{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/137561"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/137561","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Cellular and Molecular Mechanisms Governing Intramuscular Fat Development and Growth in Cattle","abstract":"Intramuscular fat (IMF), the adipose tissue deposited between skeletal muscle fibers, plays a critical role in enhancing beef flavor, juiciness, and tenderness. Compared to subcutaneous fat (SF), IMF exhibits delayed development and slower growth. What causes IMF to develop later and grow more slowly than SF in cattle remains incompletely understood. In this dissertation project, I investigated the differences between IMF and SF in morphology, fatty acid uptake, transcriptome, adipogenesis, composition and developmental stage of adipose progenitor cells, and origin of preadipocytes. In addition, I tested the hypothesis that the delayed development and slower growth of IMF is in part due to inhibition by signaling proteins secreted by the surrounding muscle tissue. I conducted four studies. In the first study, I compared the histology, ability to take up long-chain fatty acids, and gene expression profiles of IMF and SF from six adult cattle as well as the adipogenic potential of their stromal vascular fractions (SVFs). Histological analysis revealed that IMF adipocytes were significantly smaller than SF adipocytes. Functional assays showed that IMF had reduced capacities for incorporating long-chain fatty acids into triglycerides. RNA-seq analysis identified over 1,000 differentially expressed genes between IMF and SF. Several genes upregulated in IMF compared to SF, such as FOXO6, SLC27A1, HDAC9, and PIK3C2A, were associated with negative regulation of adipogenesis, while several genes downregulated in IMF, such as FABP4, AGPAT2, and PLIN2, were key promoters of adipocyte differentiation. Functional enrichment analysis revealed that genes downregulated in IMF were enriched in the PPAR signaling pathway and adipogenesis-related processes, whereas genes regulated in IMF were enriched in Wnt signaling. Adipogenic differentiation assays confirmed that the IMF-derived SVF cells exhibited lower adipogenic potential than the SF-derived SVF cells. Together, the findings from this study suggest that the delayed and slower deposition of IMF is associated with reduced fatty acid binding capacity, reduced triglyceride synthesis, and decreased adipogenesis. In the second study, I investigated whether differences in adipogenic progenitor cells (APCs) contribute to the developmental and growth differences between IMF and SF. Single-cell RNA sequencing (scRNA-seq) was performed on SVF cells from IMF and SF of adult Angus crossbred steers and mononuclear cell fractions (MCFs) from skeletal muscle of newborn Angus crossbred bull calves, with each tissue type collected from two animals. A total of 14,802 cells from six animals were sequenced. Clustering analysis identified ten major cell types, including APCs, muscle satellite cells (MuSCs), myoblasts, smooth muscle cells, and several immune populations. APCs were significantly more abundant in the SF- than in the IMF-derived SVF. Notably, APCs were not detectable in the MCFs from neonatal calves. Subclustering of APCs revealed six transcriptionally distinct subpopulations (C0–C5). Among these, subclusters C3 and C5 were absent in the IMF-derived SVF, and C1 was markedly less abundant in the IMF-derived SVF compared to the SF-derived SVF. Gene set variation analysis (GSVA) and pseudotime trajectory analysis showed that C1 and C3 represented more differentiated APCs, characterized by elevated expression of adipogenesis-related genes such as PPARG, ADAM12, and PPARGC1A. Together, the findings from the second study reveal two key differences in APCs between IMF and SF: (1) IMF contains fewer APCs than SF and (2) IMF-derived APCs are less differentiated, providing a plausible explanation for the delayed development and slower growth observed in IMF. The absence of APCs in the muscle of newborn calves suggests that intramuscular fat development occurs postnatally, challenging the widely held belief that it begins before birth. In the third study, I compared the transcriptome and differentiation potential of preadipocytes between adult steer IMF and SF. Through single-cell cloning followed by adipogenic differentiation assay, I generated dozens of single-preadipocyte clones from IMF- or SF-derived SVF that exhibited adipogenic potential, which was evidenced by lipid accumulation and increased expression of adipocyte markers (PPARG, CEBPA, FABP4, ADIPOQ) in cells differentiated from these clones. Four single-cell clones derived from IMF preadipocytes and four from SF preadipocytes, each isolated from a single steer, were selected for further characterization. Transcriptomic profiling revealed over 2,000 differentially expressed genes between four IMF- and four SF-derived preadipocyte clones. Both IMF- and SF-derived preadipocyte clones expressed classical preadipocyte markers, such as PDGFRA, DLK1, PPARG, CD34, and ZNF423, validating their preadipocyte identity. Notably, IMF-derived clones maintained high expression of muscle-specific genes (e.g., MYOG, CKM, MYH2, MYH3), even after adipogenic differentiation. Functional enrichment analysis showed that genes upregulated in IMF-derived preadipocyte clones were involved in PI3K-Akt, MAPK, and calcium signaling, as well as muscle development and neuromuscular junction pathways. Differentiation assays confirmed that IMF-derived SVF cells lacked myogenic potential, while bovine skeletal muscle-derived satellite cells (bSCs) exhibited both myogenic and adipogenic potential. Together, these results suggest that preadipocytes in bovine IMF may originate from bSCs or share a common progenitor with them, whereas preadipocytes in bovine SF likely arise from distinct progenitor cells. In the fourth study, I investigated whether skeletal muscle regulates IMF development and growth via secreted factors. I found that both extracellular fluid from bovine skeletal muscle and protein extracts from bovine skeletal muscle promoted lipolysis in IMF explants and viability of IMF-derived SVF cells while suppressing their differentiation into adipocytes. To further examine whether these effects were mediated by muscle-secreted factors, I assessed the effects of medium conditioned with myotubes formed from bSCs on lipolysis in IMF explants and adipogenic differentiation of IMF-derived SVF cells. Myotube-conditioned medium promoted lipolysis in IMF explants, enhanced cell proliferation or survival, but inhibited adipogenic differentiation in IMF-derived SVF cells. Together, the results from this fourth study suggest that muscle-derived protein factors, likely myokines, promote lipolysis and inhibit adipogenic differentiation, thereby inhibiting intramuscular fat growth in cattle.","abstract_html":"Intramuscular fat (IMF), the adipose tissue deposited between skeletal muscle fibers, plays a critical role in enhancing beef flavor, juiciness, and tenderness. Compared to subcutaneous fat (SF), IMF exhibits delayed development and slower growth. What causes IMF to develop later and grow more slowly than SF in cattle remains incompletely understood. In this dissertation project, I investigated the differences between IMF and SF in morphology, fatty acid uptake, transcriptome, adipogenesis, composition and developmental stage of adipose progenitor cells, and origin of preadipocytes. In addition, I tested the hypothesis that the delayed development and slower growth of IMF is in part due to inhibition by signaling proteins secreted by the surrounding muscle tissue. I conducted four studies. In the first study, I compared the histology, ability to take up long-chain fatty acids, and gene expression profiles of IMF and SF from six adult cattle as well as the adipogenic potential of their stromal vascular fractions (SVFs). Histological analysis revealed that IMF adipocytes were significantly smaller than SF adipocytes. Functional assays showed that IMF had reduced capacities for incorporating long-chain fatty acids into triglycerides. RNA-seq analysis identified over 1,000 differentially expressed genes between IMF and SF. Several genes upregulated in IMF compared to SF, such as FOXO6, SLC27A1, HDAC9, and PIK3C2A, were associated with negative regulation of adipogenesis, while several genes downregulated in IMF, such as FABP4, AGPAT2, and PLIN2, were key promoters of adipocyte differentiation. Functional enrichment analysis revealed that genes downregulated in IMF were enriched in the PPAR signaling pathway and adipogenesis-related processes, whereas genes regulated in IMF were enriched in Wnt signaling. Adipogenic differentiation assays confirmed that the IMF-derived SVF cells exhibited lower adipogenic potential than the SF-derived SVF cells. Together, the findings from this study suggest that the delayed and slower deposition of IMF is associated with reduced fatty acid binding capacity, reduced triglyceride synthesis, and decreased adipogenesis. In the second study, I investigated whether differences in adipogenic progenitor cells (APCs) contribute to the developmental and growth differences between IMF and SF. Single-cell RNA sequencing (scRNA-seq) was performed on SVF cells from IMF and SF of adult Angus crossbred steers and mononuclear cell fractions (MCFs) from skeletal muscle of newborn Angus crossbred bull calves, with each tissue type collected from two animals. A total of 14,802 cells from six animals were sequenced. Clustering analysis identified ten major cell types, including APCs, muscle satellite cells (MuSCs), myoblasts, smooth muscle cells, and several immune populations. APCs were significantly more abundant in the SF- than in the IMF-derived SVF. Notably, APCs were not detectable in the MCFs from neonatal calves. Subclustering of APCs revealed six transcriptionally distinct subpopulations (C0–C5). Among these, subclusters C3 and C5 were absent in the IMF-derived SVF, and C1 was markedly less abundant in the IMF-derived SVF compared to the SF-derived SVF. Gene set variation analysis (GSVA) and pseudotime trajectory analysis showed that C1 and C3 represented more differentiated APCs, characterized by elevated expression of adipogenesis-related genes such as PPARG, ADAM12, and PPARGC1A. Together, the findings from the second study reveal two key differences in APCs between IMF and SF: (1) IMF contains fewer APCs than SF and (2) IMF-derived APCs are less differentiated, providing a plausible explanation for the delayed development and slower growth observed in IMF. The absence of APCs in the muscle of newborn calves suggests that intramuscular fat development occurs postnatally, challenging the widely held belief that it begins before birth. In the third study, I compared the transcriptome and differentiation potential of preadipocytes between adult steer IMF and SF. Through single-cell cloning followed by adipogenic differentiation assay, I generated dozens of single-preadipocyte clones from IMF- or SF-derived SVF that exhibited adipogenic potential, which was evidenced by lipid accumulation and increased expression of adipocyte markers (PPARG, CEBPA, FABP4, ADIPOQ) in cells differentiated from these clones. Four single-cell clones derived from IMF preadipocytes and four from SF preadipocytes, each isolated from a single steer, were selected for further characterization. Transcriptomic profiling revealed over 2,000 differentially expressed genes between four IMF- and four SF-derived preadipocyte clones. Both IMF- and SF-derived preadipocyte clones expressed classical preadipocyte markers, such as PDGFRA, DLK1, PPARG, CD34, and ZNF423, validating their preadipocyte identity. Notably, IMF-derived clones maintained high expression of muscle-specific genes (e.g., MYOG, CKM, MYH2, MYH3), even after adipogenic differentiation. Functional enrichment analysis showed that genes upregulated in IMF-derived preadipocyte clones were involved in PI3K-Akt, MAPK, and calcium signaling, as well as muscle development and neuromuscular junction pathways. Differentiation assays confirmed that IMF-derived SVF cells lacked myogenic potential, while bovine skeletal muscle-derived satellite cells (bSCs) exhibited both myogenic and adipogenic potential. Together, these results suggest that preadipocytes in bovine IMF may originate from bSCs or share a common progenitor with them, whereas preadipocytes in bovine SF likely arise from distinct progenitor cells. In the fourth study, I investigated whether skeletal muscle regulates IMF development and growth via secreted factors. I found that both extracellular fluid from bovine skeletal muscle and protein extracts from bovine skeletal muscle promoted lipolysis in IMF explants and viability of IMF-derived SVF cells while suppressing their differentiation into adipocytes. To further examine whether these effects were mediated by muscle-secreted factors, I assessed the effects of medium conditioned with myotubes formed from bSCs on lipolysis in IMF explants and adipogenic differentiation of IMF-derived SVF cells. Myotube-conditioned medium promoted lipolysis in IMF explants, enhanced cell proliferation or survival, but inhibited adipogenic differentiation in IMF-derived SVF cells. Together, the results from this fourth study suggest that muscle-derived protein factors, likely myokines, promote lipolysis and inhibit adipogenic differentiation, thereby inhibiting intramuscular fat growth in cattle.","abstract_has_math":false,"creators":["Tan, Zhendong"],"institution":"Virginia Tech","degree_name":"Doctor of Philosophy","degree_level":"doctoral","degree_discipline":"Animal and Poultry Sciences","degree_department":"Animal and Poultry Sciences","school":null,"contributors":[],"advisors":[],"committee_chairs":["Jiang, Honglin"],"committee_members":["Corl, Benjamin A.","Liu, Dongmin","Greiner, Scott P."],"year":2025,"date_issued":"2025-08-22","date_published":"2025-08-22","updated_at":"2026-07-22T22:20:37Z","subjects":["Intramuscular fat","Adipogenesis","Cattle"],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44593"],"render_values":[{"text":"vt_gsexam:44593","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/10919/137561","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Jiang, Honglin"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Corl, Benjamin A.","Liu, Dongmin","Greiner, Scott P."]},{"key":"dc:contributor.department","label":"Department","values":["Animal and Poultry Sciences"]},{"key":"dc:creator","label":"Author","values":["Tan, Zhendong"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-08-23T08:01:02Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-08-23T08:01:02Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-08-22"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Animal and Poultry Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Intramuscular fat","Adipogenesis","Cattle"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44593"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10919/137561"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Intramuscular fat (IMF), the adipose tissue deposited between skeletal muscle fibers, plays a critical role in enhancing beef flavor, juiciness, and tenderness. Compared to subcutaneous fat (SF), IMF exhibits delayed development and slower growth. What causes IMF to develop later and grow more slowly than SF in cattle remains incompletely understood. In this dissertation project, I investigated the differences between IMF and SF in morphology, fatty acid uptake, transcriptome, adipogenesis, composition and developmental stage of adipose progenitor cells, and origin of preadipocytes. In addition, I tested the hypothesis that the delayed development and slower growth of IMF is in part due to inhibition by signaling proteins secreted by the surrounding muscle tissue. I conducted four studies. In the first study, I compared the histology, ability to take up long-chain fatty acids, and gene expression profiles of IMF and SF from six adult cattle as well as the adipogenic potential of their stromal vascular fractions (SVFs). Histological analysis revealed that IMF adipocytes were significantly smaller than SF adipocytes. Functional assays showed that IMF had reduced capacities for incorporating long-chain fatty acids into triglycerides. RNA-seq analysis identified over 1,000 differentially expressed genes between IMF and SF. Several genes upregulated in IMF compared to SF, such as FOXO6, SLC27A1, HDAC9, and PIK3C2A, were associated with negative regulation of adipogenesis, while several genes downregulated in IMF, such as FABP4, AGPAT2, and PLIN2, were key promoters of adipocyte differentiation. Functional enrichment analysis revealed that genes downregulated in IMF were enriched in the PPAR signaling pathway and adipogenesis-related processes, whereas genes regulated in IMF were enriched in Wnt signaling. Adipogenic differentiation assays confirmed that the IMF-derived SVF cells exhibited lower adipogenic potential than the SF-derived SVF cells. Together, the findings from this study suggest that the delayed and slower deposition of IMF is associated with reduced fatty acid binding capacity, reduced triglyceride synthesis, and decreased adipogenesis. In the second study, I investigated whether differences in adipogenic progenitor cells (APCs) contribute to the developmental and growth differences between IMF and SF. Single-cell RNA sequencing (scRNA-seq) was performed on SVF cells from IMF and SF of adult Angus crossbred steers and mononuclear cell fractions (MCFs) from skeletal muscle of newborn Angus crossbred bull calves, with each tissue type collected from two animals. A total of 14,802 cells from six animals were sequenced. Clustering analysis identified ten major cell types, including APCs, muscle satellite cells (MuSCs), myoblasts, smooth muscle cells, and several immune populations. APCs were significantly more abundant in the SF- than in the IMF-derived SVF. Notably, APCs were not detectable in the MCFs from neonatal calves. Subclustering of APCs revealed six transcriptionally distinct subpopulations (C0–C5). Among these, subclusters C3 and C5 were absent in the IMF-derived SVF, and C1 was markedly less abundant in the IMF-derived SVF compared to the SF-derived SVF. Gene set variation analysis (GSVA) and pseudotime trajectory analysis showed that C1 and C3 represented more differentiated APCs, characterized by elevated expression of adipogenesis-related genes such as PPARG, ADAM12, and PPARGC1A. Together, the findings from the second study reveal two key differences in APCs between IMF and SF: (1) IMF contains fewer APCs than SF and (2) IMF-derived APCs are less differentiated, providing a plausible explanation for the delayed development and slower growth observed in IMF. The absence of APCs in the muscle of newborn calves suggests that intramuscular fat development occurs postnatally, challenging the widely held belief that it begins before birth. In the third study, I compared the transcriptome and differentiation potential of preadipocytes between adult steer IMF and SF. Through single-cell cloning followed by adipogenic differentiation assay, I generated dozens of single-preadipocyte clones from IMF- or SF-derived SVF that exhibited adipogenic potential, which was evidenced by lipid accumulation and increased expression of adipocyte markers (PPARG, CEBPA, FABP4, ADIPOQ) in cells differentiated from these clones. Four single-cell clones derived from IMF preadipocytes and four from SF preadipocytes, each isolated from a single steer, were selected for further characterization. Transcriptomic profiling revealed over 2,000 differentially expressed genes between four IMF- and four SF-derived preadipocyte clones. Both IMF- and SF-derived preadipocyte clones expressed classical preadipocyte markers, such as PDGFRA, DLK1, PPARG, CD34, and ZNF423, validating their preadipocyte identity. Notably, IMF-derived clones maintained high expression of muscle-specific genes (e.g., MYOG, CKM, MYH2, MYH3), even after adipogenic differentiation. Functional enrichment analysis showed that genes upregulated in IMF-derived preadipocyte clones were involved in PI3K-Akt, MAPK, and calcium signaling, as well as muscle development and neuromuscular junction pathways. Differentiation assays confirmed that IMF-derived SVF cells lacked myogenic potential, while bovine skeletal muscle-derived satellite cells (bSCs) exhibited both myogenic and adipogenic potential. Together, these results suggest that preadipocytes in bovine IMF may originate from bSCs or share a common progenitor with them, whereas preadipocytes in bovine SF likely arise from distinct progenitor cells. In the fourth study, I investigated whether skeletal muscle regulates IMF development and growth via secreted factors. I found that both extracellular fluid from bovine skeletal muscle and protein extracts from bovine skeletal muscle promoted lipolysis in IMF explants and viability of IMF-derived SVF cells while suppressing their differentiation into adipocytes. To further examine whether these effects were mediated by muscle-secreted factors, I assessed the effects of medium conditioned with myotubes formed from bSCs on lipolysis in IMF explants and adipogenic differentiation of IMF-derived SVF cells. Myotube-conditioned medium promoted lipolysis in IMF explants, enhanced cell proliferation or survival, but inhibited adipogenic differentiation in IMF-derived SVF cells. Together, the results from this fourth study suggest that muscle-derived protein factors, likely myokines, promote lipolysis and inhibit adipogenic differentiation, thereby inhibiting intramuscular fat growth in cattle."]},{"key":"dc:description.abstractgeneral","label":"General Abstract","values":["Intramuscular fat (IMF), also known as marbling, is the adipose tissue deposited between muscle fibers and plays a key role in beef quality and value. Compared to subcutaneous fat (SF), which is the adipose tissue located under the skin, IMF develops later and grows more slowly. The reasons for these differences are not fully understood, and a better understanding of these differences may help develop new strategies to control IMF deposition in cattle and improve beef quality. First, I compared the adipocyte size, function, and gene expression profile of IMF and SF from mature steers. IMF had smaller adipocytes and lower capacity to synthesize triglycerides, and showed weaker ability to form new adipocytes compared to SF. IMF had lower expression of genes promoting fatty acid binding and adipocyte formation and higher expression of genes inhibiting these processes compared to SF. Next, I compared the gene expression profiles of individual cells in IMF and SF from mature steers as well as muscle from newborn calves using a new technology called single-cell RNA sequencing. This study revealed that adipose progenitors were more abundant and developmentally more advanced in SF than in IMF, and that they were undetectable in newborn calf muscle. I also compared the gene expression profiles of preadipocytes, the immediate precursor cells for adipocytes, in bovine IMF and SF using single-cell cloning followed by RNA sequencing. This analysis indicated that compared to SF-derived single-preadipocyte clones, IMF-derived single-preadipocyte clones expressed many muscle-related genes in addition to adipose tissue-related genes. This finding suggests that unlike SF preadipocytes, IMF preadipocytes may arise from muscle stem cells or a closely related origin. Finally, I tested the possibility that skeletal muscle-secreted factors inhibit IMF development and growth. This test revealed that protein factors released by muscle cells suppressed adipocyte formation and promoted fat breakdown in IMF tissue. Overall, these studies suggest that the delayed development and slower growth of IMF compared to SF in cattle may be mainly attributed to a lower abundance and a reduced differentiation potential of adipose progenitor cells in the former, possibly influenced by factors secreted by the surrounding muscle tissue."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["ETD"]},{"key":"dc:title","label":"Title","values":["Cellular and Molecular Mechanisms Governing Intramuscular Fat Development and Growth in Cattle"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Jiang, Honglin"],"dc:contributor.committeemember":["Corl, Benjamin A.","Liu, Dongmin","Greiner, Scott P."],"dc:contributor.department":["Animal and Poultry Sciences"],"dc:creator":["Tan, Zhendong"],"dc:date.accessioned":["2025-08-23T08:01:02Z"],"dc:date.available":["2025-08-23T08:01:02Z"],"dc:date.issued":["2025-08-22"],"dc:description.abstract":["Intramuscular fat (IMF), the adipose tissue deposited between skeletal muscle fibers, plays a critical role in enhancing beef flavor, juiciness, and tenderness. Compared to subcutaneous fat (SF), IMF exhibits delayed development and slower growth. What causes IMF to develop later and grow more slowly than SF in cattle remains incompletely understood. In this dissertation project, I investigated the differences between IMF and SF in morphology, fatty acid uptake, transcriptome, adipogenesis, composition and developmental stage of adipose progenitor cells, and origin of preadipocytes. In addition, I tested the hypothesis that the delayed development and slower growth of IMF is in part due to inhibition by signaling proteins secreted by the surrounding muscle tissue. I conducted four studies. In the first study, I compared the histology, ability to take up long-chain fatty acids, and gene expression profiles of IMF and SF from six adult cattle as well as the adipogenic potential of their stromal vascular fractions (SVFs). Histological analysis revealed that IMF adipocytes were significantly smaller than SF adipocytes. Functional assays showed that IMF had reduced capacities for incorporating long-chain fatty acids into triglycerides. RNA-seq analysis identified over 1,000 differentially expressed genes between IMF and SF. Several genes upregulated in IMF compared to SF, such as FOXO6, SLC27A1, HDAC9, and PIK3C2A, were associated with negative regulation of adipogenesis, while several genes downregulated in IMF, such as FABP4, AGPAT2, and PLIN2, were key promoters of adipocyte differentiation. Functional enrichment analysis revealed that genes downregulated in IMF were enriched in the PPAR signaling pathway and adipogenesis-related processes, whereas genes regulated in IMF were enriched in Wnt signaling. Adipogenic differentiation assays confirmed that the IMF-derived SVF cells exhibited lower adipogenic potential than the SF-derived SVF cells. Together, the findings from this study suggest that the delayed and slower deposition of IMF is associated with reduced fatty acid binding capacity, reduced triglyceride synthesis, and decreased adipogenesis. In the second study, I investigated whether differences in adipogenic progenitor cells (APCs) contribute to the developmental and growth differences between IMF and SF. Single-cell RNA sequencing (scRNA-seq) was performed on SVF cells from IMF and SF of adult Angus crossbred steers and mononuclear cell fractions (MCFs) from skeletal muscle of newborn Angus crossbred bull calves, with each tissue type collected from two animals. A total of 14,802 cells from six animals were sequenced. Clustering analysis identified ten major cell types, including APCs, muscle satellite cells (MuSCs), myoblasts, smooth muscle cells, and several immune populations. APCs were significantly more abundant in the SF- than in the IMF-derived SVF. Notably, APCs were not detectable in the MCFs from neonatal calves. Subclustering of APCs revealed six transcriptionally distinct subpopulations (C0–C5). Among these, subclusters C3 and C5 were absent in the IMF-derived SVF, and C1 was markedly less abundant in the IMF-derived SVF compared to the SF-derived SVF. Gene set variation analysis (GSVA) and pseudotime trajectory analysis showed that C1 and C3 represented more differentiated APCs, characterized by elevated expression of adipogenesis-related genes such as PPARG, ADAM12, and PPARGC1A. Together, the findings from the second study reveal two key differences in APCs between IMF and SF: (1) IMF contains fewer APCs than SF and (2) IMF-derived APCs are less differentiated, providing a plausible explanation for the delayed development and slower growth observed in IMF. The absence of APCs in the muscle of newborn calves suggests that intramuscular fat development occurs postnatally, challenging the widely held belief that it begins before birth. In the third study, I compared the transcriptome and differentiation potential of preadipocytes between adult steer IMF and SF. Through single-cell cloning followed by adipogenic differentiation assay, I generated dozens of single-preadipocyte clones from IMF- or SF-derived SVF that exhibited adipogenic potential, which was evidenced by lipid accumulation and increased expression of adipocyte markers (PPARG, CEBPA, FABP4, ADIPOQ) in cells differentiated from these clones. Four single-cell clones derived from IMF preadipocytes and four from SF preadipocytes, each isolated from a single steer, were selected for further characterization. Transcriptomic profiling revealed over 2,000 differentially expressed genes between four IMF- and four SF-derived preadipocyte clones. Both IMF- and SF-derived preadipocyte clones expressed classical preadipocyte markers, such as PDGFRA, DLK1, PPARG, CD34, and ZNF423, validating their preadipocyte identity. Notably, IMF-derived clones maintained high expression of muscle-specific genes (e.g., MYOG, CKM, MYH2, MYH3), even after adipogenic differentiation. Functional enrichment analysis showed that genes upregulated in IMF-derived preadipocyte clones were involved in PI3K-Akt, MAPK, and calcium signaling, as well as muscle development and neuromuscular junction pathways. Differentiation assays confirmed that IMF-derived SVF cells lacked myogenic potential, while bovine skeletal muscle-derived satellite cells (bSCs) exhibited both myogenic and adipogenic potential. Together, these results suggest that preadipocytes in bovine IMF may originate from bSCs or share a common progenitor with them, whereas preadipocytes in bovine SF likely arise from distinct progenitor cells. In the fourth study, I investigated whether skeletal muscle regulates IMF development and growth via secreted factors. I found that both extracellular fluid from bovine skeletal muscle and protein extracts from bovine skeletal muscle promoted lipolysis in IMF explants and viability of IMF-derived SVF cells while suppressing their differentiation into adipocytes. To further examine whether these effects were mediated by muscle-secreted factors, I assessed the effects of medium conditioned with myotubes formed from bSCs on lipolysis in IMF explants and adipogenic differentiation of IMF-derived SVF cells. Myotube-conditioned medium promoted lipolysis in IMF explants, enhanced cell proliferation or survival, but inhibited adipogenic differentiation in IMF-derived SVF cells. Together, the results from this fourth study suggest that muscle-derived protein factors, likely myokines, promote lipolysis and inhibit adipogenic differentiation, thereby inhibiting intramuscular fat growth in cattle."],"dc:description.abstractgeneral":["Intramuscular fat (IMF), also known as marbling, is the adipose tissue deposited between muscle fibers and plays a key role in beef quality and value. Compared to subcutaneous fat (SF), which is the adipose tissue located under the skin, IMF develops later and grows more slowly. The reasons for these differences are not fully understood, and a better understanding of these differences may help develop new strategies to control IMF deposition in cattle and improve beef quality. First, I compared the adipocyte size, function, and gene expression profile of IMF and SF from mature steers. IMF had smaller adipocytes and lower capacity to synthesize triglycerides, and showed weaker ability to form new adipocytes compared to SF. IMF had lower expression of genes promoting fatty acid binding and adipocyte formation and higher expression of genes inhibiting these processes compared to SF. Next, I compared the gene expression profiles of individual cells in IMF and SF from mature steers as well as muscle from newborn calves using a new technology called single-cell RNA sequencing. This study revealed that adipose progenitors were more abundant and developmentally more advanced in SF than in IMF, and that they were undetectable in newborn calf muscle. I also compared the gene expression profiles of preadipocytes, the immediate precursor cells for adipocytes, in bovine IMF and SF using single-cell cloning followed by RNA sequencing. This analysis indicated that compared to SF-derived single-preadipocyte clones, IMF-derived single-preadipocyte clones expressed many muscle-related genes in addition to adipose tissue-related genes. This finding suggests that unlike SF preadipocytes, IMF preadipocytes may arise from muscle stem cells or a closely related origin. Finally, I tested the possibility that skeletal muscle-secreted factors inhibit IMF development and growth. This test revealed that protein factors released by muscle cells suppressed adipocyte formation and promoted fat breakdown in IMF tissue. Overall, these studies suggest that the delayed development and slower growth of IMF compared to SF in cattle may be mainly attributed to a lower abundance and a reduced differentiation potential of adipose progenitor cells in the former, possibly influenced by factors secreted by the surrounding muscle tissue."],"dc:description.degree":["Doctor of Philosophy"],"dc:format.medium":["ETD"],"dc:identifier.other":["vt_gsexam:44593"],"dc:identifier.uri":["https://hdl.handle.net/10919/137561"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Intramuscular fat","Adipogenesis","Cattle"],"dc:title":["Cellular and Molecular Mechanisms Governing Intramuscular Fat Development and Growth in Cattle"],"dc:type":["Dissertation"],"thesis:degree_discipline":["Animal and Poultry Sciences"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:20:37Z"}