Isl1 is required for multiple aspects of motor neuron development

Abstract

The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.

Fig. 1

Reduced Isl1 expression in regions of the central nervous system in Isl1 compound mutant. Isl1 is expressed in various regions of the central nervous system, including the outer layer of the striatum (St) and diencephalon (Di) (A, B, I), the motor neurons (MNs) of the ventral hindbrain (A, B, C, K), cranial ganglia/nucleus (shown are V, VII–XII) (A, B, J) and the spinal MNs (A, D, L) and dI3 interneurons (dI3 IN) (D). In Isl1 compound mutants (comp), the level of Isl1 expression and the number of Isl1 expressing cells are significantly reduced as revealed by Xgal staining (E–H) and Isl1 antibody staining (M–P, comp).

Fig. 2

Reduced Isl1 expression in Isl1 compound mutant leads to increased apoptosis and abnormal development of cranial ganglia. Wholemount neurofilament staining of control (ctrl) (A), Isl1 hypomorphic (hypo) (B), Isl1 compound mutant (comp) (C) and Nestin-Cre; Isl1 conditional knockout (cko) (D) embryos at E11.5. Compared to control embryos, Isl1 hypomorphic mutant embryos displayed a relative normal size of cranial ganglia III, V, VII/VIII, X and XI (B). However, ectopic axonal projections from the cranial ganglion IX were observed (B, white arrow). Axonal projections of XI and XII ganglia were significantly reduced and blunted (B, bracket and arrowhead). In Isl1 compound mutant (C), further reduction in Isl1 expression led to reductions in the size of the cranial ganglia (V, VII/VIII, IX and X). Reductions in axon projections were observed in ganglia V (ophthalmic, maxillary and mandibular branches), VII, IX, X, XI and XII (C). Nestin-Cre; Isl1 conditional knockout embryos displayed an overall developmental defect with severe reduction in the size of cranial ganglia and their axon projections (D). TUNEL staining at E11.5 revealed significantly increased apoptosis in the cranial ganglia V, VII, IX and XI of Isl1 compound mutant compared to control embryo (E–H and E′–H′).

Fig. 3

Decreased MN genesis in Isl1 compound mutant embryos. At E9 (18 Somites), a few Isl1 expressing MNs are generated in the anterior segments of the control spinal cord, a subpopulation of which coexpress Hb9 (A). However, in Isl1 compound mutant spinal cord, significantly fewer Isl1 expressing MNs are generated and none of which expresses Hb9 (B). At E11.5, compared to that of control mice (C, E), there is a dramatic reduction in the number of MNs marked by the expression of Isl1, Isl2 and Hb9 in Isl1 compound mutant (D, F). At E11.5, Lhx1 expression in the spinal cord of Isl1 compound mutant is comparable to that of control embryo (G, H). Lhx3 is expressed in V2 INs, and the MNs of the medial motor column where it is coexpressed with HB9 (I). However, the number of Lhx3 expressing cells in the spinal cord of Isl1 compound mutant embryos is significantly increased at E11.5 (J, L—red). Ectopic Lhx3 expressing cells were observed to invade into more ventrolateral region, a few of which coexpress HB9 (J). However, at E10.5 the number of Lhx3 expressing cells is comparable in mutant and control samples (K, L—blue).

Fig. 4

Reduced Isl1 expression leads to conversion of prospective MNs to V2a INs. A significant increase in the number of Chx10 expressing V2a interneurons (INs) was observed in the spinal cord of Isl1 compound mutant embryos at E11.5 (A–D, M). These Chx10 cells found ectopically in more ventrolateral position, mixed with remaining MNs (B, D, below the bar). At E10, a few Chx10+ V2a INs start to emerge in the domain dorsal to that of Hb9+ MNs (E). A significant increase in the number of Chx10+ INs was readily detectable at this stage, which assumed a more ventral position and was mixed with Hb9 MNs (F, L). Some mutant cells started to express Chx10 immediately after they left the ventricular zone and started to differentiate (F, arrowhead). No coexpression of V2a IN marker Chx10 with MN marker Isl1 (A, B) and Hb9 (C–F) were observed in both Isl1 compound mutant and control embryos. Chx10 is never expressed in Isl1 lineage (YFP) in the normal spinal cord (G). Isl1-βgal is not coexpressed with Chx10 in control embryos (H). However, a significant number of ventral ectopic Chx10 V2a INs in Isl1 compound mutant embryos coexpress Isl1-βgal (45.4+/−23.4 per section) (I). Intraspinal cord labeling revealed that Chx10 V2a INs were similarly labeled by Rhodamine-dextran (Rhd) in both control (16.6 ± 0.5% of total Chx10+ cells) and Isl1 compound mutant (17.2 ± 3.44%) (J, K, arrowhead and N-a). A significant number of ectopic V2a INs in Isl1 compound mutant, marked by the coexpression of Chx10 and Isl1-βgal, were also labeled by Rhodamine-dextran (K-arrow and N-b). a: Rhd+/Chx10+; b: Rhd+/Chx10+/βgal+.

Fig. 5

Defects in MN migration, motor column formation and axon growth in Isl1 hypomorphic embryos. Despite significant reduction in Isl1 expression in spinal MNs of Isl1 hypomorphic mutant, the level of Hb9 expression and the number of Hb9 expressing MNs during development are not changed (A, B and C). At E16.5, Hb9+ MNs are segregated into medial (MMC) and lateral (LMC) motor columns at the cervical spinal cord, or MMC at the thoracic spinal cord of control mice (D, F). However, in Isl1 hypomorphic embryos, Hb9+ MNs are scattered and ectopically positioned or abnormally clustered and fail to form distinct motor columns (E, G). In Isl1 compound mutant spinal cord, MNs were frequently observed to migrate along ventral roots out of neural tube (H, I arrow). In Hb9-GFP transgenic mice, GFP expression marks the axons of the spinal MNs, which after exit from vertebral column, are divided into dorsal branch (db) and ventral branch (vb) (J). In Isl1 hypomorphic mice that have been crossed onto Hb9-GFP transgenic background, the dorsal branch of the spinal motor axons appeared to be blunt (K, arrow) and frequent ectopic motor projections were observed (K, arrowhead). Wholemount neurofilament immunostaining and Alexa 488-α-bungarotoxin (BTX) staining revealed motor innervation of diaphragm by phrenic nerve and the formation of neuromuscular junctions on the diaphragm of control mice (L, N). However, diaphragm of Isl1 hypomorphic mutant mice were deinnervated and no functional neuromuscular junctions were detected (M, O).