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Lipofibroblast Specific Transcription Factor Required for Lung Alveolar Development Mānoa Horizons Volume 3 | Issue 1 Article 26 11-15-2018 Lipofibroblast Specific rT anscription Factor Required for Lung Alveolar Development Ga Young (Sally) Park University of Hawaiʻi at Mānoa Follow this and additional works at: https://kahualike.manoa.hawaii.edu/horizons Part of the Cell and Developmental Biology Commons Recommended Citation Park, Ga Young (Sally) (2018) "Lipofibroblast Specific rT anscription Factor Required for Lung Alveolar Development," Mānoa Horizons: Vol. 3 : Iss. 1 , Article 26. Available at: https://kahualike.manoa.hawaii.edu/horizons/vol3/iss1/26 This Article is brought to you for free and open access by Kahualike. It has been accepted for inclusion in Mānoa Horizons by an authorized editor of Kahualike. For more information, please contact [email protected]. Lipofibroblast Specific Transcription Factor Required for Lung Alveolar Development Ga Young (Sally) Park Undergraduate Research Opportunities Program Mentor: Dr. Michelle D. Tallquist Respiratory Distress Syndrome occurs in preterm births. Premature lungs lack pulmo- nary surfactant produced by alveolar type II (ATII) cells. Understanding signaling pathways that promote maturation of ATII cells could reduce time spent in neonatal care units. Tcf21, a bHLH transcription factor, is expressed during lung development, and loss of Tcf21 leads to respiratory failure and dysregulation of Wnt signaling. It is hypothesized that lipofibroblasts secrete Wnt ligands to induce ATII cell differentiation. Using genetically engineered mice that permit isolation of ribosomes from Tcf21 lineage cells, the profile of Wnt ligand RNA expressions were determined. To confirm whether Tcf21 is sufficient to stimulate Wnt ligands, Tcf21 was overexpressed in primary lung fibroblasts. The results showed increased expressions of Wnt 2, 2b, 4, and 5a, suggesting that Tcf21 promotes expression of these Wnt ligands. The expression of Wnt by Tcf21 was investigated in vivo by isolating ribosomal associated RNA from Tcf21 control and null cells. The quantitative PCR demonstrated that Wnt2 and 5a were enriched within the lipofibroblast. Furthermore, data from Tcf21 mutant lungs demonstrated a loss of lipofibroblasts with subsequent reduction in ATII cells. The main findings from these studies indicate that lipofibroblasts are necessary for ATII cell differentiation and that Wnt ligands may be involved. late gestation to help open up their lungs to breathe. The lung Introduction & Background is composed up of three types of fibroblast: myofibroblast, PDGFRα expressing fibroblast, and lipofibroblasts. Lipofibro- Respiratory distress syndrome (RDS) is a breathing disorder blasts, fibroblasts containing lipid vesicles, play a vital role in in premature babies. Their immature lungs lack pulmonary transferring lipid for active supply for lung pulmonary surfac- surfactant that is produced by alveolar type II (ATII) cells in tant production as well as for alveolus protection against injury. I am a recent graduate from the University of Hawai‘i at Mānoa (UHM) with a B.A. in Biology and Korean with the Korean Language Flagship Program. I hope to attend the John A. Burns School of Medicine to pursue MD and become a Korean American physician in Hawai‘i. My submission to Manoa Horizons is a research project that I had also done for the Undergraduate Research Opportunities Program at UHM. I was able to work with Dr. Tallquist’s lab, which is part of the Center for Cardiovascular Research at John A. Burns School of Medicine. This is my first research project and it had really enriched me with knowledge in biology and research tech- niques. I was able to gain knowledge about Respiratory Distress Syndrome in premature babies and the lung development process. The experiments and research of my project is to understand more about the lipofi- broblast specific transcription factor: Tcf21 and Wnt signaling pathways that may play a larger role in giving us more insights to cure or even prevent lung disorders in earlier stages of development. Mānoa Horizons, Vol. 3, 2018, pp. 128–131 Copyright © 2018 by the University of Hawaiʻi at Mānoa 128 Manoa Horizons Online 3.indb 128 10/10/2018 10:54:17 PM Park Lipofibroblast Specific Transcription Factor Required for Lung Alveolar Development 129 Tcf21, a bHLH (basic helix-loop-helix protein structure) tran- scription factor, is detected in lung mesenchyme. Dr. Tallquist’s Methods lab shows that Tcf21 lineage cells give rise to a majority of lipofi- Tcf21 Inducible Mouse Model (Figure 1) broblasts during development. Loss of Tcf21 results in decreased lipofibroblast number and impaired ATII cell differentiation. In Cre is fused with the Estrogen Receptor, creating Cre-ER pro- addition, Wnt signaling, an active pathway required for organo- tein that is controlled by a tissue-specific promoter: Cre mcre genesis is dysregulated in lungs by Tcf21 loss. Tcf21 promoter. In the absence of Tamoxifen, Cre-ER is bound by the HeatShock Protein 90 (HSP 90), which keeps them in the cytosol. Before the Tamoxifen binds to the ER of Cre- Significance ER protein, the liver converts Tamoxifen into its active form: 4-hydroxytamoxifen. As the 4-hydroxytamoxifen binds to the The discovery of signaling pathways that promote maturation ER, the Cre-ER is released from HSP 90 retention in the cy- of alveolar type II cells could identify alternative therapeutic toplasm and results in a conformational change. The trans- target for premature babies with RDS. The identified specific formed Cre-ER is then transported to the nucleus where the Wnt ligand expressed by Tcf21 expressing cells will further elu- Cre recognizes the loxP sites surrounding the Tcf21. It cuts the cidate whether lipofibroblast Wnt ligand secretion is required loxP sites in half and excises the intervening Tcf21, creating a for alveolar type II cell differentiation. circular DNA with a loxP site and Tcf21 exons and another lin- ear DNA with a single loxP site. These are identified as Tcf21 recombined alleles. Research Question Ribotag system for isolation of “Does Tcf21 regulate Wnt ligand expression for alveolar devel- ribosome-associated mRNAs from Tcf21 opment?” expressing cells in tissues (Figure 2, left) Ribotag mouse with ribosomal protein L22 (Rpl 22) and Hem- Objectives agglutinin A tagged Rpl 22 was used. Cre recombinase gen- erated genes that have a single loxP site with the HA tagged • Aim 1. Determine gene expression of Wnt ligands with Rpl22. The gene was then processed through transcription and increased Tcf21 translation to create ribosomes with multiple RNAs of itself. • Aim 2. Identify lipofibroblast-specific Wnt ligands These RNAs are fibroblast specific RNA that is being translat- • Aim 3. Determine the effect of deletion of Tcf21 on lipofi- ed and the target tissues with these ribosomes were then ho- broblast and ATII cell development mogenized. HA specific antibodies precoupled with magnetic Figure 1 Tcf21 Inducible Mouse Model: Tamoxifen Treatment. Manoa Horizons Online 3.indb 129 10/10/2018 10:54:18 PM 130 Mānoa Horizons Vol. 3, Fall 2018 Figure 2 Ribotag system for isolation of ribosome-associated mRNAs from Tcf21 expressing cells in tissues. beads were used to pull out the ribosomes with the HA tags. Enrichment of Wnt 2 and Wnt 5a They were then purified through elution and Tcf21 expressing expression in lipofibroblasts (Figure 4) RNAs were later used for gene expression and analysis. qPCR analysis of Wnt transcripts comparing lipofibroblast Rpl22 genotyping PCR (Figure 2, right) RNA (IP RNA) versus total lung RNA (input RNA). Tamoxifen at P2 and lungs isolated at P14. Data shows mean ± SD, N=3. DNA ladder lane 1; Rpl22HA allele, lanes 2,3,5,7; Rpl22 wild type (wt) allele, lanes 4,6,8,9 mCrem/+; tdT/+ HA Postnatal Tcf21 loss results in decreased Tcf21 R26R mice that contained Rpl22 were lipofibroblast and alveolar type II cells then selected for immunoprecipitation and quantitative poly- (Figure 5) merase chain reaction (qpcr). Immunohistochemistry for ProSP-C (alveolar type II cells) and Adfp (lipofibroblasts) in the indicated genotypes. (A) Upper Data and Results panels ProSP-C, lower panels DAPI (B) Representative images Expression of Wnt 2, 2b, 4, and of ADFP stained Tcf21 control and mutant lungs (C) Quantita- 5a is induced by Tcf21 (Figure 3) tion of ProSP-C cells (Mean ± SD, Tcf21 control, n=4; mutant: n=2; 0.2425 p-value.) (D) Quantification of % of ADFP area. (A) qPCR detection of Tcf21 mRNA. Tcf21 1:500 treatment: (Mean ± SD, Tcf21 control: n=6; mutant: n=4; 0.0051 p-value.) 0.0148 p-value; Tcf21 1:250 treatment: 0.0604 p-value (B) **P < 0.01 mRNA Wnt genes in transduced fibroblasts adGFP (control) and adTcf21. Data shows mean ± SD, N=2; 0.0045 p-value. un- paired t-test. **P < 0.01. ND, not detected. Figure 3 Expression of Wnt ligands in Tcf21 overexpressing fi- broblasts Figure 4 Expression of Wnt ligand in lipofibroblasts Manoa Horizons Online 3.indb 130 10/10/2018 10:54:20 PM Park Lipofibroblast Specific Transcription Factor Required for Lung Alveolar Development 131 Figure 5 Tcf21 loss leads to fewer lipofibroblasts and alveolar type II cells Manipulation of Tcf21 could be the key in providing pulmonary Conclusion surfactant to premature lungs. Continuation of research on the molecular mechanism of Tcf21, developmental defect of the lung Tcf21 Overexpression: and the relationship between lung fibroblast and ATII cell differ- • Tcf21 overexpression resulted in increased Wnt 2, 2b, 4 entiation signaling pathways could reveal new insights to cure and 5a expression, suggesting that Tcf21 may regulate and prevent lung diseases and disorders in premature babies. these genes • Wnt 7b had no detection, indicating that lung fibroblasts do not express this gene References • Wnt 10a had no significant difference, suggesting that it is not controlled by Tcf21 transcriptional regulation 1. Quaggin SE, Schwartz L, Cui S, Igarashi P, Deimling J, Post M, Rossant J: The basic-helix-loop-helix protein pod1 is Wnt Ligand Expression by Lipofibroblasts: critically important for kidney and lung organogenesis.
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