Sustained Hox5 gene activity is required for respiratory motor neuron development

Abstract

Respiration in mammals relies on the rhythmic firing of neurons in the phrenic motor column (PMC), a motor neuron group that provides the sole source of diaphragm innervation. Despite their essential role in breathing, the specific determinants of PMC identity and patterns of connectivity are largely unknown. We show that two Hox genes, Hoxa5 and Hoxc5, control diverse aspects of PMC development including their clustering, intramuscular branching, and survival. In mice lacking Hox5 genes in motor neurons, axons extend to the diaphragm, but fail to arborize, leading to respiratory failure. Genetic rescue of cell death fails to restore columnar organization and branching patterns, indicating these defects are independent of neuronal loss. Unexpectedly, late Hox5 removal preserves columnar organization but depletes PMC number and branches, demonstrating a continuous requirement for Hox function in motor neurons. These findings indicate that Hox5 genes orchestrate PMC development through deployment of temporally distinct wiring programs.

Figure 1

Transcription factor expression in cervical motor neurons. (a–b) A ventromedial column of Isl1/2+ neurons in the cervical spinal cord expresses Scip and excludes FoxP1. (c–d) Scip+ neurons express Hb9 and ALCAM. (e) A ventrolateral motor neuron population can be defined by the expression of Sox5 and Lhx3. (f) Summary of distribution of motor columns in the cervical spinal cord. (g–i) Retrograde labeling of motor neurons after rhodamine dextran (RhD) injection into the phrenic nerve (arrow in g) of e12.5 embryos, the age when phrenic axons approach the diaphragm. RhD+ motor neurons express Scip (h) but exclude FoxP1 (i). (j–o) Expression of Hox proteins in cervical/brachial spinal cord at e12.5. (j–k) Scip+ PMC neurons express Hoxa5 and Hoxc5. (l–o) Scip+ PMC neurons exclude Hoxc4, Hoxc6, Hoxc8 and Hoxb5. Scale bar=25μm in (a), 100μm in (g).

Figure 2

Hox and FoxP1 activities determine PMC neuron distribution. (a–c) In Hoxc6^−/− mice there is an instrasegmental and rostrocaudal expansion of Scip+ motor neurons (MNs) at e12.5. (d–f) ALCAM expression expands in Hoxc6^−/− mice. In panels c, f, i, l, and o total PMC numbers are extrapolated from serial sections, and roman numerals below section number indicate approximate position of cervical segments. (g–h) In Foxp1^MNΔ mice there is an increase of Scip+ neurons in the rostral cervical spinal cord at e12.5 (505±51 motor neurons in wt vs 1330±45 motor neurons in Foxp1^MNΔ mice, n=3, P<0.01; see also Supplementary Fig. 2c–f). (i) Quantification of Scip+ neurons in the Hox5 domain of Foxp1^MNΔ mice. (j–k) ALCAM expression only expands within the Hox5+ domain in Foxp1^MNΔ mice. Not all ectopically generated Scip+ neurons express ALCAM. (l) Quantification of ALCAM expression in Foxp1^MNΔ mice. (m–n) Increase in the number of diaphragm projecting motor neurons in Foxp1^MNΔ mice, as determined by RhD+/Scip+ cells after tracer injection into the phrenic nerve. (o) Distribution of RhD+ motor neurons in Foxp1^MNΔ mice. Diaphragm projecting neurons are confined to the Hox5 domain. We also observed a 53% increase in the thickness of the phrenic nerve in Foxp1^MNΔ mice at e12.5, in agreement with previous observations . By e18.5 the innervation pattern of the diaphragm in Foxp1^MNΔ mice was grossly normal, although some branches overlapped (Supplementary Fig. 2g–j). (p–s) Ectopic FoxP1 expression eliminates Scip from PMC neurons in the rostral cervical spinal cord, but not from LMC neurons in the caudal cervical spinal cord. (t) Model for Hox and FoxP1 interactions controlling PMC specification. Scale bars=25μm. Error bars represent s.e.m.

Figure 3

Respiratory failure and PMC loss in Hox5 ^MNΔ mice. (a–b) All Hox5^MNΔ mice are born cyanotic and perish at birth. (c–d) Histological analysis reveals that the lungs of e18.5 Hox5^MNΔ embryos collapse before birth. (e–n) Progressive loss of Scip+ PMC neurons in Hox5^MNΔ mice. At e11.5 Scip+ neuron numbers are similar between control and Hox5^MNΔ mice but progressively decrease in the mutants. By e17.5 there are no detectable Scip+ motor neurons in Hox5^MN mice. (o) Quantification of Scip+ motor neurons in control and Hox5^MNΔ mice. At least 5 pairs of embryos were analyzed for each time point. *P<0.05, ***P<0.001 (p) Quantification of non-LMC Isl1/2+ motor neurons. There is selective loss of FoxP1− neurons in Hox5^MNΔ mice. (q–r) PMC disorganization at e12.5 in Hox5^MN mice. Isl1/2+ Scip– neurons are intercalated with PMC neurons. (s–t) ALCAM expression is reduced in Hox5^MN mice, as early as e11.5 prior to loss of Scip. (u–z) Lynx2, RTN4 and PTN are downregulated in Hox5^MN mice at e12.5. PMC position is outlined by dashed red line. Expression of Lynx2 is also lost from LMC neurons. Scale bars=25μm, except in (b)=1cm and (d)=100μm. Error bars represent s.e.m.

Figure 4

Fidelity of PMC axon projections in Hox5^MNΔ mutants. (a–f) The phrenic nerve progressively thins in Hox5^MNΔ mutants. At e12.5 phrenic nerve diameter is similar between control and Hox5^MNΔ mice (a–b). Some axons stray from the phrenic nerve in Hox5^MNΔmice (arrows in b). By e14.5 the phrenic nerves become thinner in Hox5^MNΔmice (see inserts e–f) and lack arbors seen in control nerves (arrows in e–f). (g–j) Retrogradely labeled motor neurons after RhD injection in the phrenic nerve are reduced in Hox5^MNΔ mice, but retain some aspects of their molecular identity, such as Scip expression (g–h) and FoxP1 exclusion (i–j). (k–l) Quantification of RhD retrograde transport after RhD injection in the phrenic nerve. There is a decrease in the number of RhD+ neurons in Hox5^MNΔ mice (k), as well as a decrease in the percentage of RhD+ neurons that express Scip (l). Scale bars=100μm (a–f), 25 μm (g–j). Error bars represent s.e.m., *P<0.05,**P<0.01.

Figure 5

Loss of synaptic contacts between PMC neurons and diaphragms in Hox5^MNΔ mice. (a– d) Analysis of diaphragm innervation patterns at e18.5. (a–b) Diaphragms of Hox5^MNΔ mutants display a marked reduction in terminal branches and neuromuscular synapses, as revealed by neurofilament and bungarotoxin staining (n=10). (c–d) While the phrenic nerve establishes contacts with the muscle and forms a primary branch, secondary and tertiary branches fail to form. As a consequence the number of synapses formed at the diaphragm muscle is dramatically reduced in Hox5^MNΔ mutants. Scale bar=500 μm. Images shown are tiled composites of individual panels. The pattern of forelimb innervation was not appreciably affected in Hox5^MNΔ embryos, and the biceps, a muscle supplied by Hox5+ LMC neurons, was innervated normally (Supplementary Fig. 5a–d).

Figure 6

Preventing apoptosis fails to rescue diaphragm innervation in Hox5^MNΔ mutants. (a–d) Deletion of Bax increases Scip+ motor neuron numbers both in control and in Hox5^MNΔ mice at e14.5 (P<0.001). However, in Hox5^MNΔ Bax^−/− mice the PMC still appears disorganized, despite rescue of Scip+ neuron numbers (d). (e–f) ALCAM expression is increased in Bax^−/− mice while it is only slightly recovered in Hox5^MNΔ Bax^−/− mice at e14.5 (f). (g–h) Retrograde labeling of phrenic neurons with DiI at e18.5 shows that motor neurons innervate the diaphragm in Hox5^MNΔ Bax^−/− mice. (i–l) Deletion of Bax in a Hox5^MNΔ background does not rescue the diaphragm innervation defects seen in Hox5^MNΔ mice. Both control and control Bax^−/− mice exhibit a stereotypical diaphragm innervation pattern (i,k) while Hox5^MNΔ and Hox5^MNΔ Bax^−/− mice show similar defects in diaphragm innervation at e15.5 (j,l), as seen by neurofilament staining. (m–n) Scip+ and total Isl1/2+ neuron numbers in the different strains. Scale bars=25μm (a–h), 200μm (i–l). Error bars represent s.e.m.

Figure 7

Late removal of Hox5 genes leads to PMC loss and reduced terminal branching. (a–f) In Hox5^ChATMNΔ mice Hoxa5 expression persists at e11.5 (b) but is effectively removed from motor neurons by e13.5 (d). (g–l) While Scip+ PMC neuron number is similar between control and Hox5^ChATMNΔ mice at e11.5 (g–h) and e13.5 (i–j), there is a 48% reduction by e15.5 in the mutant (k–l). The remaining motor neurons appear to remain clustered at the correct PMC position. (m–p) Diaphragms of Hox5^ChATMNΔmice exhibit grossly normal neuronal innervation, with synapses on the entire muscle. However, at high magnification, a reduction in terminal arborization is observed (p). (q) Quantification of Scip+ PMC neurons in Hox5^ChATMNΔ mice. Scale bars=25μm (a–l), 200μm (m–p). Error bars represent s.e.m.,*P<0.05