Convergent differential regulation of SLIT-ROBO axon guidance genes in the brains of vocal learners

Abstract

Only a few distantly related mammals and birds have the trait of complex vocal learning, which is the ability to imitate novel sounds. This ability is critical for speech acquisition and production in humans, and is attributed to specialized forebrain vocal control circuits that have several unique connections relative to adjacent brain circuits. As a result, it has been hypothesized that there could exist convergent changes in genes involved in neural connectivity of vocal learning circuits. In support of this hypothesis, expanding on our related study (Pfenning et al. [2014] Science 346: 1256846), here we show that the forebrain part of this circuit that makes a relatively rare direct connection to brainstem vocal motor neurons in independent lineages of vocal learning birds (songbird, parrot, and hummingbird) has specialized regulation of axon guidance genes from the SLIT-ROBO molecular pathway. The SLIT1 ligand was differentially downregulated in the motor song output nucleus that makes the direct projection, whereas its receptor ROBO1 was developmentally upregulated during critical periods for vocal learning. Vocal nonlearning bird species and male mice, which have much more limited vocal plasticity and associated circuits, did not show comparable specialized regulation of SLIT-ROBO genes in their nonvocal motor cortical regions. These findings are consistent with SLIT and ROBO gene dysfunctions associated with autism, dyslexia, and speech sound language disorders and suggest that convergent evolution of vocal learning was associated with convergent changes in the SLIT-ROBO axon guidance pathway.

Keywords: axon guidance; hummingbird; neural connectivity; parrot; songbird; vocal learning.

Figure 1

Brain pathways for vocal behavior in vocal learning and vocal nonlearning birds and mammals. A: Drawing of a zebra finch male brain section showing connectivity of posterior (HVC, RA) and anterior (LMAN, area X) song pathways. B: Drawing of a human brain section showing proposed vocal pathway connectivity including the LMC/LSC and part of the anterior striatum (ASt) that shows convergence with songbird RA and area X (Pfenning et al., 2014). Black arrows, connections and regions of the posterior vocal motor pathway; white arrows, connections and regions of the anterior vocal pathway; dashed arrows, connections between the two pathways. Red arrows show the direct projections found only in vocal learners, from vocal motor cortex regions to brainstem vocal motor neurons. C: Connectivity of a vocal nonlearning bird, chicken, showing the absence of forebrain song nuclei. D: Connectivity of vocal nonlearning primates (macaque shown), which has forebrain regions that make indirect projections to the nucleus ambiguus (Amb), although they are not necessary for vocalizations. E: Proposed mouse vocal pathway connectivity, which has a very sparse direct projection to the Amb. For abbreviations, see list. Figure panels modified from Arriaga et al., ; Petkov and Jarvis, ; and Pfenning et al., .

Figure 2

Brain expression of SLIT and ROBO genes in vocal learning and vocal nonlearning birds. A: Diagrams of the arcopallium region in coronal view in the species studied (zebra finch, a songbird; budgerigar, a parrot; Anna's hummingbird; ring dove; Japanese quail). Inset with arrows in the quail panel is a compass for orientation: d, dorsal; m, medial; v, ventral; l, lateral. B–D: mRNA expression patterns of SLIT1 (B), ROBO1 (C), and ROBO2 (D) in the RA analog and the surrounding arcopallium of avian vocal learners and vocal nonlearners. White silver grains show mRNA expression in the darkfield view; red label is cresyl violet stain. Images are expanded views of the in situ hybridizations shown in Pfenning et al. (2014), except that the zebra finch is from a different animal. E: Expression pattern of ROBO2 from another zebra finch that did not show a differential expression in RA. F,G: Expression pattern of SLIT2 (F) and SLIT3 (G) in the zebra finch. H. Quantification of SLIT1, ROBO1, and ROBO2 mRNA expression in the RA analog in vocal learners or the intermediate arcopallium in vocal nonlearners versus adjacent motor intermediate arcopallium in each species. Only SLIT1 was significantly differentially expressed in the same direction in all vocal learners versus vocal nonlearners (P < 0.05; paired t-tests; n= 3–6 per species). Error bars, SEM. For abbreviations, see list. Scale bar = 500 µm in second row (applies to rows below). Scale bar in panel B, zebra finch, applies to E–G.

Figure 3

Expression of SLIT and ROBO genes that have prominent expression in the telencephalon, in the sagittal plane in the male zebra finch brain. A: SLIT1 mRNA expression in a plane of section in which all major song nuclei are present (highlighted with lines). B: ROBO1 mRNA expression showing a mostly complementary pattern to SLIT1, except in song nuclei. The white streak is in the midbrain. C: ROBO2 mRNA expression in an adjacent section. For telencephalic subdivision abbreviations in A, see list. Scale bar = 1.0 mm in C (applies to A–C).

Figure 4

Expression of SLIT and ROBO genes in brainstem vocal (nXIIts) and neck (SSp) motor neurons. A–D: Example in situ hybridization expression patterns of SLIT1 (A), SLIT3 (B), ROBO1 (C), and ROBO2 (D) in the zebra finch brainstem motor neurons. E,F: Brightfield high magnification of ROBO1 (E) and ROBO2 (F) mRNA expression in nXIIts and SSp, showing differences of mRNA levels (measured by black silver grains) in the motor neurons. For abbreviations, see list. Scale bar = 500 µm in D (applies to A–D); 30 µm in F (applies to E,F).

Figure 5

Quantification of nXIIts:SSp gene expression ratios across species. Shown are quantifications of relative differences of four genes from the SLIT-ROBO family in the vocal motor (nXIIts) and neck motor (SSp) neurons of three vocal learning (songbird, parrot, hummingbird) and two vocal nonlearning (dove and quail) species. * P < 0.05; paired t-test between nXIIts and SPP of the same animals, indicating significantly greater or less than a ratio of 1; n = 3–4 per species. Error bars, SEM. For abbreviations, see list.

Figure 6

Expression of SLIT1 and ROBO1 in zebra finch RA during song development. A,B: In situ hybridization in darkfield view showing expression of (A) SLIT1 and ROBO1 (B) mRNA (white) in RA of male and female zebra finches during juvenile development: days 20, 35, and 65 post hatch relative to adult (>90). C: Quantification of expression in RA versus adjacent intermediate arcopallium in male (filled bars) and female (open bars) zebra finches. * P < 0.05; ** P < 0.01; *** P < 0.001 (one-tailed t-tests; n = 3 animals/group). Error bars, SD. Scale bar = 500 µm in B (applies to A,B).

Figure 7

Expression of SLIT1 in quail forebrain and zebra finch brainstem at earlier ages. A: Coronal section through a 2-day-old male quail brain, showing no region of strong differential downregulation of SLIT1. B: Expression of SLIT1 in the brainstem of the same 2-day-old quail, showing that there is no difference from that seen in the motor neurons of zebra finches. C: Expression of SLIT1 in the brainstem at in a PHD (posthatch day) 19 zebra finch. D: Expression of SLIT1 in the brainstem of a PHD61 zebra finch. For abbreviations, see list. Scale bars = 500 μm.

Figure 8

Expression of SLIT and ROBO genes in mouse putative LMC. A–D: In situ hybridization expression patterns of SLIT1 (A), SLIT2 (B), ROBO1 (C), and ROBO2 (D) in sections containing putative LMC. E: Expression of singing-driven EGR1 gene expression in a comparable section from Arriaga et al. (2012) to help identify the vocally active part of the mouse cortex (used with permission). F: Higher magnification of ROBO1 expression in layer 5 cells of the M1+M2 LMC region. For abbreviations, see list. Scale bar = 1 mm in D (applies to A–E); 200 µm in F.

Figure 9

Summary of ROBO and SLIT gene expression in forebrain motor pathway projection neurons. Shown is a simplified diagram of the cell populations that express SLIT1, ROBO1, and ROBO2 in the songbird (left) and mouse (right) brains. The ligands and receptors are labeled along the axons that express the mRNAs. We do not yet know whether the expression of these genes is in the projection neurons of each brain region or the local cells.