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. 2022 Dec 14:13:1057721.
doi: 10.3389/fphys.2022.1057721. eCollection 2022.

Comprehensive molecular and morphological resolution of blubber stratification in a deep-diving, fasting-adapted seal

J I Khudyakov  1 , K N Allen  2 , D E Crocker  3 , N S Trost  1 , A H Roberts  1 , L Pirard  4 , C Debier  4 , E R Piotrowski  1 , J P Vázquez-Medina  2
Affiliations

Comprehensive molecular and morphological resolution of blubber stratification in a deep-diving, fasting-adapted seal

J I Khudyakov et al. Front Physiol. .

Abstract

Blubber is a modified subcutaneous adipose tissue in marine mammals that provides energy storage, thermoregulation, hydrodynamic locomotion, and buoyancy. Blubber displays vertical stratification by lipid content, fatty acid composition, and vascularization, leading to the assumption that deeper blubber layers are metabolically active, while superficial layers are mainly structural and thermoregulatory. However, few studies have examined functional stratification of marine mammal blubber directly, especially in pinnipeds. We characterized morphological and transcriptional differences across blubber layers in the northern elephant seal, a deep-diving and fasting-adapted phocid. We collected blubber from seals early in their fasting period and divided blubber cores into three similarly sized portions. We hypothesized that the innermost blubber portion would have higher 1) heterogeneity in adipocyte size, 2) microvascular density, and 3) expression of genes associated with metabolism and hormone signaling than outer blubber. We found that adipocyte area and variance increased from outermost (skin-adjacent) to innermost (muscle-adjacent) blubber layers, suggesting that inner blubber has a higher capacity for lipid storage and turnover than outer blubber. Inner blubber had a higher proportion of CD144+ endothelial cells, suggesting higher microvascular density. In contrast, outer blubber had a higher proportion of CD4+ immune cells than inner blubber, suggesting higher capacity for response to tissue injury. Transcriptome analysis identified 61 genes that were differentially expressed between inner and outer blubber layers, many of which have not been studied previously in marine mammals. Based on known functions of these genes in other mammals, we suggest that inner blubber has potentially higher 1) adipogenic capacity, 2) cellular diversity, and 3) metabolic and neuroendocrine signaling activity, while outer blubber may have higher 1) extracellular matrix synthesis activity and 2) responsiveness to pathogens and cell stressors. We further characterized expression of nine genes of interest identified by transcriptomics and two adipokines with higher precision across blubber layers using targeted assays. Our study provides functional insights into stratification of blubber in marine mammals and a molecular key, including CD144, CD4, HMGCS2, GABRG2, HCAR2, and COL1A2, for distinguishing blubber layers for physiological and functional studies in seals.

Keywords: adipogenesis; blubber depth; histology; metabolism and endocrinology; seal (pinnipedia); transcriptome (RNA-seq).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Example of northern elephant seal blubber biopsies used for (A) transcriptome analyses and (B) targeted gene expression assays. The former was collected from juvenile elephant seals and dissected into three layers; the innermost and outermost layers were used for transcriptomics. The latter was collected from weaned elephant seal pups and dissected into five ∼1-cm layers (numbered 1–5 from outer-to innermost). Differences in tissue color are due to differences in blubber perfusion in individual animals at time of sampling.
FIGURE 2
FIGURE 2
(A) Longitudinally-sectioned elephant seal blubber sample stained with hematoxylin and eosin. Inset shows an example region of interest (ROI) within which adipocyte areas were measured. (B) Cross-sectional area of adipocytes in outer, middle, and inner blubber regions of four juvenile elephant seals. Different letters denote values that were significantly different (p < 0.05) between blubber regions.
FIGURE 3
FIGURE 3
Vascular area fractions in inner and outer blubber tissue layers. Sections of inner and outer blubber of juvenile northern elephant seals (n = 5) were stained with CD144 (vascular endothelial cadherin) antibody. Nuclei were counterstained with SYTOX Green. Vascular area fractions were compared between blubber layers using paired t-test. Scale bar is 50 μm.
FIGURE 4
FIGURE 4
Heatmap showing scaled relative expression levels (transcripts per million, or TPM) of 61 annotated genes that were differentially expressed between inner and outer layers of blubber in juvenile northern elephant seals (n = 3). Genes with higher expression in inner blubber are shown in yellow, while those with higher expression in outer blubber are shown in blue. Rows and columns were clustered by expression similarity (Euclidean distance).
FIGURE 5
FIGURE 5
Normalized expression (delta CT) of 11 genes of interest across five layers of blubber of northern elephant seal pups (n = 5). Target genes were (A) identified as upregulated in outer blubber, (B) identified as upregulated in inner blubber by transcriptomics, and included (C) two adipokine genes of interest. Different letters denote expression levels that were significantly different (p < 0.05) between layers. AGT, angiotensinogen; CD4, cluster of differentiation 4; COL1A2, collagen alpha-2(I) chain; ANGPTL4, angiopoietin-related protein 4; CES1, carboxylesterase 1; GABRG2, gamma-aminobutyric acid receptor subunit gamma-2; HCAR2, hydroxycarboxylic acid receptor 2; HMGCS2, hydroxymethylglutaryl-CoA synthase, mitochondrial; THBS1, thrombospondin-1; LEP, leptin; ADIPOQ, adiponectin.
FIGURE 6
FIGURE 6
CD4-positive cells in inner and outer blubber tissue layers. Sections of inner and outer blubber of juvenile northern elephant seals (n = 5) were stained with CD4 antibody. Nuclei were counterstained with SYTOX Green. Numbers of CD4+ cells were compared between blubber layers using paired t-test. Scale bar is 50 μm.
FIGURE 7
FIGURE 7
Summary of gene expression across blubber depth in elephant seals. Lines show mean normalized expression levels (delta CT) of genes shown in Figure 5 across five blubber layers. Genes are ordered by expression pattern across blubber depth, not absolute expression level. Genes shown in orange were most highly expressed in innermost blubber, those shown in yellow had highest expression in middle and inner blubber layers, and those shown in blue were expressed most highly in outermost blubber. Metric ruler was created using Vector Ruler Generator (MIT License).
See this image and copyright information in PMC

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