Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae

Abstract

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Keywords: Hox genes; co-option; goat and sheep genomics; regulatory mutation.

Fig. 1.

Polyceraty in sheep and goats and candidate genetic variants. (a) Polycerate Manx Loaghtan ram. (b) Wild-type and polycerate male goats from a local German population. These individuals represent the most common phenotype. Polycerate animals with asymetric horns and partial fusion of lateral horns are also regularly observed. (c) A 4-bp deletion causing polyceraty in sheep. Integrative Genome Viewer (IGV) screenshot with the localization of the variant with respect to HOXD1. Below is a graphical representation of nucleotide conservation at the exon 1-intron junction across 103 sarcopterigian and tetrapod species. (d) Plot of read coverage in a heterozygous polycerate goat animal carrying a deletion of 503-kb downstream the HOXD gene cluster on Chr2 and a duplication of 137 kb on Chr5. (e) FISH-mapping in a heterozygous polycerate goat with BAC clones corresponding to the region deleted in Chr2 (labeled in red) and to the segment of Chr5 inserted at the deletion site (labeled in green). Magnification: ×1,000. Sheep and goat icons were made by “Monkik” from www.thenounproject.com (last accessed November 4, 2020).

Fig. 2.

Regulation of Hoxd1 expression pattern in crest cell-derived head structures in mouse. (a) On top is the structure of the mouse HoxD gene cluster with arrows showing the timing and localization of gene expression along the body axis during development. The position of Hoxd1 is highlighted in red. Below is a 1-Mb view of the locus, with Hoxd1 in red as well as the relative positions of the POLYCERATE variants in sheep (black arrowhead) and goat (black line). Below are depicted the various murine alleles, with the lacZ insertion in Hoxd1 (blue arrowhead), the two BAC clones (thick blue lines) and the engineered deletion (black line). (b) Heads of E12.5–E13.5 mouse fetuses after X-gal staining. The dashed circle highlights the absence of Hoxd1 expression in the crown (corresponding to the localization of hornbuds in Bovidae), whereas the surrounding dermal cells are positive. The conservation of Hoxd1 expression in the back of the neck (black arrows) contrasts with the presence/absence of expression in the facial muscle precursors (white arrows) and in the eyelids (arrowhead). The comparison between the four strains indicate that Hoxd1 expression in all these cranial derivatives is controlled by regulatory elements located in a region orthologous to the proximal portion of the segment deleted in polycerate goats. Sheep, goat and mouse icons were made by “Monkik” from www.thenounproject.com (last accessed November 4, 2020).

Fig. 3.

RT–qPCR gene expression analyses in sheep and goat fetuses. (a and b) Schemes of the tissues sampled at stage 70 dpc in goat (a) and 76 dpc in sheep (b) in four control (+/+) and four heterozygous (+/−) polycerate fetuses within each species. bs: skin from the back of the head; hb: skin from the hornbud; h1: skin from the lower horn bud; and h2: skin from the upper horn bud in polycerate specimens; fs: frontal skin; el: eyelids. RT–qPCR gene expression analyses in these tissues are shown below (means and standard errors of the means from triplicate experiments). Gene expression was normalized using GAPDH, H2AFZ, and HPRT1 as reference genes. *P < 0.05, **P < 0.01 (Welch two-sample t-test with the alternative hypothesis that the means are not equal). For the sake of clarity, the symbols # and @ were also used to show significant differences (P < 0.05) between distant bars. (c) Schematic representation of the ovine HOXD1 gene and corresponding wild-type and putative mutant proteins. The localizations of the amplicons studied in (b) are indicated with double arrows. HD: Homeodomain.

Fig. 4.

Results of 3D geometric morphometric analyses of 61 ovine and 19 caprine skulls. (a) Distribution of the specimens along the first two axes of the PCA. The proportion of variance explained by the main principal components is indicated on each axis. Green dots: polycerate sheep with a distance between lateral horns (dlh) larger than the distance between upper horns (duh); light blue: polycerate sheep with a dlh≤duh; blue: polycerate sheep with at least two lateral horns partially fused at their basis; purple: wild-type sheep; black: polycerate goats; and red: wild-type goats. Representative specimens illustrate each cluster and symbols are used to indicate their respective locations in the PCA analysis (see supplementary fig. 10, Supplementary Material online, for intraspecies analyses and further information). (b) Number of heterozygous (+/−) and homozygous (−/−) polycerate rams among groups of live animals with different dlhl (dlh on the left side) and duh relative sizes (see supplementary table 12, Supplementary Material online, for further information); ***P value = 3.5 × 10⁻⁷ (Fisher’s exact test). (c) Theoretical shapes associated with the maximum (upper three) and minimum values (lower three) of PC1 axis for a sheep skull. Red dots correspond to anatomical landmarks, whereas the other dots correspond to sliding semilandmarks; light blue and purple dots highlight the sites of division of lateral horns. (d) Shape differences for the sliding semilandmarks located at the basis of the left horn. Light blue and purple dots correspond to the maximum and minimum values of PC1 axis, respectively. Dashed squares indicate the estimated position of dissected tissues in figure 3 (bs: skin from the back of the head; fs: frontal skin) in which HOXD1 expression was observed in fetuses.