).

Fig 7. Schematic representation of Tbx5 buffering the inherent LPM L/R asymmetry.

Bilateral optimal Tbx5 expression reaches above a threshold level that buffers the inherent asymmetry in the left and right LPM, which includes the future forelimb-forming regions. Bilateral, suboptimal Tbx5 expression levels that fail to reach threshold levels leads to abnormalities in limb formation and cannot buffer the inherent asymmetry in the LPM and as a result the left limb is more severely affected by lowered Tbx5 activity than the right limb.