Fasciculation and guidance of spinal motor axons in the absence of FGFR2 signaling.

During development, fibroblast growth factors (FGF) are essential for early patterning events along the anterior-posterior axis, conferring positional identity to spinal motor neurons by activation of different Hox codes. In the periphery, signaling through one of four fibroblast growth factor receptors supports the development of the skeleton, as well as induction and maintenance of extremities. In previous studies, FGF receptor 2 (FGFR2) was found to interact with axon bound molecules involved in axon fasciculation and extension, thus rendering this receptor an interesting candidate for the promotion of proper peripheral innervation. However, while the involvement of FGFR2 in limb bud induction has been extensively studied, its role during axon elongation and formation of distinct nervous projections has not been addressed so far. We show here that motor neurons in the spinal cord express FGFR2 and other family members during the establishment of motor connections to the forelimb and axial musculature. Employing a conditional genetic approach to selectively ablate FGFR2 from motor neurons we found that the patterning of motor columns and the expression patterns of other FGF receptors and Sema3A in the motor columns of mutant embryos are not altered. In the absence of FGFR2 signaling, pathfinding of motor axons is intact, and also fasciculation, distal advancement of motor nerves and gross morphology and positioning of axonal projections are not altered. Our findings therefore show that FGFR2 is not required cell-autonomously in motor neurons during the formation of initial motor projections towards limb and axial musculature

<i>FGFR2</i> is expressed in motor neurons of the LMC during forelimb innervation.

<p>(<b>A</b>, <b>A</b>’) At E10.5, spinal motor neurons in the ventral horn of the brachial spinal cord that will form the medial and lateral aspect of the LMC are identified by FoxP1 and/or Isl1 immunohistochemistry. A subset of these motor neurons shows expression of FGFR2 (arrows). (<b>B</b>, <b>B</b>’) At E11.5, motor neurons have segregated into two distinct sub-columns of the LMC; namely the LMCm (FoxP1⁺/Isl1⁺, green dashed line) and the LMCl (FoxP1⁺/Isl1⁻, red dashed line). <i>FGFR2</i> mRNA is found in the LMC and MMC (FoxP1^−/Isl1⁺, cyan dashed line). (<b>C</b>, <b>C</b>’) <i>In situ</i> hybridization against <i>FGFR2</i> shows a higher number of motor neurons that express the FGF receptor in the LMCm (FoxP1⁺/Isl1⁺, green dashed line, arrows) when compared to dorsally projecting motor neurons of the LMCl (FoxP1⁺/Isl1⁻, red dashed line). (<b>D</b>) Quantification of <i>FGFR2</i> mRNA expression in motor neurons of the LMCm and LMCl showed a significantly higher number of ventrally projecting motor neurons that expressed the FGF receptor. Scale bar in (<b>C</b>’) equals 25 µm for (<b>A</b>), 40 µm for (<b>B</b>) and 50 µm for (<b>C</b>).</p

Ablation of <i>FGFR2</i> from motor neurons does not impair fasciculation, extension and gross morphology of nerve projections.

<p>Immunohistochemical staining of wholemount embryo preparations against <i>Hb9::eGFP</i> (green, motor nerves) and Neurofilament (red, motor and sensory nerves). At E10.5, tightly fasciculated spinal nerves have formed the brachial plexus at the base of the limb of control embryos (<b>A</b>) and in <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos (<b>B</b>). (<b>C, D</b>) Quantification of the pre-plexus fasciculation of the 6 spinal nerves that form the brachial plexus shows no differences between control (0.25±0.017 SEM) and <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos (0.24±0.013, p = 0,65). (<b>E</b>) Quantification of the individual thickness of spinal nerve branches that contribute fibers to the brachial plexus showed no significant difference between control and mutant embryos (p¹ = 0,63, p² = 0,99, p³ = 0,47, p⁴ = 0,20, p⁵ = 0,89, p⁶ = 0,32). (<b>F, G</b>) At E11.5, both in control and mutant embryos, first target specific fascicles have entered the limb mesenchyme. (<b>H</b>) Motor and sensory innervation of control embryo forelimbs. 1 =  branch of the radial nerve, 2 =  radial nerve, 3 =  median nerve, 4 =  ulnar nerve. (<b>I</b>) Gross morphology of motor and sensory innervation to the forelimb is not altered in embryos where <i>FGFR2</i> was ablated in motor neurons by <i>Olig2-Cre</i>. (<b>J, K</b>) The distal advancement of the median nerve is not impaired in <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos (0.67±0.02) when compared to control littermates (0.68±0.01, p = 0,36). (<b>L</b>) Quantification of the individual thickness of the 4 major motor nerves shows not significant differences between control and mutant embryos in fasciculation (p¹ = 0,24, p² = 0,99, p³ = 0,47, p⁴ = 0,19). Innervation of intercostal muscles at thoracic levels forms tightly fasciculated nerve branches in control (<b>M</b>) and <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos (<b>N</b>). Also innervation of epaxial musculature by the ascending branch (empty arrowheads) of MMC projections is established normally in <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos (<b>P</b>) when compared to control embryos (<b>O,</b> arrowhead points to descending branch which innervates intercostal musculature). Scale bar in (<b>P</b>) equals 100 µm for <b>A, B, F</b> and <b>G,</b> 500 µm for <b>H</b> and <b>I,</b> and 200 µm for M and N, and 100 µm for <b>O</b> and <b>P.</b></p

Expression analysis of <i>FGFR1–4</i> and <i>Sema3A</i>.

<p>(<b>A</b>’–<b>J</b>’) The subdivisions of the LMC and the MMC are identified by immunohistochemistry against FoxP1 and Isl1. Motor neurons in the LMCm are FoxP1⁺/Isl1⁺ (green dashed lines), LMCl motor neurons are FoxP1⁺/Isl1⁻ (red dashed lines) and MMC motor neurons are FoxP1⁻/Isl1⁺ (cyan dashed lines). (<b>A</b>, <b>B</b>) <i>FGFR1</i> is expressed by motor neurons in the LMCm, LMCl and MMC of control and <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos, respectively. (<b>C</b>) <i>FGFR2</i> mRNA is found in motor neurons of the LMCm and subpopulations of LMCl and MMC motor neurons in control embryos. (<b>D</b>) Expression of Cre recombinase under the <i>Olig2</i> promotor tissue-specifically ablates <i>FGFR2</i> expression in motor neurons of the LMC and MMC, while in the ventricular zone <i>FGFR2</i> mRNA is still detected (compare arrows in <b>C</b> and <b>D</b>). (<b>E</b>, <b>F</b>) <i>FGFR3</i> mRNA is detected in motor neurons of the LMC and MMC in both control and <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos. (<b>G</b>, <b>H</b>) <i>In situ</i> hybridization against <i>FGFR4</i> shows expression of the FGF receptor gene in the ventral horn of the spinal cord of control and <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos. (<b>I</b>) <i>Sema3A</i> is expressed by motor neurons in the LMCm, LMCl and MMC, respectively, in control embryos. (<b>J</b>) <i>Sema3A</i> is expressed by motor neurons of the LMCm, LMCl and MMC, respectively, in <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos. (<b>K</b>, <b>L</b>) Expression of <i>FGFR2</i> in sensory neurons is not affected by ablation of <i>FGFR2</i> by <i>Olig2-Cre</i>. (<b>K</b>’, <b>L</b>’) Immunohistochemistry against Isl-1/2 to illustrate sensory neurons in the DRG. (<b>M</b>) Quantification of expression levels reveals a significant decrease of <i>FGFR2 in situ</i> hybridization signal in the LMC of <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos, while expression levels of <i>FGFR1, FGFR3</i> and <i>FGFR4</i> in the LMC remain unchanged (p<i>^FGFR1</i> = 0,52; p<i>^FGFR3</i> = 0,45, p<i>^FGFR4</i> = 0,78). (<b>N</b>) Also in the MMC, expression levels of <i>FGFR1, FGFR3</i> and <i>FRGR4</i> remain unchanged upon loss of <i>FGFR2</i> in motor neurons, while a significant decrease of <i>FGFR2 in situ</i> hybridization signal is observed in <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos when compared to control littermates (p<i>^FGFR1</i> = 0,95; p<i>^FGFR3</i> = 0,60, p<i>^FGFR4</i> = 0,20). Scale bar in <b>J</b>’ equals 45 µm for all panels.</p

Guidance decision of ventrally projecting motor axons is not affected by loss of FGFR2 signaling in motor neurons.

<p>(<b>A</b>) Retrograde tracing with dextrane-conjugated Rhodamine from dorsal limb musculature labels Isl1⁻ motor neurons in the LMCl of control embryos. (<b>B</b>) Retrograde tracing from dorsal limb musculature labels Isl1⁻ motor neurons in the LMCl, while no Isl-1⁺ motor in the LMCm show a Rhodamine labeling. (<b>C</b>) Quantification of misprojecting, Isl1⁺/Rhodamine⁺ motor neurons after retrograde tracing from dorsal limb musculature shows no significant differences between control and <i>FGFR2^flox/flox;Olig2-Cre⁺</i> mutant embryos. Scale bar in <b>B</b> equals 50 µm.</p