Novel insights into the bovine polled phenotype and horn ontogenesis in Bovidae

Abstract

Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.

Figure 1. Frequency of the most frequent haplotypes across 17 Western European breeds for sliding windows of 79 markers at the beginning of BTA01.

Only windows located in the first three megabases of BTA01, according to the UMD3.1 bovine genome assembly, are shown. Window 509, for which the maximal frequency is found (68.48%), corresponds to the segment located between positions 1,693,164 and 2,018,403.

Figure 2. Accurate mapping of the Polled locus and identification of candidate causative mutations.

(A) Localization on BTA01 of the intervals (gray boxes) containing the Polled mutations associated with different haplotypes based on Illumina BovineSNP50 beadchip genotyping data. Upper and lower double arrows indicate the region in which the most informative recombination occurred at the left and right border of the interval, respectively. (B) Gene content of the Polled intervals. (C) Localization of the candidate mutations for the Celtic (blue) and Friesian (green) Polled alleles, and details of the coding and non-coding genes in their close vicinity. LincRNA: Long intergenic non-coding RNA. LincRNA#1 and 2 correspond, respectively, to EST sequences n° AW356369, AW357421, BF654718, BM105296, BM254775, BM254845 and BC122836, DT831326, DT837875, DY200702, DY169884, EH130782, EH138227, EV606908, EV693397 in Genbank. Positions on BTA01 are based on the UMD3.1 bovine genome assembly. Polymorphism g.1855898G>A is located within an intron of LincRNA#2. Red bars indicate the two sequence variations between the original and the duplicated 80-kb segments.

Figure 3. Details of the eyelash and eyelid phenotypes associated with polledness.

Eyes of a wild-type bull (A), three P_C/p Charolais bulls (B, D and E), and a P_F/p Holstein cow (C). Note the typical eyelid hypertrichosis and accessory rows of lashes in polled animals, with additional hair showing pronounced variability in size and orientation and darker coloration in pigmented animals (C). Arrows show additional hair growing on the inner part of the eyelid (i.e., distichiasis).

Figure 4. Histological analyses of horn buds and forehead skin from wild-type fetuses and fetuses affected by different horn-defect syndromes.

(A), (D), (G), (J) and (L) Histological sections of horn buds of a wt, two TWIST1+/−, a P_C/p and a PMS fetus, respectively. (B) and (C), (E) and (F), and (H) and (I) Magnifications (X10 and X3, respectively) of (A), (D) and (G). (K) and (M) Magnifications (X10) of (J) and (L). (N) Histological section of the forehead skin of a wt fetus. (O) Magnification (X10) of (N). Scale bars in (A), (D), (G), (J), (L) and (N) represent 1 mm, whereas scale bars in (B), (C), (E), (F), (H), (I), (K), (M) and (O) represent 100 µm.

Figure 5. RT-qPCR gene expression analyses in tissues from wild-type and P_C/p 90 dpc fetuses.

HB: horn buds+frontal bone, FS: frontal skin+frontal bone. *p<0.05, **p<0.01, ***p<0.001, and a: p = 0.052 (Fischer’s test).

Figure 6. RT-qPCR gene expression analyses in tissues from wild-type, P_C/p, TWIST1+/− and PMS 90 dpc fetuses.

HB: horn buds+frontal bone, FS: frontal skin+frontal bone. *p<0.05 and **p<0.01 (Fischer’s test). P-values were not calculated for expression levels in tissues from TWIST1+/− and PMS fetuses due to the small number of individuals investigated.