REPUBLIC OF THE PHILIPPINES

BATANGAS STATE UNIVERSITY

THE NATIONAL ENGINEERING UNIVERSITY

ALANGILAN CAMPUS

COLLEGE OF ENGINEERING

MECHANICAL ENGINEERING DEPARTMENT

SCI-402

MODERN BIOLOGY

REVIEW OF A JOURNAL ARTICLE

AGBAY, RENNIER

MARFA, JOAN

MARTINEZ, SETH

ROMASANTA, JOHN GABRIEL

ME - 1302

RED, ERICKA M.

INSTRUCTOR
 Distribution of Task

Group 6 ME-1302

I. Title: John Gabriel Romasanta

II. Authors: Rennier Agbay
III. Year of Publication: Seth Martinez
IV. Main Hypothesis: Joan Marfa
V. Objectives: Joan Marfa
VI. Procedures employed: Joan Marfa
VII. Findings: John Gabriel Romasanta/ Rennier Agbay
VIII. Conclusion: Joan Marfa/ Seth Martinez
IX. Questions

General questions on quality and value, suitability, and presentation

Q1-Q2: John Gabriel Romasanta

Q3-Q5: Rennier Agbay

Q6-Q8: Seth Martinez

Questions on Data Assessment

Q1-Q3: Joan Marfa

Q4-Q5: Rennier Agbay

Q6-Seth Martinez

Detailed Comments and Suggestions

All of us
College of Engineering- Department of Mechanical Engineering

Review Of A Journal Article

I. Title of the Article: Single-cell RNA sequencing reveals the developmental program
underlying proximal–distal patterning of the human lung at the embryonic stage

II. Author/s: Shangtao Cao, Huijian Feng, Hongyan Yi, Mengjie Pan, Lihui Lin, Yao Santo
Zhang, Ziyu Feng, Weifang Liang, Baomei Cai, Qi Li, Zhi Xiong, Qingmei Shen, Minjing Ke,
Xing Zhao, Huilin Chen, Qina He, Mingwei Min , Quanyou Cai, He Liu, Jie Wang, Duanqing
Pei, Jiekai Chen and Yanlin Ma

III. Year of Publication: 2023

IV. Main Hypothesis

The main hypothesis of the study is that single-cell RNA sequencing can reveal the
developmental program responsible for the proximal-distal patterning of the human lung during
the embryonic stage. The researchers hypothesize that by analyzing the gene expression patterns
of individual cells in the developing lung, they can identify distinct cell populations, characterize
their gene expression profiles, and gain insights into the molecular mechanisms underlying the
establishment of the proximal-distal axis.
They expect that cells in the proximal region of the lung will express genes associated
with airway development and ciliated cell differentiation, while cells in the distal region will
exhibit gene expression patterns related to alveolar cell differentiation and surfactant production.
The hypothesis also suggests that there may be transitional cell populations with intermediate
gene expression profiles, indicating a stepwise process of cell fate determination during lung
development.
The study was conducted to analyze the development program underlying the
proximal–distal patterning of the human lung during the embryonic stage. Specifically, the
authors aimed to use single-cell RNA sequencing to observe the transcriptional regulation of this
process to gain insight into their development. The researchers examined how spatial and
temporal transcriptional regulation can contribute to the proximal–distal patterning of the human
lung, as well as the specific roles of transcriptional factors and regulatory pathways in the
formation of different components of the human lung. They also aimed to identify any potential
causative genes involved in this process.
Overall, the researchers anticipate that their single-cell RNA sequencing approach will
provide a comprehensive understanding of the developmental program and gene regulatory
networks involved in proximal-distal patterning of the human lung at the embryonic stage.
 V. Objectives

The objectives of the study are as follows:

● Characterize cell populations: The study aims to identify and characterize the different
cell populations present in the developing human lung during the embryonic stage. By
analyzing individual cells using single-cell RNA sequencing, the researchers seek to
classify and define distinct cell types within the lung tissue.
● Investigate gene expression patterns: The researchers aim to examine the gene
expression patterns of the identified cell populations. By analyzing the transcriptomes of
individual cells, they seek to identify genes that are differentially expressed in specific
cell types and regions of the developing lung.
● Understand proximal-distal patterning: The study focuses on understanding the
molecular mechanisms underlying the proximal-distal patterning of the human lung. The
researchers aim to determine how gene expression patterns vary along the respiratory
tract, from the proximal to the distal regions, and identify the genes and pathways
involved in this patterning process.
● Explore transitional cell populations: The study also aims to investigate transitional
cell populations that may exist between the proximal and distal regions of the developing
lung. By analyzing the gene expression profiles of these transitional cells, the researchers
hope to gain insights into the stepwise process of cell fate determination and
differentiation during lung development.
● Gain insights into lung development: Overall, the study seeks to provide a
comprehensive understanding of the developmental program underlying the
proximal-distal patterning of the human lung. By elucidating the gene expression patterns
and cell populations involved, the researchers aim to enhance our knowledge of lung
development and potentially uncover new avenues for studying lung-related diseases and
regenerative medicine approaches.

VI. Procedures Employed

● Sample collection and preparation: Human embryonic lung tissue samples were
collected. Single cells were isolated from the tissue using enzymatic digestion and
mechanical dissociation methods. The isolated cells were then processed for single-cell
RNA sequencing.
● Single-cell RNA sequencing: The isolated cells underwent single-cell RNA sequencing,
which involved capturing the RNA molecules present in each individual cell and
converting them into complementary DNA (cDNA). This step allowed for the
amplification and sequencing of the transcriptomes of the individual cells.
● Data analysis: The obtained sequencing data were analyzed to identify and classify
distinct cell populations within the developing lung. The transcriptomes of individual
cells were compared and analyzed to determine differential gene expression patterns
across different cell types and regions.
● Cell type classification: Through the analysis of gene expression patterns, the
researchers classified the identified cells into different cell types based on their molecular
profiles. They characterized the gene expression signatures associated with each cell
type.
● Proximal-distal patterning analysis: The researchers examined the gene expression
patterns along the proximal-distal axis of the developing lung. They analyzed how gene
expression varied in cells located at different positions along this axis, from the proximal
to the distal regions.
● Identification of transitional cell populations: The study also aimed to identify
transitional cell populations that exhibited intermediate gene expression profiles between
the proximal and distal regions. These transitional cells were further characterized to
understand the stepwise process of cell fate determination during lung development.
● Gene Expression Patterns: The main variable measured in the study was gene
expression patterns at the single-cell level. The researchers measured the transcriptomes
of individual cells to identify and analyze differential gene expression across different
cell types and along the proximal-distal axis of the developing lung. The study did not
involve direct manipulation of variables since it primarily focused on observational
analysis of gene expression patterns in naturally occurring cells during lung development.
● Human embryonic lung samples were collected and dissected to investigate the
development and cellular composition of the human lung. The samples were obtained
from legal elective abortions performed at the First Affiliated Hospital of Hainan Medical
University, with informed consent obtained from all participants. The protocol for sample
collection was approved by the ethics committee of the hospital. The embryonic lung
samples were obtained from different gestational stages, ranging from CS12 to CS23.
These stages were determined based on established morphological landmarks and the
gestational time. The lung tissues were dissected from the embryos and processed for
further analysis.
● For single-cell RNA sequencing (scRNA-seq) the dissected lung tissues were
dissociated into single cells. The tissues were digested with enzymes like Papain and
DNase I to obtain a single-cell suspension. The cells were then filtered, washed, and
resuspended in a buffer for scRNA-seq library construction. This allowed the researchers
to analyze the gene expression profiles of individual cells and explore the cellular
heterogeneity and developmental trajectories of the lung cells.
● Spatial transcriptomics and smiFISH (single-molecule fluorescence in situ
hybridization) is used to investigate the spatial distribution and gene expression patterns
 within the embryonic lung tissues. Tissue cryosection preparation was performed to
obtain thin sections of the lung tissues, which were then used for smiFISH analysis. The
smiFISH technique allows for the visualization and detection of specific RNA molecules
within the tissue sections.

VII. Main Findings

The results of the study show that during human lung development, the major
components necessary for lung formation are present early on, specifically at the initiation of
lung formation. The study utilized single-cell RNA sequencing (scRNA-seq) to analyze human
embryonic lungs from Carnegie stage 12 to 21, covering the embryonic and early
pseudoglandular stages.

The scRNA-seq analysis identified six major cell clusters in the embryonic lungs,
including lung stromal cells, epithelial progenitors, neural crest progenitors, endothelial
progenitors, proerythroblasts, and macrophages. These cell types were identified based on their
developmental signatures. Stromal cells were found to be the most abundant cell type throughout
the sampling period, followed by epithelial cells, neural crests, and endothelial cells.

Within the epithelial cell cluster, which is the second largest cluster, the study focused
on the proximal-distal patterning of epithelial cells, which is crucial for branching
morphogenesis in lung development. By integrating their dataset with publicly available datasets,
the researchers identified transcription factors (TFs) that play a role in proximal and distal cell
fate decisions. Known TFs involved in proximal and distal cell fates were validated, and novel
TFs associated with these cell fates were also identified. The study further revealed the
expression patterns of these TFs and their downstream targets in the developing epithelial cells.

The study also examined the role of stromal cells in influencing lung epithelial
development. Seven subtypes of stromal cells were identified, including proliferating stromal
cells and subtypes associated with cartilage development, Notch signaling regulation, and
mesenchymal differentiation. The spatial properties of these stromal cells were investigated,
revealing their proximity to epithelial cells and potential interactions between these cell types.

Here is a breakdown of the key findings and their significance:

● Cellular Composition: The researchers identified six major cell types in the developing
human embryonic lung: stromal cells, epithelial progenitors, neural crest progenitors,
endothelial progenitors, proerythroblasts, and macrophages. Stromal cells were the most
abundant cell type throughout the studied period. These findings provide insights into the
cellular architecture of the developing lung.
 ● Proximal-Distal Patterning: Lung lobes develop from tracheal buds through a process
called branching morphogenesis. The epithelial cells, which undergo branching
morphogenesis, were analyzed in detail. By integrating their dataset with publicly
available datasets covering later stages of lung development, the researchers observed
that most progenitors of proximal (basal and ciliated cells) and distal (alveolar type 1 and
type 2 cells) trajectories had emerged between weeks 4-8 of embryonic development.
They identified specific transcription factors (TFs) that regulate the proximal and distal
cell fates, including both known and novel TFs. This finding sheds light on the molecular
mechanisms involved in the development of different regions of the lung.
● Initiation of Proximal-Distal Patterning: The researchers investigated the initiation of
proximal-distal patterning in the human lung epithelium. They found that the proximal
and distal lineages could be traced back to proliferating lung epithelial progenitors as
early as week 4 of development. The expression patterns of specific markers indicated
that the proximal-distal specification starts around week 6. These findings suggest that
the proximal-distal patterning occurs early in lung development, even before the
morphological signs of distal epithelial differentiation.
● Conservation of Proximal-Distal Patterning: The study also compared the identified
TFs in human lung development with a mouse model. The analysis revealed both shared
and species-specific TFs involved in proximal-distal patterning, indicating a conserved
core transcriptional regulation network. This finding highlights the importance of
studying human lung development to understand the specificities of human biology.
● Stromal Cell Influence: The stromal cells, derived from the mesoderm, play a crucial
role in shaping lung epithelial morphogenesis. The researchers identified different
subtypes of stromal cells and their contributions to epithelial development. These stromal
subtypes exhibited diverse molecular signatures and functions, including regulation of
cartilage development, Notch signaling, and mesenchymal differentiation. Understanding
the interactions between stromal and epithelial cells provides insights into the
microenvironment necessary for proper lung development.

Overall, this study provides a comprehensive analysis of the cellular composition,

molecular dynamics, and interactions between different cell types during early human lung
development. These findings contribute to our understanding of the complex processes involved
in lung organogenesis and may have implications for studying lung development disorders and
diseases.
VIII. Conclusions

The study concluded that distinct expression patterns of specific genes from three
proximal-distal domains of the embryonic human respiratory system helped to define the
spatio-temporal developmental and morphogenetic process of the lung. Additionally, it identified
signals driving the differentiation of cells in the three discrete domains, demonstrating cell
identity and function.

The study also demonstrated that proximal–distal patterning of the human lung at the
embryonic stage is regulated by a complex network of transcription factors and signaling
pathways. Furthermore, it identified more than 5,000 genes specifically expressed in distal
epithelium, suggesting they could play critical roles in lung morphogenesis. In conclusion, this
study not only revealed the underlying developmental program of the human lung at the
embryonic stage, but also provided a platform to study other organs in terms of their
development. Understanding these developmental programs could lead to new insights in the
diagnosis and treatment of human diseases.

IX. Questions

General questions on quality and value, suitability, and presentation

(1) Is the submission original?

● This journal plainly states that it is a review. That is, it is based on an original
composition. Its goal is to look at an existing research of the embryonic development
program that underlies the proximal-distal patterning of the human lung.
(2) Is the research sound and evidenced?
● Yes, the research given in the publication is sound and supported by evidence. The
authors' study methods and decisions, such as the utilization of single-cell RNA
sequencing and real-time quantitative PCR, have been discussed. They also talked about
the approach they employed to study the proximal-distal patterning process. Furthermore,
the authors acknowledged the study's flaws and offered suggestions for further research.
Overall, the results emphasize the importance of gene expression patterns in lung
development.
(3) Does it help to expand or further research in this subject area?
● Yes, expanding and conducting further research in this subject area can have significant
benefits. By studying the development of the human embryonic lung and identifying the
key transcription factors involved in proximal-distal patterning, researchers can acquire a
better knowledge of the molecular mechanisms underlying lung development. This
information has potential applications for various fields, such as regenerative medicine,
respiratory diseases, and developmental biology.
(4) Do you feel that the significance and potential impact of the paper is high or low?
● It appears to have a high significance and potential impact. The study focuses on the
development of the human embryonic lung and identification of key transcription factors
involved in lung epithelial differentiation. This research can help us to understand lung
development and might lead to advances in regenerative medicine and therapies for
lung-related diseases. Moreover, this research opens doors to further investigations and
advancements that can positively impact human health.

(5) Would the paper be of interest to the readership of the journal?

● Yes, the paper would certainly be of great interest to the readership of the journal. These
topics align with the focus and interests of the journal’s readership, making the paper
relevant and valuable to them. Also, the experts and enthusiasts in these areas would find
the paper highly relevant and informative.

(6) Is the writing style clear and appropriate to the readership?

● As for the journal, the writing style is made clear and appropriate for the readership. The
information that is already stated in the research is detailedly discussed. Moreover, the
data are organized and even include the illustrations of cell types that emerges at the
initiation of human lung embryonic development, the progression of the cells, as well as
the proportions of different types of cells, and the results of the findings.

(7) Are all relevant accompanying data, citations, or references given by the author?
● Yes, the author includes the relevant accompanying data that supports the research. The
authors of the research provided many information sources and made sure that there were
no information gaps and that it is thoroughly researched. The authors also included
additional references for more thorough explanations of single-cell RNA sequencing.

(8) Does the paper contain the appropriate referencing to provide adequate context for the
present work?
● Yes, the paper appropriately contains references from various sources and provides
adequate context necessary for the paper. The references included in the paper allow the
readers to be aware of the context of the paper that they are reading. This also allows the
readers to redirect to the sources of the provided evidence and even makes the reading
better and clearer for the audience. Additionally, the research includes graphics and data
collected and presented in an orderly manner. The figures provided by the authors are
labeled with letters. Each figure included in the paper is arranged specifically to match
the flow of description using letters as labels.
Questions on data assessment

(1) Are the data selective? Why have the authors included some results and not others?

● The data presented in the article is selective, as it only focuses on the genes involved in
the proximal-distal patterning of the human lung at the embryonic stage. The authors
selected specific data to include depending on the research question they were trying to
answer. For example, they included gene expression data to identify signals driving cell
differentiation. Additionally, they chose to focus only on genes specific to the distal
domain of the lung in order to study its morphogenesis.

(2) Are the conclusions drawn from the data reasonable?

● Yes, the conclusions derived from the data presented in the paper are reasonable.The
authors discussed their research techniques and judgments, as well as the study's flaws.
The data is well-structured and provides important insights into the role of gene
expression patterns in lung development.
(3) Have the authors explained why they chose certain values (e.g. quantities of chemicals)?

● Yes, the authors have provided explanations as to why they used specific values in their
research. For example, they explain that they used single-cell RNA sequencing to study
gene expression patterns in the three distinctive domains of the embryonic human lung.
Additionally, they explain why they chose to focus on the distal domain by selecting
genes specifically expressed in this domain. The authors also explain why they chose to
use real-time quantitative PCR (qPCR) to validate the expression patterns of the genes
they identified. Additionally, they detail the strategies used to study the proximal–distal
patterning process, such as the use of hierarchical clustering and principal component
analysis. Finally, the authors discuss several limitations to their study and how these
limitations could be addressed in future research.

(4) Do the authors give enough information about the experiment that it could be
reproduced?
● Yes, the authors provide sufficient information about the experiment so that it could be
reproduced. They describe the specific methods used, such as bioinformatics analysis,
single-cell and spatial transcriptome data analysis, smiFISH validation, isolation of
human developing tissues, and tissue digestion for single-cell suspension. They also
discuss the people involved in collecting human embryos, preparing single-cell samples,
and performing library construction and spatial transcriptomic experts. Therefore, this
detail allows other researchers to understand and replicate the experimental procedures.
(5) Are the values reported with a sufficient degree of accuracy?
● Yes, the values reported in the study are presented with a sufficient degree of accuracy.
The authors explain why they utilized certain values in their research, such as single-cell
RNA sequencing and real-time quantitative PCR (qPCR) to analyze gene expression
patterns. They also discuss the techniques they used to evaluate the proximal-distal
patterning process, such as hierarchical clustering and principal component analysis.

(6) Is the paper easy to follow?

● Yes, the research showed the organization and sectioning of subtopics, results, and
methods with their respective explanations and findings. The figures provided by the
authors are labeled with letters and are organized orderly. The detailed explanations for
each figure are organized accordingly to match the order of the figures. This made the
paper easy to follow and understand.

Detailed Comments and Suggestions

(1) Any unclear points in the paper that need further elaboration?
● Upon reviewing the paper, it becomes evident that there are several points that require
further elaboration in order to enhance clarity and provide a more comprehensive
understanding of the topic at hand. One such point pertains to the time frame of the
human lung development process that was studied by the authors. While the paper
provides some information on this aspect, there is a need for additional details regarding
the specific stages and duration of the developmental process. Elucidating the precise
timeline would enable readers to contextualize the findings within the broader scope of
lung development and better comprehend the implications of the study.
Additionally, the paper would benefit from a more thorough discussion of the type of cell
differentiation that was investigated during this research and how different types of cell
differentiation may contribute to lung morphogenesis. While the authors touch upon this
topic, further elaboration is needed to provide a deeper understanding of the mechanisms
involved and their significance in the overall developmental process. Exploring the
interplay between various cell types and their contributions to lung morphogenesis would
shed light on the complexity of the process and its implications for human health.
Furthermore, the authors could provide more explicit details on the types of experiments
used to validate the gene expression patterns identified in their study. While the paper
mentions the validation of gene expression, additional information on the specific
experimental techniques employed and their rationale would be valuable. Clarifying the
experimental methodologies, including the selection of appropriate markers and
techniques for identifying cell function during the differentiation process, would bolster
 the credibility of the findings and facilitate replication and further investigation by other
researchers.
Moreover, it is important to emphasize that the developmental process of the human lung
at the embryonic stage is a dynamic one, characterized by intricate interplay between
different temporal expression patterns. To capture this complexity, the authors could
explain why a three-dimensional approach is necessary. By delving into the rationale
behind this methodological choice and providing examples of how it enhances our
understanding of the dynamic nature of lung development, the authors would strengthen
the overall argument and broaden the impact of their research.
Finally, the significance of the experiments conducted to validate the expression patterns
of the identified genes deserves further elucidation. The paper briefly touches upon the
experiments, but a more detailed discussion of their purpose, methodology, and
implications would enrich the reader's interpretation. By highlighting the significance of
these experiments and demonstrating how they contribute to our understanding of lung
development, the authors would underscore the broader implications of their findings and
inspire further research
in this field.
In conclusion, the paper would benefit from providing more comprehensive information
on various aspects, including the time frame of human lung development, the types of
experiments used for gene expression validation, the necessity of a three-dimensional
approach for capturing temporal patterns, and the significance of the experiments
conducted. Additionally, discussing any limitations or constraints that may have affected
the research and identifying potential avenues for future investigation would enhance the
clarity and scope of the topic, fostering scholarly discourse and promoting the
advancement of knowledge in the subject area.

(2) How can the author improve the clarity, succinctness, and overall quality of the
presentation of his/her paper?
● To enhance the clarity and quality of the paper, the author should provide clear and
detailed descriptions of the experiments and strategies employed, while removing
unnecessary details. A more concise focus on key points would improve the paper's
succinctness. Adding context to the introduction, incorporating figures and tables for
easier data interpretation, and including more citations to support claims and conclusions
would enhance the overall quality. The author should also explore the wider implications
of the study for disease diagnosis and treatment, consider adding an analysis and
discussion section, and include additional references to demonstrate their position in the
field and build upon previous research.
Improving the paper's intelligibility can be achieved by offering detailed explanations of
experiments, eliminating extraneous information, and emphasizing core themes. The
 introduction should be expanded to include relevant material, accompanied by figures,
tables, and citations to support arguments and conclusions. Addressing the study's
implications for disease diagnosis and treatment, incorporating an analysis and discussion
section, and providing additional references would further enhance the paper's
intelligibility.
To enhance the clarity, succinctness, and overall quality of the paper, the author should
provide a step-by-step explanation of the experimental techniques in a clear and concise
manner. Organizing the content using headings and subheadings would improve the
paper's structure, allowing readers to navigate and locate specific sections of interest.

(3) State your personal review (comments and/or recommendations) for the paper.

● The paper under review provides an extensive and comprehensive analysis of the
developmental program underlying proximal-distal patterning of the human lung at the
embryonic stage. Through the use of single-cell RNA sequencing, the authors offer novel
insights into the intricate gene expression patterns that drive the process of lung
morphogenesis. The presented data is well-structured, accompanied by clear
explanations, and serves as a valuable resource for researchers in the field of human lung
development. The depth and breadth of the analysis make this paper an excellent
contribution to the current body of research in this area.

The authors have successfully utilized single-cell RNA sequencing to unravel the
complex gene expression patterns that orchestrate the development of the human lung.
By examining gene expression at the single-cell level, they have been able to elucidate
the dynamic changes that occur during proximal-distal patterning, shedding light on the
molecular mechanisms underlying this critical process. This approach provides a
high-resolution view of the cellular heterogeneity and transcriptional dynamics during
lung development, opening up new avenues for further exploration.

The presented data is carefully organized and effectively communicated, allowing readers
to follow the authors' analysis with clarity. The paper includes detailed descriptions of
experimental methods, facilitating reproducibility and enabling other researchers to build
upon this work. The authors have also taken care to discuss the limitations of their study,
acknowledging the challenges and potential biases inherent in the experimental
techniques employed. This transparency strengthens the integrity of the research and
enhances the overall quality of the paper.

Moreover, the authors demonstrate a commendable attention to context by providing an

expanded introduction that sets the stage for the study. This contextual information aids
in understanding the significance of the findings within the broader field of human lung
development. However, further elaboration on the time frame of the development process
 and the specific types of cell differentiation studied would enrich the paper and provide
additional insights.

To improve the overall clarity and succinctness of the paper, the authors could consider
refining their descriptions of experimental methods and strategies. By focusing on the
key points and removing any unnecessary details, the paper could become more concise
without sacrificing the essential scientific information. Additionally, the inclusion of
relevant figures and tables would enhance the interpretation of the data, making it easier
for readers to grasp the presented findings.

In conclusion, this paper represents an excellent resource for researchers interested in the
developmental program of the human lung. The authors' utilization of single-cell RNA
sequencing to investigate gene expression patterns during proximal-distal patterning is
commendable, and the data presented is well-structured and clearly explained. By
addressing the suggested areas for improvement, such as providing further elaboration on
the time frame and types of cell differentiation studied, refining descriptions,
incorporating relevant figures and tables, adding citations, and including an analysis and
discussion section, the authors can enhance the overall quality, comprehensibility, and
impact of their paper.

Link of the Article:

Cao, S., Feng, H., Yi, H., Pan, M., Lin, L., Yao Santo Zhang, Feng, Z., Liang, W., Cai, B., Li, Q.,
Xiong, Z., Shen, Q., Ke, M., Zhao, X., Chen, H., He, Q., Min, M., Cai, Q., Liu, H., & Wang, J.
(2023). Single-cell RNA sequencing reveals the developmental program underlying
proximal–distal patterning of the human lung at the embryonic stage. 33(6), 421–433.
https://doi.org/10.1038/s41422-023-00802-6