<i>Tbx5</i> Buffers Inherent Left/Right Asymmetry Ensuring Symmetric Forelimb Formation

<div><p>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 <i>TBX5</i>, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor <i>Tbx5</i> in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of <i>Tbx5</i> 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 <i>Tbx</i> hypomorphic mutants maintained on an <i>INV</i> mutant background, with <i>situs inversus</i>, 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 <i>Tbx5</i> are required to overcome this asymmetry to ensure symmetric forelimb formation.</p></div

<i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx1-Tbx;INV/INV</i> mutants with <i>situs inversus</i> have right biased asymmetric forelimb defects.

<p><b>A,</b><i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx-Tbx</i> mutant embryo at E14.5. The heart is on left (asterisk). <b>B,</b> Skeletal preparation. Right forelimb has 4 digits, while left forelimb has 3 digits (red arrow). <b>C,</b> <i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx1-Tbx;INV/INV</i> mutant embryo at E14.5. The heart is on the right (asterisk), indicating <i>situs inversus</i>. The right forelimb is more severely affected than the left (black arrow). <b>D,</b> 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).</p

Marker gene expression in left and right forelimb buds of <i>Tbx</i> hypomorphs.

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

A mouse <i>Tbx</i> hypomorph mutant produces left-biased asymmetric forelimb defects.

<p><b>A-B,</b> E17.5 control (A) and <i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx1-Tbx</i> mutant (B). <b>C,</b> <i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx1-Tbx</i> 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). <b>D,</b> A transverse section at the level of the forelimb bud of a E9.5 <i>Z/AP/+;Prx1Cre</i> embryo. <b>E,</b> qPCR analysis of left and right forelimb buds of <i>Prx1Cre</i> embryos. <b>F,</b> qPCR analysis of the left and right forelimb buds of <i>Prx1-Tbx</i> transgenic embryos. <b>G-J.</b> 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). <b>K.</b> Numbers of patients showing left-biased, right-biased and symmetrical forelimb defects.</p

Schematic representation of <i>Tbx5</i> buffering the inherent LPM L/R asymmetry.

<p>Bilateral optimal <i>Tbx5</i> 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 <i>Tbx5</i> 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.</p

Comparison of Cre activity in <i>Prx1Cre</i> and <i>Prx1Cre(98)</i> transgenic deleter lines.

<p><b>A-F</b>, Lateral views of right side of embryos are shown. LacZ staining of <i>Rosa26RLacZ/+;Prx1Cre</i> embryos (A, C and E) and <i>Rosa26RLacZ/+;Prx1Cre(98</i>) embryos (B, D, and F). In the <i>Prx1Cre</i> 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 <i>Rosa26RLacZ/+;Prx1Cre(98)</i> 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 <i>Rosa26RLacZ/+;Prx1Cre</i> embryos (C). At this stage cre is active in the nascent forelimb bud of <i>Prx1Cre(98)</i> embryo in a ‘salt and pepper’ mosaic manner (D). At E10.5 strong staining is observed throughout the forelimb buds of <i>Rosa26RLacZ/+; Prx1Cre</i> (E, arrowhead) and in a mosaic manner in the forelimb buds of <i>Rosa26RLacZ/+;Prx1Cre(98)</i> embryo (F, arrowhead). <b>G,</b> qPCR analysis of left and right forelimb buds of <i>Prx1Cre(98)</i> embryos. Cre mRNA is expressed at a similar level on both sides. <b>H</b>, All the elements of the forelimb have failed to form in E17.5 <i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre embryo</i> (arrow). <b>I</b>, Abnormal forelimbs are formed in E17.<i>5</i> ^{<i>Tbx5lox/lox</i>}<i>;Prx1Cre(98)</i> embryo (arrow).</p

<i>Fgf10</i> expression at an optimal level in the forelimb LPM cannot rescue asymmetric defects of <i>Tbx5</i>^{<i>lox/lox</i>}<i>; Prx1Cre; Prx1-Tbx</i> mutants.

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