Unformatted text preview: 1.The earliest study concerning liver induction was performed by Willer and Rawles (1931), who
observed that chick explants with two hears (cardia bifida) often developed two livers, but never
developed a second liver in the absence of cardia bifida.
A) With the benefit of hindsight, what was the major inductive molecule secreted by the hearts?
B) What embryonic tissue is stimulated by this molecule, and what is the earliest known marker of
-FGF4 induces ventral foregut endoderm directly adjacent to the cardiac mesoderm to become
hepatic endoderm. The transcription factor, Hhex, is the earliest known marker of the region.
2. Endoderm development contains examples of inductive signaling and permissive signaling.
Give an example of both using pancreatic development as an example.
-Inductive signaling: dorsal pancreatic endoderm is induced by notochord (without it, you do not
get Pdx1 expression; this is because it suppresses Shh in the endoderm in this region). Another
example: dorsal (and likely ventral) pancreas are induced by endothelial cells (the dorsal aorta in
the case of dorsal; likely the vitelline veins in the case of ventral).
-Permissive signaling: pancreatic buds require mesenchyme, as shown by the classic
experiments of Golosow and Grobstein. A strong line of evidence that this is permissive is that
pancreatic endoderm is specified well before the appearance of pancreatic mesoderm (before 14
somites vs. after 20 somites respectively in mice).
3. In 1968, Nicole Le Dourain showed that the septum transversum mesenchyme can be
substituted for a variety of different mesenchymal tissues and still promotes migration
A) What tissue was she referring to when she said 'migration'?
-The liver bud, which delaminates and undergoes an epithelial to mesenchymal transformation
into the mesenchyme
B) In the context of more modern experiments, what is a possible molecular explanation for her
-All mesenchyme will be vascularized and thus have this important proliferative signal for the
4. Compare and contrast the origin initial induction of the hepatic vs. pancreatic endoderm.
-The liver forms from three populations of ventral foregut: two lateral populations and one medial
population that come together when the foregut tube forms. The ventral pancreas also has two
medial populations, located caudally to the hepatic precursor regions, but no ventral region.
Unlike the liver, two distinct pancreas are induced (dorsal and ventral) that will eventually fuse.
The dorsal pancreas has a different origin, dorsal foregut endoderm.
5. Hhex mutant embryos have a proliferative defective in endoderm that retards their migration.
As a result, the most caudal endoderm is directly adjacent to the cardiac mesoderm. Explain why
this results in a lack of ventral pancreatic formation.
-Pancreatic endoderm is specified caudal to the hepatic endoderm (it must migrate past the
cardiac mesoderm, which specifies liver and inhibit pancreatic formation). In Hhex mutants, the
tissue simply isn't any further caudal at the time of liver induction. This results in a complete loss
of ventral pancreatic specification.
6. Lammert et al. (2001) showed that co-cultures of dorsal aorta + dorsal endoderm generated
pancreatic gene expression, including insulin-positive cells, while dorsal endoderm cultured alone
did not express pancreatic markers. Propose an experiment that would allow you to identify if the
inductive signal coming from the endothelium is secreted or if it requires direct cell-cell contact
(hint: think of the Grobstein experiments).
-Using their cell culture method, insert a permeable membrane between the endoderm and dorsal
aorta. If induction of pancreatic markers still occurs, it is a secreted signal.
7. What is the difference between a transgene expressing Cre, and one expressing Cre-ER?
-A standard Cre will be active from the moment the protein is translated. Cre is normally nuclear and will go into the nucleus, and act on LoxP sites. Cre-ER proteins are fusions with a modified
estrogen receptor that sequesters Cre in the cytoplasm. When the drug Tamoxifen is added, this
triggers the translocation of the protein to the nucleus, where it will activate recombination.
Tamoxifen is usually injected into the mom, and then diffuses into the embryo through the
placenta. This is a way of adding temporal specificity to Cre (useful when timing is critical, and to
look at what is the cell fate of a Cre-expressing population over time).
8. What do you think would happen if you expressed Gremlin specifically within the interdigit
mesenchyme of mice?
-You would inhibit BMPs, therefore no apoptosis, and there would be webbing between digits
9. Why is a lungfish considered to have a fin that is somewhat similar to a tetrapod when
compared with currently living fish?
-Lungish have a single bone that connects the fin to the body. This is an ancestral stylopod.
10. What is the major difference between Tiktaalik and Eusthenopteron limbs?
-Tiktaalik is the earliest fossil related to the tetrapod lineage that has digit-like appendages.
Eusthenopteron has stylopod and zeugopodal-like bones, but lacks digit-like appendages.
11. Compare a bat wing with a bird wing. A) Is this considered to be homologous or convergent
evolutionary trait? B) What about a bat wing and a horse's limb?
-A) Convergent evolution (similar structures that developed independently during evolution); B)
Homolgous (structures that evolved from a commmon ancestor).
12. The following image contains part of a figure discussed in lecture and part of a figure that we
did not discuss. In this experiment, Carboxypeptidase A regulatory sequences were fused to
CreER to generate CPA::CreER transgenic animals. These were crossed to Rosa::LacZ reporter
mice, and Tamoxifen was administered to pregnant mice at E10.5 and E13.5. Mice were
Developmental Cell at E18.5, and pancreatic tissues were s tained as indicated below. Based on these
nt Progenitor Domain of the PancreasMultipotent Progenitor Domain of images, what do you conclude about the ability of CPA -positive cells to act as a
representative the Pancreas
progenitor population over time?
Endo, Endocrine cells, Amy,
Amylase, a mature exocrine
marker; DBA, a pancreatic duct
marker, DAPI, marks nuclei of all
cells, Bgal, beta-galactosidase
(product of LacZ). -At E11, CPA-positive cells give rise to all three populations of the pancreas (exocrine,
endocrine, duct). At E14, CPA-positive cells do not give rise to endocrine or duct cells.
They only give rise to exocrine cells (amylase positive).
pa1+ Cells Are Multipotent before E14 Figure 5. Cpa1+ Cells Are Multipotent before E14 +
Cpa1CreERT2;R26R stages. were labeled with one dose E18.5. different embryonic
;R26R embryos were labeled with one dose of TM at different embryonicembryosPancreata were harvested atof TM atCpa1+ cells pulsed stages. Pancreata were harvested at E18.5. Cpa1 cells pulsed
at E13–14, respectively) gave rise to endocrine (A–C, arrows), exocrine (E–G, hollow
.5, and E12.5 (labeling occurs at E11–12, E12–13, and E10.5, E11.5, and E12.5 (labeling occurs at E11–12, E12–13, and E13–14, respectively) gave rise to endocrine (A–C, arrows), exocrine (E–G, hollow
arrowheads), and duct (E–G, white arrowheads) progeny. In only exocrine (H, hollow
, and duct (E–G, white arrowheads) progeny. In contrast, Cpa1+ cells pulsed at E13.5 (labeling E14–15) generate contrast, Cpa1 cells pulsed at E13.5 (labeling E14–15) generate only exocrine (H, hollow
arrowheads), but not endocrine (D) or duct (H) cells. Endo, and pancreatic polypep, but not endocrine (D) or duct (H) cells. Endo, four major endocrine hormones (insulin, glucagon, somatostatin,four major endocrine hormones (insulin, glucagon, somatostatin, and pancreatic polypeptide) were simultaneously recognized with a mixture of antibodies; Amy, Amylase,
multaneously recognized with a mixture of antibodies; Amy, Amylase, a mature exocrine marker; DBA, a pancreatic duct marker. (I–L)a mature exocrine marker; DBA, a pancreatic duct marker. (I–L)
Summary of the lineage exocrine; D, duct.
the lineage of Cpa1+ cells at different embryonic stages. En, endocrine; Ex,of Cpa1 cells at different embryonic stages. En, endocrine; Ex, exocrine; D, duct. 2 three
late endocrine progenitors, as well as endo+bgal+ mature
essive days for analysis (Figure 6 and Figures successive days forprogenitors, as welland endo+bgal+ mature
late endocrine analysis (Figure 6 as Figures
day after progenies, the detected (Figures
day after TM injection, the initial population of One endocrineTM injection,wereinitial population of 6I andendocrine progenies, were detected (Figures 6I and 6L).
TM-responsive cells expresses that Cpa1+ cells appear to self-renew data suggest that Cpa1+ cells appear to self-renew
sive cells expresses both bgal and Cpa1 as exThese data suggest both bgal and Cpa1 as ex+
ure 6A, arrows). Consistent with the labeling of (Figure 6A, arrows). Consistent with the labeling of
(making more Cpa1+ cells) and lay down daughters(making more Cpa1 cells) and lay down daughters that
go through a series of differentiation steps to generate
an early multipotent progenitor cell differentiation steps to generate
go through a series of type, these cells were
ultipotent progenitor cell type, these cells were
mature pancreatic cells in vivo (Figure 6M).
mature pancreatic cells in vivo (Figure 6M).
r the early endocrine marker, Ngn3 (Figurenegative for the early endocrine marker, Ngn3 (Figure 6D),
docrine marker, Pax6 (Figure 6G), and all endo- endocrine marker, Pax6 (Figure 6G), and all endoDISCUSSION
ones (Figure 6J). Starting from 2 days after la- hormones (Figure 6J). Starting from 2 days after laÀ
observed that Cpa1À bgal+ cells appear in the we observed that Cpa1 bgal cells appear in the
Formation of the mammalian pancreas as a complex
n of the branches (Figures 6B and 6C, arrow- region of the branches (mammalian and 6C, arrow-a complex
Formation of the Figures 6B pancreas as
three-dimensional organ requires timely generation, mitips
ddition to Cpa1+bgal+ cells residing in theheads) in addition to Cpa1 bgal cells residing in thegeneration, mithree-dimensional organ requires timely tips
(Figures 6B and 6C, arrows). This observation, togethertypes gration, and differentiation of different cell types from
B and 6C, arrows). This observation, together
gration, and differentiation of different cell
multipotent progenitors. We performed genome-wide TF
ta that most Cpa1+ cells are multipotent beforethe data that most progenitors.are multipotent before
multipotent Cpa1 cells We performed genome-wide TF
5 and Figures S4), of the developing Cpa1
e 5 and Figures S4), suggests that some Cpa1+(Figureexpression analysissuggests that somepancreas andexpression analysis of the developing pancreas and disdisare capable of limited self-renewal (i.e., producing more
le of limited self-renewal (i.e., producing more
covered multiple distinct gene expression domainscovered multiple distinct gene expression domains that
indicate the presence of speciﬁc pancreatic progenitor
ltipotent tip cells as well as Cpa1À differentiated multipotent tip cells as well as Cpa1 differentiated progenitor
indicate the presence of speciﬁc pancreatic
progenies).domains. Using a combination of serial immunoﬂuoresdomains. Using a combination of serial immunoﬂuores).
Starting from E14, some progeny of Cpa1+ cells labeled we cence and genetic lineage tracing experiments, we profrom E14, some progeny of Cpa1+ cells labeled
cence and genetic lineage tracing experiments,
in to express Ngn3 (Figures 6E and 6F, arrow- begin to express Ngn3 (Figures 6E and pancreatic progenitor that one type of multipotent pancreatic progenitor
pose that one type of multipotent 6F, arrowheads), indicating that they have become committed markers be recognized by a combination of markers
dicating that they have become committed
can be recognized by a combination of
+ + + + High + ...
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