POSITIVE REGULATION OF MUSCLE GROWTH BY AN ALTERNATIVE SPLICING ISOFORM OF AVIAN MYOSTATIN GENE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1012138
• Grant No.: 2017-67015-26540
• Cumulative Award Amt.: $484,000.00
• Proposal No.: 2016-08413
• Project Start Date: May 1, 2017
• Project End Date: Apr 30, 2022
• Grant Year: 2017
• Program Code: [A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Animal Sciences

Non Technical Summary
A greater understanding of mechanisms regulating muscle growth and development is essential to improve meat production in domestic animals. Because myostatin (MSTN) has been known to inhibit muscle growth, approaches to inactivate myostatin could increase muscle mass. Recently, our laboratory identified a short isoform of avian MSTN that could bind to and inhibit the anti-myogenic function of a full-length MSTN in muscle cell culture. However, mechanisms by which short MSTN regulates muscle growth and development need to be confirmed in vivo. We hypothesize that the short isoform of MSTN is important to negatively regulate MSTN processing, thereby preventing myostatin-mediated inhibition of myogenesis in avians. The mechanism of action of short MSTN is preventing cleavage of full-length MSTN, so as to release lessor amounts of mature MSTN (the active anti-myogenic form). We will evaluate the effects of short MSTN overproduction in muscle of transgenic quail on muscle accretion and underlying differentiation and proliferation of myogenic cells; evaluate the effect of over-production of short MSTN on cleavage of full-length MSTN and generation of mature anti-myogenic MSTN; and determine the amounts of the short MSTN in different poultry breeds selected for smaller or greater body weights. Outcomes from the proposed studies will validate, and further establish, the mechanism of the short MSTN in muscle growth and development. This knowledge will reveal if the abundance of short MSTN provide a marker of superior lines of poultry for enhanced muscle growth. Long term, the short MSTN may serve as a potential target for pharmaceutical application to improve muscle growth.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3210	1020	25%
302	3220	1020	25%
303	3840	1040	25%
303	3840	1050	25%

Knowledge Area
303 - Genetic Improvement of Animals; 302 - Nutrient Utilization in Animals;

Subject Of Investigation
3210 - Egg-type chicken, live animal; 3220 - Meat-type chicken, live animal; 3840 - Laboratory animals;

Field Of Science
1040 - Molecular biology; 1020 - Physiology; 1050 - Developmental biology;

Keywords

alternative splicing

avian transgenesis

muscle growth

myostatin

poultry

proliferation

quail

Goals / Objectives
The major goal of the project is to understand the role of myostatin-B (MSTN-B) in muscle growth and development of the poultry species and to develop strategies using MSTN-B protein to enhance muscle growth.Objectives1) Evaluate the effect of overexpression of the MSTN-B gene in the muscle of transgenic quail on muscle accretion, and differentiation and proliferation of myogenic progenitor cells.2) Evaluate the effect of overexpression of the MSTN-B gene on processing of pro-MSTN to generate mature anti-myogenic MSTN.3) Determine to what extent broilers and layers, and the lines of quail selected for smaller or greater body weights, have temporal changes in total amounts of MSTN and/or ratios of MSTN-B/MSTN-A in the muscle.

Project Methods
Generation of transgenic quail expressing MSTN-B from by injecting lentiviral vector containing muscle-specific promoter and quail MSTN-B cDNA intothe stage X blastodermal quail embryosValidation of transgene expression by Western blot analysisMeasure weights of whole body, tissues and organsWeights of the breast and leg muscle will be measured to see the effect of transgene on muscle accretionThe pectoralis major muscle and gastrocnemius muscle will be fixed in 10% formalin for analysis of histomorphometric parameters including fiber sizes and numbers and for immunohistological analysis of myogenic markers. In addition, total amounts of DNA in a whole breast muscle will be measured. These methods will determine to what extent the overexpression of MSTN-B increases total fiber numbers and diameter of muscle fibers, indicating muscle hyperplasia and hypertrophy, respectively.To investigate the effect of MSTN-B overexpression on cell proliferation, tissue sections will be immunostained for proliferating cell nuclear antigen (PCNA). The data from PCNA staining will be correlated with the rate of increasing total amounts of DNA in whole breast muscle at different ages.To test the hypothesis that overexpression of MSTN-B in the skeletal muscle of transgenic quail increases muscle mass by preventing processing of pro-MSTN from releasing mature MSTN, we will compare the ratios of pro-MSTN to mature MSTN proteins in the muscle tissue between wild-type and transgenic quail by Western blot analysisTo test our hypothesis that greater growth of breast muscle is accompanied with less mature MSTN production through low levels of total MSTN expression or greater expression of MSTN-B, we will compare the gene and protein expressions of MSTN isoforms in muscle between two different lines (Commercial broilers and Leghorns) of chickens and among three different lines (LW, RBC, and HW) of quail.

Progress 05/01/17 to 04/30/22

Outputs
Target Audience:Target audiences will be researchers in the area of animal science, developmental biology, poultry science, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the pharmaceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 2 graduate students, 2 postdocs, 1 technician, and 4 undergraduate students were trained to learn new assay skills and tasks. The assay skills they learned included production of lentivirus, genotyping of transgenic quail, poultry handling, cell culture, transfection, quantitative real-time PCR, Western blot, and histology. Lab members learned the general concept of using transgenic animals to understand gene function in muscle growth and development. Lab members also learned the process of myostatin mRNA and protein in different breeds of quail and chickens, and negative relationship between serum concentrations of myostatin protein with body growth in meat-type and layer chickens. The project provided opportunities to the researchers with learning physiology, cell biology, developmental biology, and molecular biology in the area of muscle growth and development. In addition, they also grasp the importance of integration of multiple research areas in advancing our knowledge in improving animal production. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted a total of 9 manuscripts. The results from the projects were also included in the thesis or dissertation to be available to the scientific community in the area of animal science. The results from the project have been presented in the annual meetings at the American Society of Animal Science, Ohio Agricultural Research and Development Center, and several universities and research institutes, if the PD is invited. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Myostatin (MSTN) negatively regulates in muscle growth and development. Among alternative splicing isoforms of avian MSTN, MSTN-A has anti-myogenic activities and MSTN-B functions as a pro-myogenic factor. For this project, three transgenic quail lines overexpressing MSTN-B form have been generated. Among three independent transgenic lines, two independent lines that overexpressed MSTN-B form were further investigated. These transgenic quail lines were designed to produce exogenous MSTN-B form only in muscle tissue by using the chicken skeletal muscle alpha actin 1 (cACTA1) promoter which resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior but not the pectoralis major muscle. Overexpression of the MSTN-B form increased leg muscle weight about 15% compared with control wild-type quail in both male and female quail by increasing myofiber numbers (10.3-11.1%). However, there were no differences in myofiber sizes between transgenic and control quail. In conclusion, MSTN-B form increased muscle mass by increasing myofiber numbers and not by size. Analysis of expression of myogenic regulatory factors revealed that PAX7 expression was prolonged in both lines of transgenic embryos until 15 days of embryonic age (E15), whereas PAX7 expression in non-transgenic embryos was low at E15. PAX7 expression levels at E15 were 3.0-3.3 folds greater in both transgenic lines than their respective non-transgenic counterparts (p < 0.01). During embryonic development, prolonged expression of PAX7 in the transgenic embryos possibly allowed for greater proliferation of myogenic precursor cells to support enhanced muscle accretion in the transgenic quail lines. The objectives of the current study are to compare temporal expression of the MSTN isoforms in pectoralis major muscle (PM) among three quail lines and two chicken breeds and to relate MSTN expression with temporal changes in muscle growth. First, two selected lines of Japanese quail were used: a random bred control (RBC) and a heavy weight (HW) quail line with muscle hypertrophy. Gains of body weight (BW) and PM weight were greater until post-hatch day (D) 28 (P<0.001), and the fold increases in total DNA contents of PM were greater in the HW line compared with the RBC line during D7 to D28 (P<0.05). PCR analysis showed that MSTN-A expression was greater at 14D (E14) of embryonic age (P<0.01), D7 (P=0.052), and D14 (P<0.01) in the RBC line compared with the HW line. At D28 and D75, expression of MSTN-A was greater in the HW line compared with the RBC line (P<0.05). MSTN-B expression was barely detectable from E14 to D14 and measurable from D28 to D75 in the muscle of both lines. Ratios of the MSTN-B/-A form ranging from 0.15 to 0.29 indicate a minor expression of the B form. Taken together, the lesser expression levels of MSTN-A at E14, D7, and D14 are associated with the fast growth of PM, and greater MSTN-A expression at D28 and D75 are associated with a slowdown of PM growth in the HW line. These data indicate a negative association of MSTN expression with PM growth and provide a scientific basis for potential usage of MSTN expression as a selection marker for greater muscle growth in poultry. Genetic selection of quail for a low body weight for more than 80 generations established a low-weight (LW) Japanese quail line that has been previously characterized to have a muscle hypoplasia phenotype. The aim of this study is to investigate the relationship of temporal expression levels of myostatin (Mstn) and myogenic regulatory factors (MRFs) with hypoplastic muscle growth in the LW line. During embryonic day (E) 13 to 15, gain of embryo weight was 2-fold lower (P<0.001) in the LW line than that in the random bred control (CON). Gains in body weight and pectoralis muscle weight from hatch to posthatch day (P) 28 were also significantly lower (P<0.01) in the LW line but increased by 4-fold (P<0.05) during P42 to P75. PCR analysis showed that expression levels of Mstn were greater in the LW at embryonic stage (E12 to E14, P<0.05), but there was no difference after hatch. In addition, expression levels of Pax7 and myogenin (MyoG) at E12 were 23-fold (P<0.05) and 3.4-fold (P<0.05) lesser in the LW line, respectively. At E14, expression of Pax3, Pax7, and MyoG gene was 3.5-fold (P<0.05), 6.5-fold (P=0.065), and 4.4-fold (P<0.01) less than that in the CON. Taken together, high expression levels of Mstn and low expression of MRFs during embryonic stages can be associated with development of muscle hypoplasia and delayed muscle growth in the LW quail line. These data provide evidence that genetic selection for a low body weight resulting in an avian model with muscle hypoplasia has altered the expression profiles of myogenic factors. Two different lines of chicken were used: a layer and a broiler line with muscle hypertrophy and hyperplasia. The expression of the Mstn isoforms and the sizes of myofibers and muscle bundles during development were analyzed in broiler and layer chicken breeds. There was no difference in the expression levels of total Mstn (Mstn-A and -B forms) during embryonic development and at D33 between the two breeds. Interestingly, the ratios of Mstn-B to -A were significantly higher in the broiler compared to the layer at most ages. In pectoralis major muscle (PM) tissue, the cross-sectional area (CSA) of muscle fiber was significantly greater in the broiler. The broiler also showed greater bundle CSA and a similar fiber number per bundle compared to the layer at D5 and D33. In serum, levels of mature form of MSTN protein were higher in layers compared with broilers. Results from these studies demonstrated higher ratios of Mstn-B/-A expression in the muscles of broiler chickens, suggesting that the development of strategies to enhance the expression of Mstn-B will lead to increased muscle growth and poultry production.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chen PR, Suh S, Shin S, Woodfint RM, Hwang S, Lee K. 2019. Exogenous Expression of an Alternative Splicing Variant of Myostatin Prompts Leg Muscle Fiber Hyperplasia in Japanese Quail. International Journal of Molecular Sciences. 20(18):4617.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kim DH, Choi YM, Suh Y, Shin S, Lee J, Hwnag S, Lee K. 2020. Association of temporal expression of myostatin with hypertrophic muscle growth in selected Japanese quail lines. Poultry Science. 99:2926-2930
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Kim DH, Choi YM, Suh Y, Shin S, Lee J, Hwang S, Lee SS, Lee K. 2021. Increased myostatin expression and decreased expression of myogenic regulatory factors in embryonic ages in a quail line with muscle hypoplasia. Poultry Science. 100(4):100978.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahn J, Wu H, Lee K. 2019. Integrative analysis revealing human adipose-specific genes and consolidating obesity loci. Scientific Reports 9(1):3087.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Woodfint R, Hamlin E, Lee K. 2018. Avian Bioreactor Systems: A Review. Molecular Biotechnology, 60(12):975983
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lee JB, Ma J, Lee K. 2019. Direct delivery of adenoviral CRISPR/Cas9 vector into quail blastoderms for generation of knockout birds. Proceedings of the National Academy of Sciences of the USA 116:13288-13292.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahn J, Woodfint R, Lee J, Wu H, Ma J, Suh Y, Hwang S, Cressman M, Lee K. 2019. Comparative identification and nutritional and physiological regulation of chicken liver-specific genes. Poultry Science 98(7):3007-3013.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahn J, Suh Y, Lee K. 2019. Adipose-specific expression and developmental and nutritional regulation of the gene encoding retinol-binding protein 7 (RBP7) in pigs. Lipids 54:359-367.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Kim DH, Choi YM, Lee J, Shin S, Kim S, Suh Y, Lee K. 2022. Differential Expression of MSTN Isoforms in Muscle between Broiler and Layer Chickens. Animals 12(5):539.

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the pharmaceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 1 postdoc, 1 technician, and 1 undergraduate student were trained to learn new assay skills and tasks. The assay skills they learned included poultry handling, quantitative real-time PCR, and Western blot. Lab members also learned the process of myostatin protein in different breeds of chickens and to understand its function in muscle growth and development. The project provided opportunities to the researchers with learning muscle biology, developmental biology, and molecular biology in the area of muscle growth and development. In addition, they also grasp the importance of integration of multiple research areas in advancing our knowledge in improving animal production. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the projects were published in scientific journals to be available to the scientific community in animal and poultry sciences. The results from the project were presented in the virtual scientific meetings at the Ohio Agricultural Research and Development Center and local meetings. What do you plan to do during the next reporting period to accomplish the goals?For the last part of the objective 3, we will work on comparison of expression of MSTN isoforms at the protein and mRNA levels during muscle development between Broiler and Leghorn chickens.

Impacts
What was accomplished under these goals? Myostatin (MSTN) negatively regulates in muscle growth and development. Among alternative splicing isoforms of avian MSTN, MSTN-A has anti-myogenic activities and MSTN-B functions as a pro-myogenic factor. For the objective 3, different lines of Japanese quail were used: a random bred control (RBC) and a heavy weight (HW) quail line with muscle hypertrophy. The objectives of the current study are to compare temporal expression of the MSTN isoforms in pectoralis major muscle (PM) between two quail lines and to relate MSTN expression with temporal changes in muscle growth and total amounts of DNA in PM. Gains of body weight (BW) and PM weight were greater until posthatch day (D) 28 (P < 0.001), and the fold increases in total DNA contents of PM were greater in the HW line compared with the RBC line during D7 to D28 (P < 0.05). PCR analysis showed that MSTN-A expression was greater at 14 D (E14) of embryonic age (P < 0.01), D7 (P = 0.052), and D14 (P < 0.01) in the RBC line compared with the HW line. At D28 and D75, expression of MSTN-A was greater in the HW line compared with the RBC line (P < 0.05). MSTN-B expression was barely detectable from E14 to D14 and measurable from D28 to D75 in the muscle of both lines. Ratios of the MSTN-B/-A form ranging from 0.15 to 0.29 indicate a minor expression of the B form. Taken together, the lesser expression levels of MSTN-A at E14, D7, and D14 are associated with the fast growth of PM, and greater MSTN-A expression at D28 and D75 are associated with a slowdown of PM growth in the HW line. These data indicate a negative association of MSTN expression with PM growth and provide a scientific basis for potential usage of MSTN expression as a selection marker for greater muscle growth in poultry.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Kim DH, Choi YM, Suh Y, Shin S, Lee J, Hwang S, Lee SS, Lee K. 2021. Increased myostatin expression and decreased expression of myogenic regulatory factors in embryonic ages in a quail line with muscle hypoplasia. Poultry Science. 100(4):100978.

Progress 05/01/19 to 04/30/20

Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the pharmaceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 1 postdoc, 1 technician, and 1 undergraduate student were trained to learn new assay skills and tasks. The assay skills they learned included poultry handling, quantitative real-time PCR, and Western blot. Lab members also learned the general concept of using transgenic animals to understand gene function in muscle growth and development. The project provided opportunities to the researchers with learning physiology, cell biology, developmental biology, and molecular biology in the area of muscle growth and development. In addition, they also grasp the importance of integration of multiple research areas in advancing our knowledge in improving animal production. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the projects were also included in the thesis or dissertation to be available to the scientific community in the area of animal science. The results from the project were presented in the annual meetings at the Ohio Agricultural Research and Development Center and local invited meetings. What do you plan to do during the next reporting period to accomplish the goals?For the extension of objective 3, we will work on expression of MSTN isoforms at the protein level during muscle development in poultry.

Impacts
What was accomplished under these goals? Genetic selection of quail for a low body weight for more than 80 generations established a low weight (LW) Japanese quail line that has been previously characterized to have a muscle hypoplasia phenotype. The goal of this study is to investigate the relationship of temporal expression levels of myostatin (Mstn) and myogenic regulatory factors ( MRFs ) with hypoplastic muscle growth in the LW line. During embryonic day (E) 13 to 15, gain of embryo weight (EW) was 2-fold lower (p < 0.001) in the LW line compared to the random bred control (CON). Gains in body weight (BW) and pectoralis muscle weight (PMW) from hatch to 28 days of post-hatch (P) were also significantly lower (p < 0.01) in the LW line, but were increased by 4-fold (p < 0.05) during P42 to P75. PCR analysis showed that expression levels of Mstn were greater in the LW at embryonic stage (E12 to E14, p < 0.05), but there was no difference after hatch. In addition, expression levels of Pax7 and myogenin (MyoG) at E12 were 23-fold (p < 0.05) and 3.4-fold (p < 0.05) less in the LW line, respectively. At E14, expression of Pax3, Pax7, and MyoG gene were 3.5-fold (p < 0.05), 6.5-fold (p = 0.065), and 4.4-fold (p < 0.01) less compared to the CON. Taken together, high expression levels of Mstn and low expression of MRFs during embryonic stages can be associated with development of muscle hypoplasia and delayed muscle growth in the LW quail line. These data provide evidence that genetic selection for a low body weight resulting in an avian model with muscle hypoplasia has altered the expression profiles of myogenic factors.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chen PR, Suh S, Shin S, Woodfint RM, Hwang S, Lee K. Exogenous Expression of an Alternative Splicing Variant of Myostatin Prompts Leg Muscle Fiber Hyperplasia in Japanese Quail. International Journal of Molecular Sciences. 20(18):4617.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kim DH, Choi YM, Suh Y, Shin S, Lee J, Hwnag S, Lee K. Association of temporal expression of myostatin with hypertrophic muscle growth in selected Japanese quail lines. Poultry Science. 99:2926-2930
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Kim DH, Choi YM, Suh Y, Shin S, Lee J, Hwnag S, Lee SS, Lee K. Increased myostatin expression and decreased expression of myogenic regulatory factors in a quail line with muscle hypoplasia. Poultry Science.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Paula Chen. Muscle Fiber Hyperplasia in Leg Muscle of Transgenic Quail Overexpressing an Alternative Splicing Variant of Myostatin. MS thesis. The Ohio State University.

Progress 05/01/18 to 04/30/19

Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the pharmaceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 2 postdoc, 1 technician, and 2 undergraduate students were trained to learn new assay skills and tasks. The assay skills they learned included genotyping of transgenic quail, poultry handling, histology, quantitative real-time PCR, and Western blot. Lab members also learned the general concept of using transgenic animals to understand gene function in muscle growth and development. The project provided opportunities to the researchers with learning physiology, cell biology, developmental biology, and molecular biology in the area of muscle growth and development. In addition, they were also able to grasp the importance of integrating multiple research areas in advancing our knowledge in improving animal production. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we recently submitted two manuscripts. The results from the projects were also included in the thesis or dissertation to be available to the scientific community in the area of animal science. The results from the project were presented in the annual meetings at Ohio Agricultural Research and Development Center, and at several universities and research institutes. What do you plan to do during the next reporting period to accomplish the goals?We will complete the proposed experiments for all objectives for the 3rd year and prepare manuscripts to be submitted to high impact journals.

Impacts
What was accomplished under these goals? Among three independent transgenic lines, two independent lines that overexpressed MSTN-B form were further investigated. The chicken skeletal muscle alpha actin 1 (cACTA1) promoter resulted in restricted exogenous MSTN-B protein expression to certain skeletal muscles, such as the gastrocnemius and tibialis anterior but not the pectoralis major muscle. MSTN-b form increased muscle mass (14.6%) by increasing myofiber numbers (10.3-11.1%). Analysis of expression of myogenic regulatory factors revealed that PAX7 expression was prolonged in both lines of transgenic embryos until 15 days of embryonic age (E15), whereas PAX7 expression in non-transgenic embryos was low at E15. PAX7 expression levels at E15 were 3.0-3.3 folds greater in both transgenic lines than their respective non-transgenic counterparts (p < 0.01). During embryonic development, prolonged expression of PAX7 in the transgenic embryos possibly allowed for greater proliferation of myogenic precursor cells. For the objective 3, two selected lines of Japanese quail for 4-week body weight for over 80 generations were used: a random bred control (RBC) and a heavy weight (HW) quail line with muscle hypertrophy. In addition, two breeds of chickens were used: Leghorn and Broiler with muscle hypertrophy and hyperplasia. The objectives of the current study are to compare temporal expression of the MSTN isoforms in pectoralis major muscle (PM) between two quail lines and two chicken breeds and to relate MSTN expression with temporal changes in muscle growth. The lesser expression levels of MSTN-A at E14, post-hatch day (P)7 and P14 are associated with the fast growth of PM, and greater MSTN-A expression at P28 and P75 are associated with a slowdown of PM growth in the HW line. In two breeds of chickens, Broiler chickens had lesser expression of MSTN in breast muscle compared with Leghorn from E17 to P11. PCR analysis revealed that ratios of the MSTN-B/-A form ranging from 0.15-0.3 indicate a minor expression of the B form. The expression of MSTN-B form was very low or absent in embryonic and early post-hatch periods but appeared after the fast growing stages in quail and chickens. Taken together, these data indicate negative association of MSTN expression with muscle growth and provide a scientific basis for potential usage of MSTN expression as a selection marker for greater muscle growth in poultry.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Chen PR, Suh S, Shin S, Woodfint RM, Hwang S, Lee K. Exogenous expression of an alternative splicing variant of myostatin prompts leg muscle fiber hyperplasia in Japanese quail. Faseb J.
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Kim DH, Choi YM, Suh Y, Shin S, Lee J, Hwnag S, Lee K. Association of temporal expression of myostatin with hypertrophic muscle growth in selected Japanese quail lines. Poultry Science
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lee JB, Ma J, Lee K. 2019. Direct delivery of adenoviral CRISPR/Cas9 vector into quail blastoderms for generation of knockout birds. Proceedings of the National Academy of Sciences of the USA 116:13288-13292.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahn J, Woodfint R, Lee J, Wu H, Ma J, Suh Y, Hwang S, Cressman M, Lee K. 2019. Comparative identification and nutritional and physiological regulation of chicken liver-specific genes. Poultry Science 98(7):3007-3013.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahn J, Suh Y, Lee K. 2019. Adipose-specific expression and developmental and nutritional regulation of the gene encoding retinol-binding protein 7 (RBP7) in pigs. Lipids 54:359-367.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahn J, Wu H, Lee K. 2019. Integrative analysis revealing human adipose-specific genes and consolidating obesity loci. Scientific Reports 9(1):3087.

Progress 05/01/17 to 04/30/18

Outputs
Target Audience:Target audiences will be researchers in the area of animal science, developmental biology, poultry science, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the pharmaceutical industry. Changes/Problems:For the generation of transgenic bird expressing MSTN-B form in muscle tissue, alpha-actin promoter was used. The expression levels of the transgene were about 2-fold greater than those of the control wild type quail, resulting in about a 10% increase in leg muscle weight. This marginally increased muscle mass may be due to the low expression of the transgene. Therefore, we have replaced the alpha-actin promoter with the stronger CbH promoter (CMV element + chicken β-actin promoter) to drive high expression of the MSTN-B transgene that will have greater muscle mass of the transgenic quail. After microinjection of the new transgene cassette into 150 quail embryos, 15 chimeric founder quail recently hatched. The progenies (G1 generation) from these founder lines will be screened for germ line transmission of the transgene. The positive G1 offspring will be propagated for generation of G2 offspring that will be used to confirm the dose-dependent relationship between expression levels of transgene and increase in muscle mass. What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 1 postdoc, 1 technician, and 1 undergraduate student were trained to learn new assay skills and tasks. The assay skills they learned included production of lentivirus, genotyping of transgenic quail, poultry handling, cell culture, transfection, quantitative real-time PCR, and Western blot. Lab members also learned the general concept of using transgenic animals to understand gene function in muscle growth and development. The project provided opportunities to the researchers with learning physiology, cell biology, developmental biology, and molecular biology in the area of muscle growth and development. In addition, they also grasp the importance of integration of multiple research areas in advancing our knowledge in improving animal production. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we will prepare and submit manuscripts. The results from the projects will be also included in the thesis or dissertation to be available to the scientific community in the area of animal science. The results from the project will be presented in the annual meetings at the American Society of Animal Science, Ohio Agricultural Research and Development Center, and several universities and research institutes, if the PD is invited. What do you plan to do during the next reporting period to accomplish the goals?We will start the proposed experiment for objective 2 and 3 for the 2nd year and prepare a manuscript for the results from the 1st objective to be submitted to high impact journals.

Impacts
What was accomplished under these goals? For this project, transgenic quail overexpressing MSTN-b form have been previously generated. These transgenic quail lines were designed to produce exogenous MSTN-b form only in muscle tissue, which was confirmed by Western blot analysis. During the first year, as we planned in the proposal, we focused on characterizing two selected lines of the transgenic quail. Overexpression of the MSTN-b form increased leg muscle weight about 10-12% compared with control wild-type quail in both male and female quail. The increases in leg muscle weight are associated with higher (~10%) numbers of total myofiber in the leg muscle compared with the control quail. However, there were no differences in myofiber sizes between transgenic and control quail. In conclusion, MSTN-b form increased muscle mass by increasing myofiber numbers and not by size.

Publications