Gene expression supports a single origin of horns and antlers in hoofed mammals

Abstract

Horns, antlers, and other bony cranial appendages of even-toed hoofed mammals (ruminant artiodactyls) challenge traditional morphological homology assessments. Cranial appendages all share a permanent bone portion with family-specific integument coverings, but homology determination depends on whether the integument covering is an essential component or a secondary elaboration of each structure. To enhance morphological homology assessments, we tested whether juvenile cattle horn bud transcriptomes share homologous gene expression patterns with deer antlers relative to pig outgroup tissues, treating the integument covering as a secondary elaboration. We uncovered differentially expressed genes that support horn and antler homology, potentially distinguish them from non-cranial-appendage bone and other tissues, and highlight the importance of phylogenetic outgroups in homology assessments. Furthermore, we found differentially expressed genes that could support a shared cranial neural crest origin for horns and antlers and expression patterns that refine our understanding of the timing of horn and antler differentiation.

Fig. 1. Cranial appendage tissue composition and phylogenetic relationships of living and extinct ruminants.

Living and entirely extinct (†) ruminant families (even-toed hoofed mammals; Artiodactyla, Ruminantia), showing cranial appendage types and tissue composition. 1: Tragulidae, 2: Antilocapridae (pronghorns), 3: Climacoceratidae†, 4: Giraffidae (ossicones), 5: Palaeomerycidae†, 6: Cervidae (antlers), 7: Moschidae, 8: Bovidae (horns). Tragulidae and Moschidae lack cranial appendages. Phylogeny based on Hassanin et al..

Fig. 2. Top 50 differentially expressed genes for cattle horn buds.

Heatmaps of log fold changes for the top 50 significantly differentially expressed genes in each cattle horn bud comparison, focusing on genes expressed in a different direction or not significant in cattle skin and including the log fold changes for the genes in the deer antler and pig tissues. Heat scales in each heatmap are log fold change. a Combined cattle horn bud analysis (2- and 4-month-old cattle horn buds tested together). b Two-month-old cattle horn bud analysis. c Four-month-old cattle horn bud analysis. Figure panels display combined cattle horn buds (Comb.), 2-month-old cattle horn bud (2 mo), 4-month-old cattle horn bud (4 mo), bulk deer antler tip (Ant), bulk deer antler pedicle (Ped), isolated deer antler mineralized cartilage (MC), isolated deer antler cartilage (CA), isolated deer antler transition zone (TZ), isolated deer antler pre-cartilage (PC), isolated deer antler reserve mesenchyme (RM), and control cartilage (Cart.), skin (Skin), and bone (Bone) unrelated to cranial appendages.

Fig. 3. Homologous gene heatmap and shared proportions between cattle horn buds and deer antlers.

a Heatmap of log fold changes (log FC) for the differentially expressed genes that mapped as homologous (expressed in the same direction in at least one bovid horn bud and cervid antler tissue and expressed in the opposite direction or not significant in pig tissues; genes expressed in the same direction as within-species skin or bone controls excluded). b Proportions of homologously expressed genes shared between each cattle horn bud analysis (combined, 2-month-old, and 4-month-old) and each deer antler tissue. Figure panels display combined cattle horn buds (Comb.), 2-month-old cattle horn bud (2 mo), 4-month-old cattle horn bud (4 mo), bulk deer antler tip (Ant), bulk deer antler pedicle (Ped), isolated deer antler mineralized cartilage (MC), isolated deer antler cartilage (CA), isolated deer antler transition zone (TZ), isolated deer antler pre-cartilage (PC), isolated deer antler reserve mesenchyme (RM), and skin (Skin) and bone (Bone) unrelated to cranial appendages.

Fig. 4. Highly ranked gene set barcode plots.

Barcode plots of highly ranked gene sets with homologous expression patterns in cattle or deer tissues relative to pig tissues (either genes that tended to be expressed in a different direction or were not ranked). Each barcode plot represents the log fold changes (log FC) of genes in the set as vertical bars, regardless of significant differential expression (colored regions represent the range of significant differential expression log fold changes for each tissue). Lines above each barcode show local enrichment of these genes and their neighbors to represent the expression trends for genes in the set. a The gene set “Regulation of mesodermal cell fate specification” (GO:0042661) was significantly ranked and overexpressed in three tissues, including a cattle horn bud and deer antler tissue; this gene set was unranked in any of the pig tissues. b The Molecular Signatures Database (MSigDB) Hallmark gene set “Complement,” representing genes that are expressed as part of the complement system of innate immune response. Cattle and deer cranial appendage tissues and deer bone tended to underexpress the genes in this set, while pig cartilage and bone overexpressed them. C The MSigDB Hallmark gene set “IL6 JAK STAT3 signaling” was significantly ranked and underexpressed in all three cattle horn bud comparisons, two isolated deer antler tissues, and deer bone, but overexpressed in pig cartilage. Figure panels display combined cattle horn buds (Comb.), 2-month-old cattle horn bud (2 mo), 4-month-old cattle horn bud (4 mo), bulk deer antler pedicle (Ped), isolated deer antler transition zone (TZ), isolated deer antler pre-cartilage (PC), isolated deer antler reserve mesenchyme (RM), and control cartilage (Cart.), skin (Skin), and bone (Bone) unrelated to cranial appendages.

Fig. 5. Self-organizing map summary figures.

Self-organizing map (SOM) of log counts per million for 9433 genes across cattle, deer, and pig transcriptome analyses, showing mapping quality, distance, and clustering metrics. a heat scale is quality scores based on the average sum of squares distance of the count profile for each gene in the SOM unit to the “codebook” vector of the unit; lower scores (lighter colors) represent shorter distances between a gene’s expression profile and the codebook vector for the unit; b heat scale is average sum of squares distances of each SOM unit from its neighbors; lighter regions show units that are closer to their neighbors, and thus have genes with more similar gene expression patterns; c heat scale is the number of genes mapped to each SOM unit; this metric is expected to be similar between units across the map, and most units on our map had 20–30 genes. d The map showing the four k-medoid clusters of SOM units. Genes in clusters 1 and 3 exhibited patterns that support cranial appendage homology: Cluster 1 genes tended to have average log counts per million higher in cattle (Bovidae) and deer (Cervidae) cranial appendage tissues than in pig outgroup tissues, and genes in Cluster 3 tended to have lower average counts in cranial appendage tissues than in pig outgroup tissues. The contribution to the SOM by each tissue and taxon is in Fig. 6.

Fig. 6. Tissue contributions to the self-organizing map.

Each tissue’s contribution to the overall self-organizing map (SOM), showing the average log counts per million for genes mapping to each SOM unit. Bold lines mark the boundaries of k-medoid clusters, of which clusters 1 and 3 (see inset from Fig. 5D, where numbers correspond to cluster numbers, and Table 3) show patterns that differentiate cranial appendage (cattle horn and deer antler) tissues from outgroup pig skin, cartilage, and bone. Heat scale is log counts per million; gray units had zero counts per million for that tissue and species. Figure panels display 2-month-old cattle horn bud (2 mo), 4-month-old cattle horn bud (4 mo), bulk deer antler tip (Ant), bulk deer antler pedicle (Ped), isolated deer antler mineralized cartilage (MC), isolated deer antler cartilage (CA), isolated deer antler transition zone (TZ), isolated deer antler pre-cartilage (PC), isolated deer antler reserve mesenchyme (RM), and control cartilage (Cart), skin (Skin), and bone (Bone) unrelated to cranial appendages.

Fig. 7. Schematic representation of tissue comparisons and analyses for transcriptomic homology assessment.

Homology assessments followed three core steps: 1. Gene expression, tissue specificity, and gene set ranking determined for each test tissue. Bars above or below the line here represent hypothetical gene expression patterns (not based on actual data). 2. Results compared between cranial appendage tissues and within-species test controls (cattle skin or deer skin and bone) to identify significant genes that distinguish cranial appendages from these tissues, here highlighted in dark gray. 3. Homology evaluated by comparing distinct genes from step 2 (represented in circles) across species; genes that were similar in cattle horn and deer antler, but differed from outgroup pig skin, cartilage, and bone, were evidence of homology. A: 2-month-old cattle horn bud, B: 4-month-old cattle horn bud, C: test control tissues (cattle skin; deer skin and bone; pig skin, bone, and cartilage), D: bulk deer antler tip, E: bulk deer antler pedicle, F: isolated deer antler mineralized cartilage, G: isolated deer antler cartilage, H: isolated deer antler transition zone, I: isolated deer antler pre-cartilage, J: isolated deer antler reserve mesenchyme.