Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1

Abstract

Background: Lim-HD proteins control crucial aspects of neuronal differentiation, including subtype identity and axonal guidance. The Lim-HD proteins Lhx2/9 and Lhx1/5 are expressed in the dorsal spinal interneuron populations dI1 and dI2, respectively. While they are not required for cell fate acquisition, their role in patterning the axonal trajectory of dI1 and dI2 neurons remains incompletely understood.

Results: Using newly identified dI1- and dI2-specific enhancers to trace axonal trajectories originating from these interneurons, we found that each population is subdivided into several distinct groups according to their axonal pathways. dI1 neurons project axons rostrally, either ipsi- or contra-laterally, while dI2 are mostly commissural neurons that project their axons rostrally and caudally. The longitudinal axonal tracks of each neuronal population self-fasciculate to form dI1- and dI2-specific bundles. The dI1 bundles are spatially located ventral relative to dI2 bundles. To examine the functional contribution of Lim-HD proteins to establishment of dI axonal projections, the Lim-HD code of dI neurons was altered by cell-specific ectopic expression. Expression of Lhx1 in dI1 neurons caused a repression of Lhx2/9 and imposed caudal projection to the caudal commissural dI1 neurons. Complementarily, when expressed in dI2 neurons, Lhx9 repressed Lhx1/5 and triggered a bias toward rostral projection in otherwise caudally projecting dI2 neurons, and ventral shift of the longitudinal axonal fascicule.

Conclusion: The Lim-HD proteins Lhx9 and Lhx1 serve as a binary switch in controlling the rostral versus caudal longitudinal turning of the caudal commissural axons. Lhx1 determines caudal turning and Lhx9 triggers rostral turning.

Figure 1

Characterization of the dI1 enhancer. The EdI1 enhancer element was cloned upstream of Cre recombinase and electroporated with (A) a conditional alternating mCherry/GFP (CAGG-loxP-mCherry-loxP-GFP) or (B, C) a conditional nuclear GFP (CAGG-loxP-STOP-loxP-nGFP) plasmid. Chick embryos were electroporated at stage 16 and fixed at stage 23 (B, C) or stage 26 (A). Cross-sections of electroporated neural tube were stained with dI-specific antibodies (B, C). (A) The use of the alternating mCherry/GFP allows the simultaneous detection of the electroporated cells (expressing mCherry) and cells that express the EdI1 enhancer (expressing GFP). Most of the cells, along the entire dorsal/ventral levels, express mCherry, while a subpopulation of dorsally located cells expresses GFP. (B) Cross-sections of electroporated neural tube were stained with Isl1 and Lhx2/9 antibodies. Nuclear GFP (nGFP)-expressing neurons are Lhx2/9⁺/Isl1^-. (C) Cross-sections of electroporated neural tube were stained with Isl1 and Lhx1/5 antibodies. nGFP expressing neurons are Lhx2/9⁺/Lhx1/5^-. The boxed areas in (B, C) are represented as enlargements in their different channels at the bottom of each panel. The arrows point to the nGFP-expressing cells. Scale bars: 50 μm.

Figure 2

Characterization of dI2 enhancers. The EdI2/V1 and EdI1/2 enhancer elements were cloned upstream of Cre recombinase or Gal4 and electroporated with (A) a conditional alternating mCherry/GFP plasmid (CAGG-loxP-mCherry-loxP-GFP), (B-E) a conditional nuclear GFP (nGFP) plasmid (CAGG-loxP-STOP-loxP-nGFP) and (F-H) a double conditional GFP plasmid (UAS-loxP-STOP-loxP-GFP). Chick embryos were electroporated at stage 16 and fixed at stage 23 (B-H) or stage 26 (A). (A) Embryos were co-electroporated with a EdI2/V1::Cre plasmid and a conditional alternating mCherry/GFP plasmid. Most of the cells, along the entire dorsal/ventral levels, express mCherry, while a subpopulation of dorsally and medial-laterally located cells expresses GFP. (B-E) Embryos were co-electroporated with a EdI2/V1::Cre plasmid and a conditional nGFP plasmid. Cross-sections of electroporated neural tube were stained with cell fate markers. nGFP-expressing neurons at the dorsal neural tube are dI2 neurons, as indicated by the expression of Lhx1/5⁺/Lhx2/9^- (B), Lhx2/9^-/Isl1^- (C), and Lhx1/5⁺/Pax2^- (D); at the medial neural tube, nGFP-expressing neurons are V1 neurons, as indicated by the co-expression of Lhx1/5⁺/Pax2⁺ (arrowhead in (D)) and En1⁺/Pax2⁺ (E). (F-H) Expression of GFP in neurons that co-express the EdI2/V1 and EdI1/2 enhancers. Embryos were electroporated with three plasmids: EdI2/V1::Gal4, EdI1/2::Cre and the double conditional GFP plasmid. Note that GFP is expressed in the cytoplasm, axons and dendrites. The arrows point to the center of the neurons. GFP-expressing neurons are Lhx1/5⁺/Pax^- (F), Pax^-/En1^- (G) and Lhx2/9^-/Isl1^- (H). (I) A table showing the specificity of the antibodies used in Figures 1 and 2. Boxed areas in the panels are represented as enlargements in their different channels at the bottom of each. The arrows point to the nGFP- and GFP-expressing cells. Scale bars: 50 μm.

Figure 3

Axonal projection pattern of dI1 neurons. (A) Chick embryos were electroporated at stage 16 (left side) with EdI1 enhancer along with a Cre-dependent GFP plasmid (EdI1::Cre + pCAGG-LoxP-STOP-LoxP-GFP). At E6, spinal cords were removed, fixed and analyzed as an open-book preparation. Whole neural tubes (sacral to cervical) are presented. Confocal images were taken and photomerged utilizing Photoshop software. (B) The schematic illustrates the axonal projection pattern of the dI1 neuronal population. Rostral is up in the image and the schematics. Asterisks represent the levels of the limbs. dI1 neurons have two main rostral axonal projection pathways (A): ipsi-lateral (A4, A5, yellow arrows), and contra-lateral (A, A2, A3, white arrows and arrowheads). After crossing the floor plate (FP), dI1_comm neurons elongate along the floor plate at the ventral funiculus (VF; white arrowheads) for several segments and, subsequently, turn toward the lateral funiculus (LF; white arrows). dI1_ipsi axons turn longitudinally and rostrally at the LF of the lumbar, thoracic and cervical levels (A, A4, A5). At the caudal sacral level dI1_ipsi axons project caudally (A, A1, magenta arrows). (C, D) The relative position of dI1_ipsi and dI1_comm fascicules at the LF was studied following differential labelling of dI1_ipsi and dI1_comm axons. At the LF, dI1_ipsi and dI1_comm form one fascicle (green+red arrow in (D)). (E) Schematic with boxed areas representing the frames of (C, D). Only the rostrally turning axons are illustrated in (E). c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. Scale bars: 150 μm (A, D); 100 μm (A1–A5); 300 μm (C).

Figure 4

Axonal projection pattern of contra-laterally projecting dI2 neurons. Chick embryos were electroporated at stage 16 (left side) with (A) EdI2/V1 and (B) EdI1/2 enhancers along with a Cre-dependent GFP plasmid (EdI2/V1::Cre or EdI1/2::Cre + pCAGG-LoxP-STOP-LoxP-GFP). At E6, spinal cords were removed, fixed and analyzed as an open-book preparation. Whole neural tubes (sacral to cervical) are presented. Confocal images were taken and photomerged utilizing Photoshop software. Rostral is up in the image and the schematics. At the sacral level at the contra-lateral side dI2_caud axons turn caudally (A, A1, B, B1, magenta arrows); at the caudal thoracic level axons turn either caudally or rostrally, forming a crisscross pattern (A, A2, B, B2, white and magenta crossed arrows). At the rostral two-thirds of the thoracic level, the brachial and cervical levels dI2_rost axons turn rostrally (A, A3, B, B3, white arrows). At the sacral and cervical levels axons turn from the ventral funiculus (VF) to the lateral funiculus (LF) (A, A1, A3, B). c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. Asterisks represent the level of the limbs. Scale bar: 200 μm (A); 50 μm (A1–A3); 150 μm (B); 30 μm (B1–B3).

Figure 5

Axonal projection pattern of dI2 neurons. (A, B) Chick embryos were electroporated at stage 16 with three plasmids: dI1/2::Cre, dI2/V1::Gal4 and UAS::LoxP-STOP-LoxP-GFP. The schematic (B) illustrates the axonal projection pattern of the dI2 neuronal population. At the sacral and lumbar levels on the contra-lateral side, dI2_caud axons turn caudally (A, A1, magenta arrows). At the caudal thoracic level of the contra-lateral side a crisscross pattern of axons turning either rostrally or caudally is apparent (A, A2, A3, white and magenta crossed arrows). At the rostral thoracic level, and the cervical level, dI2_rost axons turn rostrally (A, A4, white arrows). Along the entire longitudinal axis, dI2 axons initially form a fascicle at the contra-lateral ventral funiculus (VF; arrowheads in A, A1, A2) and subsequently axons are deflected to the lateral funiculus (LF; A, A4). At the ipsi-lateral side only few axons project longitudinally (A5, yellow arrow). Most of the axons grow toward the floor plate (A, A6). c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. Asterisks represent the level of the limbs. Scale bar: 150 μm (A); 75 μm (A1, A2, A4, A6); 35 μm (A3, A5).

Figure 6

Lhx9 and Lhx1 cross-repress each other. (A, C) Lhx9 and (B, D) Lhx1, cloned in pCIG plasmid, were electroporated at stage 19 into the chick neural tube. At E4, embryos were fixed and stained with antibodies (Ab) to the cognate Lim-HD proteins. A vast reduction in Lhx1/5 (A) is evident after Lhx9 ectopic expression. Lhx1 ectopic expression resulted in a reduction of Lhx2/9 (B). For quantification (C, D), the ratio between dIx-specific neurons expressing ectopic Lim-HD (Lim^ecto) plus their own Lim-HD (Lim^x) and the total number of electroporated dIx neurons is presented. Neurons co expressing Lim^ecto and Lim^x are nuclear GFP (nGFP)+/Lim^x+. For estimating the total number of electroporated dIx neurons, the number of nGFP-/Lim^x+ neurons at the electroporated side was subtracted from the number of Lim^x+ neurons at the control side. The quotient plus the number of nGFP+/Lim^x+ equals the number of electroporated dIx neurons. Scale bar: 200 μm.

Figure 7

Lhx9 and Lhx1 do not change dI cell fate. In situ hybridizations with dI-specific genes Foxd3 (dI2), and Lhx2 and Atoh1 (dI1) were preformed on sections of embryos electroporated with (A, B) Lhx1-IRES-GFP, (C, D) Lhx9-IRES-nGFP and (E-H) Lhx9-IRES-GFP. Adjacent sections were used for GFP detection (A, E, G) and for in situ hybridization (B, F, H). Alternatively, GFP detection by antibody staining and in situ hybridization were performed on the same slide (C, D). Ectopic expression of Lhx1 does not activate the expression of FoxD3 (A, B). Ectopic expression of Lhx9 down-regulates the expression of Lhx2 (C, D) and Atoh1 (E, F) and does not affect Foxd3 expression (G, H). Arrows point to dI2 neurons (B, H) and dI1 neurons (D, F). Arrowheads point to V1 neurons (B, H). Scale bar: 150 μm.

Figure 8

Lhx1 confers caudal axonal projection to the caudal dI1_comm neurons. (A, B) Lhx1 and taumyc were expressed ectopically in dI1 neurons, utilizing the Cre/lox system and the EdI1 enhancer (EdI1::Cre + pCAGG-LoxP-STOP-LoxP-Lhx1-IRES-taumyc). The following dI2-specific axonal cues are assumed by the commissural dI1^Lhx1 neurons: at the lumbosacral levels dI1^Lhx1 axons turn caudally (A, A1); at the thoracic level axons turn either rostrally or caudally, forming a crisscross pattern (A, A2); at the cervical level, axons turn rostrally (A, A3). An illustration of the phenotype of dI1^Lhx1 ectopically expressing neurons is presented in (B). c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. The asterisks represent the level of the limbs. The white arrows point to the rostrally projecting axons. The magenta arrows point to the caudally projecting axons. The crossed arrows point to the crisscross axonal pattern. The arrow-head points to the longitudinal ipsi-lateral axons. Scale bar: 150 μm (A); 75 μm (A1–A3).

Figure 9

Lhx9 mediates a caudal to rostral change in the turning of dI2 axons. Lhx9 and GFP were expressed ectopically in dI2 and V1 neurons, utilizing the Cre/lox system and the EdI2/V1 enhancer (EdI2/V1::Cre + pCAGG-LoxP-STOP-LoxP-Lhx9-IRES-GFP). (A) At the caudal sacral level the commissural dI2^Lhx9 axons turn caudally (A, A1). At the rostral sacral level axons turn rostrally and caudally, forming a crisscross pattern (A, A2). Rostral to the lumbar level dI2^Lhx9 axons turn rostrally (A, A3, A4). A longitudinal fascicle at the ventral funiculus (VF) is present at the ipsi- and contra-lateral sides of the floor plate (A, A4). (B) Schematic representation of the caudal/rostral axonal projection of three control neural tubes (dI2^GFP) and of six manipulated neural tubes (dI2^Lhx9). The vertical lines represent the location of the limbs. (C) Schematic illustration of the phenotype of dI2^Lhx9 axonal cues. c, cervical level; b, brachial level; FP, floor plate; l, lumbar level, s, sacral level; t, thoracic level. The asterisks represent the level of the limbs. The white arrows point to the rostrally projecting axons. The magenta arrows point to the caudally projecting axons. The crossed arrows point to the crisscross axonal pattern. Scale bars: 200 μm (A); 150 μm (A1, A3–A5); 100 μm (A2).

Figure 10

Lhx9 triggers a ventral shift to the longitudinal dI2^Lhx9 axons. (A, B) An open-book preparation of the neural tube in which the commissural dI1 axons express GFP and dI2 axons express taumyc (EdI1::Gal4 + UAS::GFP; EdI2/V1::Cre + pCAGG-LoxP-STOP-LoxP-taumyc). The contra-lateral side of the neural tube is shown. dI2 axons turn longitudinally, at the lateral funiculus (LF), and form a fascicle that is located dorsally to the dI1 fascicle. Ectopic expression of Lhx9 in dI2 neurons (EdI2/V1::Gal4 + UAS::GFP_UAS::Lhx9) resulted in dI2^Lhx9 axons fasciculating with dI1 axons at the contra-LF. (C) Schematic illustrations of the co-fasciculation phenotype of dI2^Lhx9 neurons. The green and red arrows point to the axons of dI1 and dI2 neurons according to the color code that is indicated in each illustration. Scale bars: 150 μm (A, C, D); 200 μm (B).