Hematopoiesis 1st Edition Emery H. Bresnick (Eds.

)
Updated 2025

https://ebookfinal.com/download/hematopoiesis-1st-edition-emery-h-
bresnick-eds/

★★★★★
4.7 out of 5.0 (52 reviews )

Instant PDF Download

ebookfinal.com
 Hematopoiesis 1st Edition Emery H. Bresnick (Eds.) Pdf
Download

EBOOK

Available Formats

■ PDF eBook Study Guide Ebook

EXCLUSIVE 2025 EDUCATIONAL COLLECTION - LIMITED TIME

INSTANT DOWNLOAD VIEW LIBRARY

We have selected some products that you may be interested in
Click the link to download now or visit ebookfinal.com
for more options!.

Vagabond Vol 29 29 Inoue

https://ebookfinal.com/download/vagabond-vol-29-29-inoue/

Shakespeare s Friends Kate Emery Pogue

https://ebookfinal.com/download/shakespeare-s-friends-kate-emery-
pogue/

Arduino Projects to Save the World 1st Edition Emery

Premeaux

https://ebookfinal.com/download/arduino-projects-to-save-the-
world-1st-edition-emery-premeaux/

Geographers Biobibliographical Studies Volume 29 1st

Edition Hayden Lorimer

https://ebookfinal.com/download/geographers-biobibliographical-
studies-volume-29-1st-edition-hayden-lorimer/
Advances in Photochemistry Volume 29 1st Edition Douglas
C. Neckers

https://ebookfinal.com/download/advances-in-photochemistry-
volume-29-1st-edition-douglas-c-neckers/

Batik Gems 29 Dazzling Quilt Projects 1st Edition Laurie

J. Shifrin

https://ebookfinal.com/download/batik-gems-29-dazzling-quilt-
projects-1st-edition-laurie-j-shifrin/

Leading for Organisational Change Building Purpose

Motivation and Belonging Jennifer Emery

https://ebookfinal.com/download/leading-for-organisational-change-
building-purpose-motivation-and-belonging-jennifer-emery/

Studies in Symbolic Interaction Vol 29 1st Edition Norman

K. Denzin (Ed.)

https://ebookfinal.com/download/studies-in-symbolic-interaction-
vol-29-1st-edition-norman-k-denzin-ed/

Renegotiating Family Relationships Divorce Child Custody

and Mediation 2nd Edition Robert E. Emery

https://ebookfinal.com/download/renegotiating-family-relationships-
divorce-child-custody-and-mediation-2nd-edition-robert-e-emery/
Hematopoiesis 1st Edition Emery H. Bresnick (Eds.)
Digital Instant Download
Author(s): Emery H. Bresnick (Eds.)
ISBN(s): 9780128033265, 0128033266
Edition: 1
File Details: PDF, 7.02 MB
Year: 2016
Language: english
 CURRENT TOPICS IN
DEVELOPMENTAL BIOLOGY
“A meeting-ground for critical review and discussion of developmental processes”
A.A. Moscona and Alberto Monroy (Volume 1, 1966)

SERIES EDITOR
Paul M. Wassarman
Department of Developmental and Regenerative Biology
Icahn School of Medicine at Mount Sinai
New York, NY, USA

CURRENT ADVISORY BOARD

Blanche Capel Susan Mango
Wolfgang Driever Philippe Soriano
Denis Duboule Cliff Tabin
Anne Ephrussi Magdalena Zernicka-Goetz

FOUNDING EDITORS
A.A. Moscona and Alberto Monroy

FOUNDING ADVISORY BOARD

Vincent G. Allfrey Dame Honor B. Fell
Jean Brachet John C. Kendrew
Seymour S. Cohen S. Spiegelman
Bernard D. Davis Hewson W. Swift
James D. Ebert E.N. Willmer
Mac V. Edds, Jr. Etienne Wolff
Academic Press is an imprint of Elsevier
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA
525 B Street, Suite 1800, San Diego, CA 92101-4495, USA
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
125 London Wall, London, EC2Y 5AS, UK
First edition 2016
Copyright © 2016 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher. Details on how to seek
permission, further information about the Publisher’s permissions policies and our
arrangements with organizations such as the Copyright Clearance Center and the Copyright
Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by
the Publisher (other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional practices,
or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described
herein. In using such information or methods they should be mindful of their own safety and
the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of
products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions, or ideas contained in the material herein.

ISBN: 978-0-12-803319-7
ISSN: 0070-2153

For information on all Academic Press publications

visit our website at https://www.elsevier.com/

Publisher and Acquisition Editor: Zoe Kruze

Editorial Project Manager: Sarah Lay
Production Project Manager: Radhakrishnan Lakshmanan
Cover Designer: Mark Rogers
Typeset by SPi Global, India
CONTRIBUTORS

E.H. Bresnick
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
K. Choi
Washington University, School of Medicine, St. Louis, MO, United States
E. de Pater
Erasmus MC, Rotterdam, The Netherlands
A.W. DeVilbiss
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
E. Dzierzak
Erasmus MC, Rotterdam, The Netherlands; MRC Centre for Inflammation Research
and MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh,
United Kingdom
X. Gao
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
M.A. Goodell
Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX,
United States
K.J. Hewitt
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
T. Hoang
Laboratory of Hematopoiesis and Leukemia, Institute of Research in Immunology and
Cancer (IRIC), University of Montreal, Montreal, QC, Canada
M. Jeong
Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX,
United States
K.D. Johnson
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
K.R. Katsumura
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
S. Keles
University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

ix
x Contributors

J.A. Lambert
Laboratory of Hematopoiesis and Leukemia, Institute of Research in Immunology and
Cancer (IRIC), University of Montreal, Montreal, QC, Canada
M.W. Maijenburg
University of Pennsylvania, Philadelphia, PA, United States
R. Martin
Laboratory of Hematopoiesis and Leukemia, Institute of Research in Immunology and
Cancer (IRIC), University of Montreal, Montreal, QC, Canada
S.C. McIver
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
D.T. Scadden
Massachusetts General Hospital, Boston; Harvard Stem Cell Institute; Harvard University,
Cambridge, MA, United States
N.A. Speck
University of Pennsylvania, Philadelphia, PA, United States
S. Sumanas
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States
N. Tanimura
UW-Carbone Cancer Center, University of Wisconsin School of Medicine and Public
Health; UW-Madison Blood Research Program, Madison, WI, United States
J. Tober
University of Pennsylvania, Philadelphia, PA, United States
V.W.C. Yu
Massachusetts General Hospital, Boston; Harvard Stem Cell Institute; Harvard University,
Cambridge, MA, United States
PREFACE

The process of developing the diverse blood cell repertoire from stem and
progenitor cells termed hematopoiesis has been subject to considerable
investigation. However, key steps in the complex process of hematopoiesis,
including hematopoietic stem cell generation during embryogenesis, hema-
topoietic stem, and progenitor cell expansion to accommodate physiological
and pathological requirements, and mechanisms that ensure hematopoietic
stem and progenitor cell phenotypic integrity remain incompletely under-
stood. Elucidating these mechanisms will yield concepts that can be extrap-
olated to diverse biological realms, and given the vital importance of the
hematopoietic system, future efforts will invariably lead to medical break-
throughs that advance human health.
This issue of Current Topics in Developmental Biology reviews cutting-edge
research on mechanistic and biological aspects of hematopoiesis. The initial
chapters lay the groundwork for understanding the cellular origin of
hematopoietic stem cells (Dzierzak and de Pater) and the pivotal role of
the microenvironment or “niche” in non-cell-autonomously regulating
hematopoietic stem cell genesis and function (Yu and Scadden). The next
series of chapters probe into molecular mechanisms governing the develop-
ment of hematopoietic stem and progenitor cells and for maintaining their
unique phenotypes. Whereas a plethora of studies have used loss-of-function
approaches to delineate proteins with activities essential for hematopoiesis,
Hewitt et al. describe a new strategy aimed at discovering noncoding DNA
sequences or cis-regulatory elements with nonredundant activities to control
hematopoiesis. Editing specific cis-regulatory elements out of the mouse
genome disrupts specific steps of hematopoiesis, yields instructive animal
models, and provides unique systems for decoding the global cis-regulatory
element network governing hematopoietic stem/progenitor cell transitions
termed a “Hematopoietic Stem/Progenitor Cell Cistrome.” Upstream of
GATA-2, the generation and identity of endothelial cells with the unique
capacity to form hematopoietic stem cells (hemogenic endothelial cells)
requires an intriguing ETS transcription factor termed ETV2/ER71/Etsrp.
Sumanas and Choi describe foundational studies on this critical protein, pro-
vide a unique perspective on how ETV2 functionally interfaces with a
cohort of other ETS factors, and highlight the promise of forging ETV2-
dependent regenerative medicine strategies. Having emerged from studies

xi
xii Preface

on mechanisms underlying leukemogenesis, the transcription factor Runx1,

previously deemed Acute Myeloid Leukemia protein 1, has multiple crucial
roles in the hematopoietic hierarchy. Tober et al. describe its activities to
control hematopoietic stem and progenitor cell transitions and its relation-
ship to a host of other components implicated in specific steps in hemato-
poiesis. A huge challenge is to assemble and decipher the complex protein
networks governing hematopoiesis, and the basic helix-loop-helix protein
Stem Cell Leukemia (SCL)/T-cell Acute Lymphocytic Leukemia Protein
1 (TAL1) is integral to such networks. SCL/TAL1 commonly colocalizes
with GATA factors on chromatin and functions at multiple levels of the
hematopoietic hierarchy. Hoang et al. provide a comprehensive perspective
of how SCL/TAL1 controls normal hematopoiesis and is deregulated
in leukemia. Moreover, they highlight how the mechanistic insights have
illuminated new avenues to reprogram cellular phenotypes. Transcription
factors commonly engage non-DNA binding coregulators that mediate
activation or repression through enzymatic or nonenzymatic biochemical
mechanisms. In certain cases, the same coregulator mediates activation
and repression, often through elusive context-dependent mechanisms.
DeVilbiss et al. describe concepts related to how GATA-1 negotiates core-
gulator ensembles to control the development and function of specific blood
cell types. They highlight the concept of a “coregulator matrix,” which, in
effect, constitutes a code for how a given transcription factor functions in a
specific environment—a code that exhibits fluidity dictated by the overall
regulatory milieu. Progress on the role of noncoding regulatory RNAs in
biological regulation has exploded in recent years, and this area is ripe for
making discoveries to understand and modulate hematopoiesis. Jeong and
Goodell provide an insightful overview of noncoding RNAs implicated
in hematopoiesis and discuss prospective mechanisms. Noncoding RNA-
dependent mechanisms are expected to function at all levels of the hemato-
poietic hierarchy and to converge upon the mechanisms involving ETV2,
GATA-2, RUNX1, SCL/TAL1, and GATA-1 discussed in this volume.
For the most part, integrating noncoding regulatory RNA mechanisms with
the emerging factor-dependent pathways remains virgin territory.
In aggregate, this series of chapters highlights compelling mechanistic
and biological advances, and the progress to date has emphasized the need
to increase the depth of investigation into these problems—to get beyond
limitations of dissecting complex mechanisms in cell populations, rather
than in individual cells, to develop definitive insights as to whether murine
mechanisms can be seamlessly extrapolated to humans, and to devise novel
Preface xiii

strategies for dissecting mechanisms within microenvironments impacted by

diverse non-cell-autonomous regulatory inputs. It will be particularly
instructive to determine how the respective mechanisms react to dynamic
alterations in the microenvironment and systemic changes associated
with pathologies and aging. The resulting knowledge will yield a veritable
Pandora’s box of scholarly insights that fill mechanistic/biological voids
and transform existing concepts. Future mechanistic leveraging will yield
desperately needed new strategies for treating nonmalignant (eg, anemia)
and malignant hematopoietic disorders (eg, leukemia) and has potential
to yield economically viable pipelines for engineering therapeutic blood
products.
EMERY H. BRESNICK
 CHAPTER ONE

Regulation of Blood Stem Cell

Development
E. Dzierzak*,†,1, E. de Pater*
*Erasmus MC, Rotterdam, The Netherlands
†
MRC Centre for Inflammation Research and MRC Centre for Regenerative Medicine, University of
Edinburgh, Edinburgh, United Kingdom
1
Corresponding author: e-mail address: [email protected]

Contents
1. Introduction 2
1.1 HSC Generation 5
1.2 Tracking Hematopoietic Cell Generation from Embryonic Endothelium 6
1.3 Regulation of Hemogenic Endothelium 8
1.4 Hemogenic Transcriptional Program 10
1.5 Reprogramming 12
References 15

Abstract
Understanding how the blood system is formed is an ongoing fundamental research
challenge. Developmental biology has provided many insights into the molecules
and processes that affect the formation of the blood tissues, both in health and disease.
It is of particular interest for clinical transplantation therapies to understand how hema-
topoietic stem cells (HSCs)—the self-renewing purveyors of the adult blood system that
produce over 10 different functionally specialized cell lineages and over 1011 cells
daily—are generated during embryonic stages. Recent successes to reprogram the fate
of adult differentiated cells to pluripotency and to other cell lineages now highlight the
importance of identifying the cells and molecules that affect the in vivo developmental
initiation of rare and robust transplantable HSCs. The close association of the developing
hematopoietic and vascular system, hematopoietic cell mobility through the circulation,
and the essential role of the embryonic hematopoietic system in adult hematopoietic
cell development make this a formidable study. This chapter reviews the advances, con-
troversies, and current state of our knowledge of the growing field of hematopoietic
development, with a special focus on the regulation of the natural transdifferentiation
of endothelial cells to HSCs within the developing embryo.

Current Topics in Developmental Biology, Volume 118 # 2016 Elsevier Inc. 1

ISSN 0070-2153 All rights reserved.
http://dx.doi.org/10.1016/bs.ctdb.2016.01.001
2 E. Dzierzak and E. de Pater

1. INTRODUCTION
The dramatic and unexpected demonstration by Yamanaka and col-
leagues that fully differentiated skin cells could be reprogrammed to
pluripotency using pivotal transcription factors changed a major paradigm
in mammalian developmental biology—that of the fixed cell identity
(Takahashi & Yamanaka, 2006). Since this major breakthrough, many
researchers have been able to reprogram differentiated cells to lineage-
specific stem or progenitor cells (reviewed by Ieda, 2013). Additional studies
with induced pluripotent stem (iPS) cells or embryonic stem (ES) cells
demonstrate controlled differentiation toward several adult lineages, such as
neuronal, muscle, pancreatic cells, and even hematopoietic cells of the lym-
phoid, myeloid, and erythroid lineages (reviewed in Ohnuki & Takahashi,
2015). However, to program or reprogram cells toward a hematopoietic stem
cell (HSC) has been extremely difficult and daunting challenge.
The gold-standard functional definition of a robust HSC is one that is
based on its potential for long-lasting, high-level, multilineage hematopoi-
etic repopulation in cell transplantation therapies. Clinical transplantation of
HSC-containing adult bone marrow cells was the first successful “cell
therapy” in humans. Beginning in the 1950s, this approach is currently part
of the therapy used internationally for the treatment of leukemias and severe
hematopoietic deficiencies (Takizawa, Schanz, et al., 2011). The potent
adult HSC is the only cell type capable of sustained self-renewal and produc-
tion of all types of hematopoietic cells (Yamamoto, Morita, et al., 2013).
To understand the biology of HSCs and the principles behind the
growth of HSCs and repopulation following transplantation, the mouse
has served as an excellent model. Inbred mouse strains, genetic markers,
immunophenotyping, and cell sorting, as well as the ease of genetic manip-
ulation make this the ideal model to study the biology and development of
HSCs. Experimental transplantations of cells into adult murine recipients
(irradiated so as to myeloablate the existing hematopoietic system) has
broadened our knowledge of HSCs and what constitutes their supportive
microenvironment (Copelan, 2006). In the adult, HSCs are maintained
in specialized niches of the bone marrow and also are found at lower fre-
quencies in the spleen and peripheral blood. Other vertebrate animal models
such as chick, Xenopus, and zebrafish have also been instrumental in bringing
us to our current knowledge of HSC development (Dzierzak & Speck,
2008; Orkin & Zon, 2008).
Blood Development 3

Already before the generation of the HSCs that reside in the bone mar-
row of the adult, the embryo requires blood cells for oxygenation, metab-
olite distribution, and tissue remodeling. This primitive embryonic blood is
generated in a wave of hematopoietic cell commitment occurring in meso-
dermal precursors migrating to the early yolk sac (reviewed in Palis, Malik,
et al., 2010). Cells with vascular endothelial and hematopoietic (and other)
potential (generally referred to as hemangioblasts) contribute to the yolk sac
blood islands and the production of primitive red blood cells, the first visible
hematopoietic cells in the mouse and human embryos. This primitive wave
of hematopoietic generation is not long-lived, although the primitive mac-
rophages it produces form the tissue resident macrophages of the adult
(reviewed in Davies & Taylor, 2015; Frame, McGrath, et al., 2013). More
complex hematopoietic cells are produced in definitive waves. Erythroid-
myeloid progenitors (EMP) are the first definitive multilineage progenitors
and are produced in the yolk sac (McGrath, Frame, et al., 2015). Thereafter,
more complex progenitors are generated in the yolk sac, aorta-gonad-meso-
nephros (AGM) region (Fig. 1A) and major vasculature of the embryo, the
placenta, and the vascularized regions of the embryonic head. These cells
have potential for the lymphoid lineages in addition to erythro-myeloid lin-
eages (Cumano, Dieterlen-Lievre, et al., 1996). Finally, at midgestation in
the mouse and at the first trimester of human development, the first trans-
plantable HSCs that will sustain the life-long hematopoiesis in the adult, are
generated (Bertrand, Giroux, et al., 2005; de Bruijn, Speck, et al., 2000;
Gekas, Dieterlen-Lievre, et al., 2005; Ivanovs, Rybtsov, et al., 2011;
Li, Lan, et al., 2012; Medvinsky & Dzierzak, 1996; Medvinsky, Taoudi,
et al., 2008; Ottersbach & Dzierzak, 2005; Tavian, Robin, et al., 2001).
The in vivo waves of primitive and definitive progenitor generation that
developmentally precede the HSC generating wave complicate in vitro stud-
ies, such as those that attempt to generate HSCs from ES/iPS cells and other
cell types. With the exception of primitive erythrocytes, the majority of the
differentiated blood cells produced in the early waves are similar to those in
the adult. However, they do not originate from HSCs, and as such will not
contribute in the long-term to the self-renewable adult blood system. Also
greatly complicating studies to produce HSCs in culture is the lack of an
exclusive HSC marker that enables recognition of these cells when they
are produced, and the fact that to date, HSCs from any source cannot be
cultured efficiently in vitro. The de novo generation of HSCs occurs in the
embryo during a very restricted window of time from embryonic day
E10.5 to E12.5 in the mouse (Zovein, Hofmann, et al., 2008) and gestational
4 E. Dzierzak and E. de Pater

Fig. 1 The AGM, hematopoietic clusters and the process of endothelial-to-hematopoi-

etic transition (EHT). (A) Graphical representation of EHT in the mouse. An E10.5 mouse
embryo is depicted on the left where the square indicates the level of the cross-section
shown in the middle panel. The boxed area indicates the ventral side of the dorsal aorta.
The right panel shows where hemogenic endothelial cells in purple give rise to clusters
of hematopoietic cells and HSCs indicated in pink. These shed into the lumen of the
dorsal aorta and eventually populate the bone marrow. (B) and (C) z projection of a con-
focal image of (B) a wild-type (WT) mouse embryo and (C) a Gata2f/f:Vec-Cre embryo.
Endothelial cells are stained for CD31 (red) and hematopoietic cluster cells are stained
for c-Kit (green). Note the size of WT clusters in (B) and the flat c-Kit positive cluster cells
in (C), indicating that not only the number of clusters is severely reduced upon loss of
Gata2 but also the process of EHT is affected. (D) Graphical representation of EHT in
zebrafish. A schematic diagram of a Tg(Fli:GFP) zebrafish embryo (top panel) where
the dorsal aorta (DA) and the posterior cardinal vein (PCV) are marked in green in a
embryo. The bottom panel shows the boxed area where an endothelial cell bulges out-
ward, rounds up to close the gap in the DA and sheds as a hematopoietic cell into the
abluminal space between the DA and the PCV. (E) Single z scans of time-lapse confocal
imaging of the process of EHT in vivo using a Tg(Fli:GFP) embryo. The DA and PCV are
indicated in the first frame and the arrow indicates the cell that is undergoing EHT in
each frame. The timing is indicated in the bottom left corner of each frame and the entire
process of EHT is completed after 4 h. (F) Schematic representation of a cell undergoing
EHT after knockdown of Runx1 in the zebrafish embryo. EHT is greatly reduced, but the
occasional cell undergoing EHT lyses halfway through the process. E, embryonic day;
DA, dorsal aorta; AGM, aorta-gonad-mesonephros region; HC, hematopoietic cell;
HEC, hemogenic endothelium; HSC, hematopoietic stem cell; PCV, posterior cardinal
vein.
Blood Development 5

week 4–10 in the human (Ivanovs et al., 2011; Tavian et al., 2001). There-
fore to understand which regulators are important for reprogramming and
differentiating cells to HSCs, we first need to understand how HSCs are gen-
erated during embryonic development.

1.1 HSC Generation

The AGM is the first site of HSC generation in the mouse (Fig. 1A),
identified through transplantations of dissected embryonic tissues into adult
irradiated recipient mice (Medvinsky & Dzierzak, 1996; Muller, Medvinsky,
et al., 1994). More refined dissections localized HSCs to the major vascula-
ture—aorta, vitelline, and umbilical arteries at mouse embryonic day E10.5
(de Bruijn et al., 2000; North, Gu, et al., 1999), and more specifically to the
ventral wall of the dorsal aorta (de Bruijn, Ma, et al., 2002; Taoudi &
Medvinsky, 2007). Beginning at E11, HSCs are also found in other
tissues—the yolk sac, placenta, and most recently the embryonic head
(Gekas et al., 2005; Li et al., 2012; Medvinsky et al., 2008; Muller et al.,
1994; Ottersbach & Dzierzak, 2005). Enrichment for HSCs in the AGM
and these other tissues using antibodies (or other markers) that define the adult
BM and/or fetal liver (FL) HSC in flow cytometric sorting, showed that long-
term repopulating AGM HSCs are c-Kit+CD34+ and Ly6a (Sca1)+, but not
CD150+ (de Bruijn et al., 2002; Ma, Robin, et al., 2002; McKinney-
Freeman, Cahan, et al., 2012; Sanchez, Holmes, et al., 1996; Wood, May,
et al., 1997). Further enrichment of AGM HSCs includes the use of hemato-
poietic cell markers CD41lo, CD45, and Mac1 and markers typically defining
endothelial cells, such as VE-cadherin, Flk1, CD31, and Tie2 (Li et al., 2012;
Newman, Berndt, et al., 1990; Robin, Ottersbach, et al., 2011; Rybtsov,
Sobiesiak, et al., 2011; Sanchez et al., 1996; Taoudi, Gonneau, et al.,
2008; Yokomizo & Dzierzak, 2010). Knockin/knockout (North, de
Bruijn, et al., 2002; North et al., 1999) and transgenic strategies (Ly6a
(Sca1) GFP) (de Bruijn et al., 2002; Ma et al., 2002) identified other markers
of HSCs in the AGM and in other tissues (Ottersbach & Dzierzak, 2005).
Immunostaining of embryo sections of the AGM region localized the
expressing cells to the hematopoietic cluster cells and/or the endothelial
cells lining the aorta (Fig. 1B). In situ hybridization studies examining hema-
topoietic transcription factor expression and transcription factor reporter
knockin or transgenic reporter mice showed expression of Runx1, SCL,
Gata2, Gata3, Notch, and other hematopoietic transcription factors in
the cells of the AGM (de Bruijn et al., 2002; Fitch, Kimber, et al., 2012;
6 E. Dzierzak and E. de Pater

Marks-Bluth, Khanna, et al., 2015; North et al., 2002, 1999; Porcher, Swat,
et al., 1996; Robert-Moreno, Espinosa, et al., 2005). Again, the expressing
cells were localized in the hematopoietic cluster cells closely associated with
aortic endothelium and/or within the ventral endothelial cell layer. Indeed,
all amniote embryos examined to date have clusters of round hematopoietic-
like cells associated with the aorta at the critical time in development when
definitive hematopoiesis begins (reviewed in Jaffredo, Nottingham, et al.,
2005). The developmental acquisition (c-Kit, CD45) and loss of other
markers (Flk1), and the localization of the cells within the endothelium
and the clusters gave further support for a fate change and differentiation
to the hematopoietic lineage (Yokomizo & Dzierzak, 2010). These evi-
dence add up to suggest that in the embryo, the vascular endothelial cells
lining the major arteries possess hemogenic potential and generate hemato-
poietic progenitor and stem cells.

1.2 Tracking Hematopoietic Cell Generation from Embryonic

Endothelium
The first experimental evidence linking hematopoietic cells in the clusters
lining the dorsal aorta with aortic endothelial cells came from grafting
and dye/retroviral marking studies in chick embryos ( Jaffredo, Gautier,
et al., 1998, 2000; Pardanaud & Dieterlen-Lievre, 1993). Until this time,
microscopic observations over a period of about 100 years reported the close
physical association of aortic hematopoietic cluster and endothelial cells
(reviewed in Jaffredo et al., 2005). Jaffredo and colleagues were able to trace
origins of the emergent hematopoietic cluster cells by the presence of
markers that were used to exclusively label endothelial cells lining the aorta
at a developmental time prior to hematopoietic cell appearance.
In the mouse, genetic lineage tracing studies with Cre:Lox recombina-
tion markers established the relationship between vascular endothelial cells
(vascular endothelial cadherin (VEC)+) with emerging hematopoietic cluster
cells. Stable and temporally controlled recombination marking in VEC-Cre:
Rosa lox stop reporter and VEC-Cre ERT:Rosa lox stop reporter mice, respec-
tively, showed that the adult hematopoietic system and HSC arise from
endothelial cells and that the cells with hemogenic potential are present
in the embryonic vasculature from about E8 to E11 (Chen, Yokomizo,
et al., 2009; Zovein et al., 2008). Moreover, when pivotal transcription fac-
tors such as Runx1 or Gata2 were deleted in VEC expressing embryonic cells
(Fig. 1B), no vascular hematopoietic clusters and no HSCs were detected
Blood Development 7

(Chen et al., 2009; de Pater, Kaimakis, et al., 2013; Lim, Hosoya, et al.,
2012; Ruiz-Herguido, Guiu, et al., 2012). Similar genetic marking studies,
using another endothelial gene, Tie2 as the Cre driver for reporter recom-
bination, confirmed the precursor–progeny relationship of embryonic vas-
cular endothelial cells with emerging hematopoietic cells (Liakhovitskaia,
Gribi, et al., 2009; Schlaeger, Mikkola, et al., 2005; Tang, Harrington,
et al., 2010). Additional support was provided from transgenic reporter
mouse strategies. Enhancer elements that drive expression of transcription
factors such as Runx1 (+23) in definitive HPSCs (hematopoietic stem and
progenitor cells), facilitated not only the isolation of HPSC but also hemo-
genic endothelial cells that can be shown in OP9 cocultures to generate
hematopoietic cells (Swiers, Baumann, et al., 2013).
Confocal microscopy and live-imaging facilitated the strongest proof of
endothelial-to-hematopoietic transition (EHT). The physiologic evidence
that EHT occurs in vivo in the major vasculature of the mouse embryo at
the time of HSC generation was provided by Boisset and colleagues
(Boisset, van Cappellen, et al., 2010). The time-lapse imaging, performed
through thick sections of the E10.5 mouse AGM, visualized
Ly6aGFP+CD34+ aortic hemogenic endothelial cells transitioning to
Ly6aGFP+CD34+ hematopoietic cells that reside alone/in clusters on the
luminal wall of the aorta. These cells coexpress additional markers, like
c-Kit, that characterize functional AGM HSCs. Quantitation of EHT events
revealed only two emerging hematopoietic cells per aorta, corresponding to
the number of transplantable AGM HSCs at this developmental time point
(Taylor, Taoudi, et al., 2010), thus indicating (along with/and verifying
genetic data) that endothelial-to-HSC transition occurs in vivo. The process
of EHT was also captured on video in zebrafish embryos (Bertrand, Chi,
et al., 2010; Kissa & Herbomel, 2010). The zebrafish embryo shows round
hematopoietic cells emerging from aortic endothelial cells in an abluminal
direction, rather than in a luminal direction as in the mouse embryo
(Fig. 1D and E). The emerged cells then migrate and enter into the circu-
lation of the posterior cardinal vein. This was reported previously (Kissa,
Murayama, et al., 2008) and counterintuitive at the time because in mouse
and chick, clear luminal clusters were identified and it was assumed that the
process would be similar in zebrafish. As in the mouse embryo, knockdown
of Runx1 severely affects the process of EHT (Fig. 1F). Taken together,
these data confirm that the generation of hematopoietic progenitors and
stem cells from endothelial cells is a normal in vivo physiologic process.
8 E. Dzierzak and E. de Pater

The latest refinement in understanding the birth of HSCs has been the
identification of several populations of pre-HSCs and precursors. These
studies showed that populations of endothelial cells harvested at E9.5 (before
the generation of the first HSCs in mouse) are expressing VEC and CD41lo,
but not CD45 and CD43. Under specialized conditions in which AGM cells
are reaggregated and cultured for several days, they transition to CD43+ and
CD45+ HSCs that yield high-level hematopoietic chimerism following
transplantation (Rybtsov, Batsivari, et al., 2014; Rybtsov et al., 2011;
Taoudi et al., 2008). This in vitro system holds promise to reveal some of
the relevant cell–cell interactions and molecular regulators of EHT.

1.3 Regulation of Hemogenic Endothelium

How are the specialized hemogenic endothelial cells that undergo EHT
formed? What makes them different from other endothelial cells? Why does
the ventral wall of the aorta in most species form hematopoietic clusters,
while the dorsal wall does not? These issues are of great interest and several
explanations are plausible, including differences in positional information
that instructs the fate of the endothelial cells; differing mesodermal origins
of the endothelial cells; and/or the effects of neighboring cells.
In chick embryos, both positional information and mesodermal origins
play a role. Factors such as EGF and TGFa that emanate from dorsal tissues
inhibit, whereas ventral tissue-emanating factors BMP4, FGF, VEGF, and
TGFb1 promote hemogenic activity (Pardanaud & Dieterlen-Lievre,
1999). Chick embryo grafting experiments also reveal that different types
of mesoderm (lateral plate and somitic) contribute to the ventral and dorsal
walls, respectively (Pardanaud & Dieterlen-Lievre, 1993), and this was
recently found in the zebrafish embryo (Pouget, Peterkin, et al., 2014).
In addition, at the time of fusion of the paired dorsal aortae, the ventral
endothelial cells express Runx1 which marks the hemogenic endothelium.
Upon hematopoietic differentiation, Notch signaling is downregulated and
hematopoiesis is enhanced (Richard, Drevon, et al., 2013). As development
proceeds, somitic cells begin to contribute to the ventral wall.
In the zebrafish embryo, the stage at which the divergence of endothelial
and hemogenic endothelial cell fate occurs is as yet unknown. In the poste-
rior lateral plate mesoderm, it is unclear whether endothelial- and hemo-
genic endothelial-cell fates are diverged yet, but several signaling
molecules are implicated in the specification of hemogenic endothelial cells.
In the lateral plate mesoderm, FGF signaling provided by the somite is
required for the start of the hemogenic program in cells which can be later
Blood Development 9

defined as hemogenic endothelial cells. Also Notch3 function in the somite

is non-cell autonomously required for HSC specification (downstream of
wnt16) (Kim, Melick, et al., 2014; Kobayashi, Kobayashi-Sun, et al.,
2014; Lee, Manegold, et al., 2014). In Xenopus, hemogenic endothelial cells
require short-range VEGFa, also provided by the somite (Leung, Ciau-Uitz,
et al., 2013). From experiments in zebrafish (and chick) we know that
hemogenic endothelial cells (and endothelial cells) migrate underneath
the somite to the midline where they receive multiple signaling molecules.
Hemogenic endothelial cells receive and require long-range VEGFa and
Notch signaling. This is facilitated through junctional adhesion molecule
1a (Jam1a) in hemogenic endothelial cells interacting with Jam2a in the
somite. This interaction facilitates the movement underneath the somite
and allows the Notch signaling to take place (also received from the somite)
(Kobayashi et al., 2014). Notch signaling is required to start the hemogenic
program in endothelial cells. Once the dorsal aorta is formed, a ventral BMP
signal further enhances the hemogenic program together with hedgehog
(HH) signaling. All HPSCs require BMP and HH signaling at this time
of their generation, and FGF signaling from the somite functions to restrict
the BMP signal and the initiation of the hematopoietic program to the ven-
tral cells of the dorsal aorta (Gering & Patient, 2005; Pouget et al., 2014;
Wilkinson, Pouget, et al., 2009). The cells undergoing EHT are then shed
into the area between the dorsal aorta and the posterior cardinal vein. For
EHT to be accomplished, Notch signaling needs to be downregulated in
hemogenic endothelial cells through activation of Gpr183 (Zhang, He,
et al., 2015).
In the mouse embryo it is as yet unclear whether there are diverse meso-
dermal origins of aortic endothelial cells. However, it is thought that hemo-
genic fate is determined as early as E8 ( Jaffredo et al., 2005; North et al.,
2002). Not all endothelial cells of the dorsal aorta are capable of generating
HSCs, and endothelial cells and hemogenic endothelial cells are intermixed
in the dorsal aorta, as suggested by Ly6a-GFP expression at E10.5 (de Bruijn
et al., 2002; Solaimani Kartalaei, Yamada-Inagawa, et al., 2015). Hemogenic
endothelial cells are fully functioning endothelial cells and are defined by a
different genetic program which sets them apart from endothelial cells
(Solaimani Kartalaei et al., 2015; Swiers et al., 2013). Positional information
is key to hemogenic and hematopoietic fate. Before the onset of HSC gen-
eration in the AGM, a coculture of this tissue with ventral gut tissue is capa-
ble of inducing HSC generation in the AGM region, while culture with
dorsal tissue inhibits the generation of HSCs even after the onset of HSC
10 E. Dzierzak and E. de Pater

generation (Peeters, Ottersbach, et al., 2009). BMP4 is a ventrally expressed

factor and plays a role in HSC generation in the mouse AGM (Durand,
Robin, et al., 2007). Only some endothelial cells and some hematopoietic
cluster cells are BMP-activated (Crisan, Kartalaei, et al., 2015). Since all
AGM HSCs are ventrally localized (Taoudi & Medvinsky, 2007), and all
HSCs at the time of their in vivo generation are BMP-activated (Crisan
et al., 2015), the ventral positional BMP4 signaling factor likely plays an
important role in hemogenic endothelial cells and their specification toward
HSCs. Notch1 and Jagged1 are also expressed in some ventral endothelial
cells in the aorta (Guiu, Bergen, et al., 2014; Robert-Moreno et al.,
2005; Robert-Moreno, Guiu, et al., 2008). Notch signaling is required
for both endothelial and hematopoietic development, but only the Notch
ligand Jagged1 is required for HSC generation. Also in mouse, Notch sig-
naling is downregulated upon the formation of hematopoietic cells, but this
is more transient (Richard et al., 2013) and Notch is again required for fetal
hematopoiesis (Gerhardt, Pajcini, et al., 2014). Unlike BMP4, HH expres-
sion is not ventrally restricted. HH affects HSC generation in AGM explants
most likely through its effects on niche cells (Peeters et al., 2009).

1.4 Hemogenic Transcriptional Program

Extrinsic molecular regulators (BMP, HH, and others) induce the hemo-
genic transcriptional program. The intrinsic hemogenic molecular program
is hallmarked by the expression of pivotal transcription factors (Solaimani
Kartalaei et al., 2015; Swiers et al., 2013), the “heptad” factors. The heptad
transcription factor complex comprised of Gata2, Runx1, Scl/Tal1, Lmo2,
Fli1, Erg, and Lyl1 (Wilson, Foster, et al., 2010) was identified by global
screening of the regulatory elements of known hematopoietic genes in a
hematopoietic progenitor cell line, HPC7. Most such hematopoietic gene
enhancers contain consensus binding sites for, and were shown to bind,
the heptad complex of factors. Hence, these factors are thought to work
in harmony in hematopoietic progenitors and stem cells. Since all heptad
factors are found to be upregulated during EHT, and the genetic deletion
of each factor individually reveals pivotal roles in EHT and HSC generation,
they are likely to act in combinatorial manner to promote EHT and hema-
topoietic progenitor/stem cell development.
Several of the heptad transcription factors belong to the Ets family of
transcription factors (Fli1, Scl/Tal1, Erg, and Lyl1). The Ets family of factors
have overlapping transcriptional targets, but due to differential expression
Blood Development 11

they regulate distinct processes in HSC generation (Dooley, Davidson, et al.,

2005; Patterson, Gering, et al., 2005; Robb, Drinkwater, et al., 1995;
Shivdasani, Mayer, et al., 1995). Scl/Tal1 and Fli1 are important for endo-
thelial formation (Bussmann, Bos, et al., 2010; Spyropoulos, Pharr, et al.,
2000; Visvader, Fujiwara, et al., 1998), and Erg and Lyl1 play an important
role in HSC maintenance (Capron, Lecluse, et al., 2006; Taoudi, Bee, et al.,
2011). Scl and Fli1 start the hemogenic program and regulate expression of
Lmo2, Gata2, and Runx1, where the program is further enhanced by Gata2
(Gao, Johnson, et al., 2013; Landry, Kinston, et al., 2008; Taoudi et al.,
2011; Zhu, Traver, et al., 2005).
Several independent studies showing the transcriptional activation and
complex formation between the individual members of the heptad support
the combinatorial function of the heptad factors (Gering & Patient, 2005;
Pimanda, Ottersbach, et al., 2007; Wilson, Foster, et al., 2010). The indi-
vidual roles of each heptad factor in HSC generation and maintenance
are beginning to be revealed through similar conditional knockout
approaches. For example, Scl and Lyl1 are highly homologous Ets transcrip-
tion factors with the same major downstream gene targets, but Scl and Lyl1
knockouts show very different phenotypes that can only be partially
explained by their expression patterns. In embryonic development, Scl
expression starts earlier than Lyl1 expression. However, the overexpression
of Lyl1 cannot rescue the hematopoietic differentiation defect seen in Scl /
ESC (Chan, Follows, et al., 2007). In contrast, Scl and Lyl1 have overlapping
functions in HSC maintenance. Lyl1 can rescue loss of Scl, thus explaining
the mild defect in Scl / HSCs (Souroullas, Salmon, et al., 2009). Also Erg
and Fli1 have a dual role in the HSC and megakaryocytic lineage, since dou-
ble heterozygous mice have a much more severe phenotype than the single
heterozygous mutant mice (Kruse, Loughran, et al., 2009).
Gata2 and Runx1 are the most widely studied transcription factors in
hematopoietic development. Gata2 expression precedes Runx1 expression
in hematopoietic cell development. Mouse germline knockouts of Gata2
die at E10.5, just before the onset of HSC generation (Tsai, Keller, et al.,
1994). These Gata2 / embryos die with FL anemia and Gata2 / ES cells
cannot contribute to definitive hematopoiesis in chimeric embryos/mice.
Furthermore, in E10.5 Gata2+/ mouse embryos there is a 90% decrease
in HSCs (Ling, Ottersbach, et al., 2004). This led to the conclusion that
Gata2 is required for HSC generation. Since Gata2 expression persists after
generation of HSCs from hemogenic endothelium, it was postulated that it
should function thereafter. A conditional knockout approach showed that
12 E. Dzierzak and E. de Pater

Gata2 functions not only in the generation of HSCs from hemogenic endo-
thelium but also after the generation of HSC for their maintenance (de Pater
et al., 2013; Gao et al., 2013). The importance of Gata2 was also shown in
the zebrafish model, where gata2b plays an important role in EHT (Butko,
Distel, et al., 2015).
Mouse germline knockouts of Runx1 die at E12.5 with FL anemia and
vascular hemorrhaging (Mukouyama, Chiba, et al., 2000; Okuda, van
Deursen, et al., 1996). Runx1 expression in the E10.5 embryo is localized
to the dorsal aorta, and Runx1 expression is used as a marker for hemogenic
endothelial cells and HSCs (North et al., 2002; Swiers et al., 2013). All AGM
HSCs express Runx1 and Runx1+/ AGMs contain only 50% of the wild-
type numbers of HSCs. Runx1 expression persists after the generation of
HSCs (Chen et al., 2009). However, unlike Gata2, the Runx1 requirement
is much more restricted to the generation of HSCs, although the exact
timing of the Runx1 requirement is still under debate (Liakhovitskaia,
Rybtsov, et al., 2014; Tober, Yzaguirre, et al., 2013). Also in zebrafish,
Runx1 is required for EHT. Upon Runx1 knockdown, EHT is very rare
and the cells which try to undergo EHT eventually lyse (Fig. 1F; Kissa &
Herbomel, 2010).
The difference in the phenotypes in the conditional knockout studies of
Runx1, Gata2, and the other factors shows that we do not fully understand
the role of each of the heptad factors in HSC generation, collectively and
individually, and leaves much to explore.

1.5 Reprogramming
The reprogramming of cells toward an HSC fate would open new doors to
future regenerative therapy strategies and provide insight into the regulatory
mechanisms of HSC generation and even leukemia. Findings in the field of
developmental biology of HSC generation have been the guide to repro-
gram cells toward an HSC fate. Two main strategies have been used to
obtain HSCs in vitro. The first uses extrinsic factors to differentiate ES cells
toward a hematopoietic fate, while the second uses the overexpression of
transcription factors to (de)differentiate cells to an HSC.
With the first strategy (ES cell differentiation; Fig. 2), the stepwise addi-
tion of a variety of factors induces stages of ES cell differentiation leading to
primitive streak, mesoderm, hemangioblast/hemogenic endothelium, and
then hematopoietic cells. Some of these factors include Nodal/Activin,
Wnt, and BMP (Irion, Clarke, et al., 2010; Nostro, Cheng, et al., 2008;
Pearson, Sroczynska, et al., 2008; Sturgeon, Ditadi, et al., 2014). BMP is
Blood Development 13

Fig. 2 Different reprogramming strategies in an effort to obtain HSCs. Two strategies

are used. The first approach differentiates ES cells or iPS cells toward a hematopoietic
progenitor and/or HSC fate via a hemogenic endothelial intermediate step through the
use of a combination of growth and developmental factors. The second approach uses a
reprogramming strategy by overexpressing combinations of transcription factors in
starting nonhematopoietic cells such as fibroblasts or hematopoietic cells such as B-cell
progenitors.

important for induction of Flk1+ cells, which are believed to serve as

hemangioblasts/hemogenic endothelial cells and is a necessary step in gen-
erating definitive hematopoietic cells in vivo. Under the influence of Wnt, a
definitive hematopoietic progenitor is formed, whereas by blocking Wnt a
primitive progenitor is formed (Sturgeon et al., 2014). Others have used
Activin and FGF to induce hemangioblast formation and Vegf to induce
committed hematopoietic precursors (Pearson et al., 2008). These studies
were unsuccessful in generating (detectable) repopulating HSCs, suggesting
that an additional factor is required for HSC generation.
With the second strategy, the reprogramming or de-differentiation
approach utilizes transcription factor transduction/expression to induce an
HSC fate in nonhematopoietic or differentiated hematopoietic cells, respec-
tively (Fig. 2). Mouse fibroblasts, when transfected with multiple transcrip-
tion factor genes—Gata2, Gfi1b, Fos, and Etv6 (Pereira, Chang, et al.,
2013)—and cultured for 35 d, were reprogrammed and found to express
a hemogenic transcriptional program. Following a further culture of
20 d, definitive hematopoietic progenitors, but no HSCs, were found.
A similar strategy with human EB-derived CD34+CD45+ progenitors
transfected with HOXA9, ERG, RORA, SOX4, and MYB resulted in
short-term repopulating progenitors (Doulatov, Vo, et al., 2013). Endothe-
lial cells, purified from human umbilical cord blood and subsequently trans-
fected with FOSB, GFI1, RUNX1, and SPI1 (and cultured up to 40 d)
Exploring the Variety of Random
Documents with Different Content
 Engineering - Exam Preparation
Fall 2025 - Center

Prepared by: Prof. Garcia

Date: July 28, 2025

References 1: Current trends and future directions

Learning Objective 1: Literature review and discussion
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Interdisciplinary approaches
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Case studies and real-world applications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 4: Learning outcomes and objectives
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Learning Objective 5: Fundamental concepts and principles
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Problem-solving strategies and techniques
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 6: Experimental procedures and results
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 7: Diagram/Chart/Graph]
Remember: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Key terms and definitions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 9: Ethical considerations and implications
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Quiz 2: Experimental procedures and results
Key Concept: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 11: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Key terms and definitions
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Key Concept: Literature review and discussion
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Note: Interdisciplinary approaches
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Ethical considerations and implications
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 16: Case studies and real-world applications
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Historical development and evolution
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Assessment criteria and rubrics
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 19: Case studies and real-world applications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 20: Diagram/Chart/Graph]
Methodology 3: Ethical considerations and implications
Practice Problem 20: Statistical analysis and interpretation
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 21: Theoretical framework and methodology
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Current trends and future directions
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Research findings and conclusions
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Experimental procedures and results
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Comparative analysis and synthesis
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Historical development and evolution
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 27: Study tips and learning strategies
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Ethical considerations and implications
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Study tips and learning strategies
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Module 4: Key terms and definitions
Key Concept: Problem-solving strategies and techniques
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 31: Interdisciplinary approaches
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Current trends and future directions
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 33: Diagram/Chart/Graph]
Definition: Study tips and learning strategies
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 34: Current trends and future directions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 35: Learning outcomes and objectives
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Case studies and real-world applications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Historical development and evolution
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Theoretical framework and methodology
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Current trends and future directions
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Module 5: Experimental procedures and results
Definition: Study tips and learning strategies
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Key terms and definitions
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Best practices and recommendations
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 43: Interdisciplinary approaches
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 44: Historical development and evolution
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Problem-solving strategies and techniques
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Case studies and real-world applications
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Problem-solving strategies and techniques
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 48: Statistical analysis and interpretation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Current trends and future directions
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 50: Diagram/Chart/Graph]
Conclusion 6: Research findings and conclusions
Note: Best practices and recommendations
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 51: Diagram/Chart/Graph]
Definition: Case studies and real-world applications
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Assessment criteria and rubrics
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Assessment criteria and rubrics
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Ethical considerations and implications
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 55: Learning outcomes and objectives
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Statistical analysis and interpretation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 57: Experimental procedures and results
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 58: Diagram/Chart/Graph]
Definition: Ethical considerations and implications
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 59: Current trends and future directions
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Appendix 7: Theoretical framework and methodology
Example 60: Literature review and discussion
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Key terms and definitions
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Case studies and real-world applications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Statistical analysis and interpretation
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Problem-solving strategies and techniques
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 65: Diagram/Chart/Graph]
Key Concept: Critical analysis and evaluation
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Learning outcomes and objectives
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 67: Historical development and evolution
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Theoretical framework and methodology
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Learning outcomes and objectives
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Results 8: Study tips and learning strategies
Definition: Study tips and learning strategies
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Critical analysis and evaluation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Learning outcomes and objectives
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Case studies and real-world applications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Key terms and definitions
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Literature review and discussion
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 77: Assessment criteria and rubrics
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 78: Historical development and evolution
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 80: Diagram/Chart/Graph]
Part 9: Critical analysis and evaluation
Practice Problem 80: Research findings and conclusions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 81: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Ethical considerations and implications
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 83: Problem-solving strategies and techniques
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Ethical considerations and implications
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 85: Best practices and recommendations
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 86: Diagram/Chart/Graph]
Definition: Theoretical framework and methodology
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Practical applications and examples
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Research findings and conclusions
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Problem-solving strategies and techniques
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Background 10: Research findings and conclusions
Key Concept: Comparative analysis and synthesis
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Literature review and discussion
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Literature review and discussion
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 93: Case studies and real-world applications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 94: Diagram/Chart/Graph]
Practice Problem 94: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Experimental procedures and results
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 96: Learning outcomes and objectives
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 97: Diagram/Chart/Graph]
Remember: Current trends and future directions
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 98: Diagram/Chart/Graph]
Important: Experimental procedures and results
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Example 99: Interdisciplinary approaches
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Methodology 11: Critical analysis and evaluation
Key Concept: Experimental procedures and results
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Experimental procedures and results
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Theoretical framework and methodology
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 103: Diagram/Chart/Graph]
Key Concept: Literature review and discussion
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Critical analysis and evaluation
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Statistical analysis and interpretation
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Critical analysis and evaluation
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 107: Diagram/Chart/Graph]
Definition: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Comparative analysis and synthesis
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 109: Diagram/Chart/Graph]
Practice Problem 109: Study tips and learning strategies
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Results 12: Key terms and definitions
Remember: Research findings and conclusions
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 111: Research findings and conclusions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Interdisciplinary approaches
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Current trends and future directions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 114: Practical applications and examples
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Problem-solving strategies and techniques
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Interdisciplinary approaches
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Ethical considerations and implications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 118: Diagram/Chart/Graph]
Example 118: Literature review and discussion
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 119: Diagram/Chart/Graph]
Practice Problem 119: Learning outcomes and objectives
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Summary 13: Problem-solving strategies and techniques
Definition: Historical development and evolution
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 121: Diagram/Chart/Graph]
Important: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Literature review and discussion
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 123: Diagram/Chart/Graph]
Remember: Fundamental concepts and principles
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Learning outcomes and objectives
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Learning outcomes and objectives
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Research findings and conclusions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 128: Diagram/Chart/Graph]
Note: Critical analysis and evaluation
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 129: Diagram/Chart/Graph]
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

ebookfinal.com