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

<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

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