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Joonhwan Jang

on 16 April 2013

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Figure 6 (D-L) Fgf9 and Wnt4 Act as Antagonisitic signals to regulate Mammalian Sex Determination JJ Jang
Stefanie Cisek Background Sertoli cells - (#7 as indicated in picture)
Nurse cells of the testis activated by FSH SRY gene - Sex-determining Region Y Figure 1 (A-F) Figure 1 (A-F) Diverge before 12.5 dpc Similar pattern and detection of Fgf9 in both XX/XY Figure 3 (G-J) Figure 1 (G-H) Figure 2 (A-D) Figure 2 (E-H) Figure 2 (I-K) Figure 3 (A-F) Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, et al. (2006). Published in PLos Biol 4(6):e187. What We Know Figure 4 (A-F) Founding Signals Initially Expressed in Both Gonads: coordinate growth, morphogenesis, differentiation Figure 4 (G-H) Fgf9: Figure 4 (I-K) fibroblast growth factor; becomes male specific after the activation of Sry null mutation causes sex reversal (male-->female) Wnt4: Figure 5 (A-C) wingless-related MMTV integration site; becomes female specific Figure 5 (D-G) null mutation causes partial sex reversal (evidence of testicular differentiation) Relationship between signals?? Figure 6 (A-C) Authors' Purpose To investigate the relationships between Sry, Sox9, Fgf9, and Wnt4 Figure (K-O) Author's Hypothesis Fgf9 functions through a feed-forward loop to the critical threshold sufficient in repressing Wnt4 --> male fate In a reciprocal manner, Wnt4 represses Sox9 and Fgf9 The sexual fate of the gonad is controlled by mutually antagonistic signals between Fgf9 and Wnt4 Figure 7 (A-D) Review of Part 1 Questions Method: antibody specific to FGF9 protein (red)
A,C,E: XX gonad
B,D,F: XY gonad Review Part 2 Questions Part 1
Figure (1-3) Part 2
Figures 4-7 Methods G&I: alkaline phosphatase staining
H&J: PECAM (green)
: Fgf9 expression (red)
J: arrowhead = testis cords formation fsss Methods A&B: EGFP bound to Sry promoter (arrowheads)
C&D: Myc-tagged Sry expression (red, arrowheads)
: Syto13 counterstain (green), nucleic acid binding Sry expression is not dependent on Fgf9
.:. Fgf9 must act in parallel and/or downstream Is Fgf9 an upstream regulator of Sry? Is Fgf9 expression within testis cords localized to Sertoli cells (excluded from germ cells)? What is the normal Fgf9 expression in normal gonads XX/XY? Is Fgf9 involved in Sox9 expression? Methods:
F,H: addition of exogenous FGF9 F: up-regulation of SOX9 Testing the local effect of FGF9 Methods: stable immobilization using beads to gonad surface
B: coated with BSA, control
F: coated with FGF9 .:.Positive interaction between Fgf9 and Sox9 Is Fgf9 required to maintain Sox9 expression? Methods: A-F: immunostaining SOX9 (red)
E: decrease in SOX9 expression
F: all expression lost .:.Fgf9 not required for SOX9 activation but required for maintenance of expression organization of Sertoli precursor cells lack of organization Investigating linear relationship:
Sry--> Sox9--> Fgf9? Methods: Crossing mice with homozygous flox (null) allele with mice carrying Cre transgene G&H: similar Sry mRNA detection
I&J: significant decrease in Fgf9 mRNA .:. Sox9 must act between Sry and Fgf9 Sox9 is essential for Fgf9 expression. Fgf9 maintains Sox9 expression = +feed-forward loop! Are other Sertoli Precursors affected? A&B: Dhh expression absent C&D: reduction of Amh expression
E&F: severe reduction of Amh expression .:. Not all Sertoli pathways are initiated Is the initial expression of Sox9 (which disappears) due to cell death? Methods: G&H: active caspase-3, apoptotic cell marker (red) No apoptosis observed in region of interest .:.There must be a different cause of decreased Sox9 expression... Is it due to disruption of FGF9/SOX9
feed-forward regulation? REMEMBER: Wnt 4 is an ovary marker .:. Because Wnt4 was up-regulated in K,
Fgf9 must be necessary for its down-regulation
in differentiating XY gonads Can Fgf9 down-regulate Wnt4? Method: A-C: cultured XX gonad with/without exogenous Fgf9
Comparing B&C: inhibition of Wnt4 by Fgf9 .:. Fgf9 is responsible for down-regulating Wnt4 Does a reduction in Wnt4 make XX gonads
more susceptible to Fgf9 "effects"? Methods: D-G: immunostaining of cultured XX gonads : Sox9 expression (red) .:.Antagonism between Fgf9 and Wnt4 under gain-of-function conditions
(in vitro) What about under loss-of-function conditions
(in vivo)? Can Fgf9 be derepressed? Methods: A-C: used antibody specific for Fgf9 (red)
C: absence of Wnt4 --> Fgf9 expression .:. Fgf9 must normally be down-regulated by Wnt4 in the XX pathway We know Fgf9 is a positive regulator of Sox9 and is derepressed in XX gonads with the absence of Wnt4. So will there be expression of Sox9 here? Methods: D-I: antibody against Sox9 (red)
D, G, J: XX, no Sox9 detection
E,H,K: normal XY expression and
F,I,L: initial Sox9 expression then down-
I&L: arrowheads- coelomic vessel

Coelomic vessel: develops on XY gonad, usually not present in XX gonads! .:.Sox9 is up-regulated in XX gonads (where Sry was not activated) through the up-regulation of Fgf9 made possible by inhibiting the antagonistic effect of Wnt4 Background Bipotential embryonic gonad-->two divergent pathways (testis and ovaries)
- underlying mechanisms conserved among species? Pathways 1- Ovary: the basic "default" pathway
2- Testis: diverts from ovary pathway by influencing the fate of the key supporting cell lineage and initiating a male-specific morphogenetic program - in its absence --> ovarian pathway
- expression leads to Sertoli cell
differentiation (as opposed to follicles)
- critical threshold to fully establish
testis differentiation Sox9 gene - male-specific up-regulation in Sertoli cell precursors - cell-autonomous activation by Sry
- expression --> testis development Future Implications Studies to quantify actual critical threshold needed
for the diverging pathways
How many cells or % of cells need to be affected in
order to commit to a single pathway? cells near surface of gonads
cells in testis cords FGF9 expression Unpublished: No signal was detected in double mutants XY Note that Fgf9 expression is higher near Sertoli cells
because their precursors secrete Fgf9! Sox9 is essential for Fgf9 expression ==> Fgf9 expression maintains Sox9 expression.

FEED-FORWARD LOOP What is Kit? 1J -mice with no germ cells and consequently no gametes Why Kit? - embryos contain agametic gonads so we are able to determine if testis cord formation can occur without germ cells Alkaline phosphatase staining If you were running an experiment to see the difference
in Fgf9 or Sox9 expression(XX vs XY), which timing would be best? A. 9 DPC
B. 10 DPC
C. 11 DPC
D. 12 DPC Stains undifferentiated pluripotent
stem cells

Used in G&I to compare normal XX
gonad and Kit gonad Exogenous FGF9 injected into a 12 DPC embryo would (XY gonad):

A. Change nothing in the cell
B. Repress WNT4
C. Repress Sox9
D. B and C
E. None of the above. Fgf9 is ___________ SOX9 expression in
XY gonads

A. unnecessary for initial
B. capable of down regulating
C. necessary for maintaining
D. A and C
E. None of the Above Is cell proliferation reduced with the loss of Sox9? Methods: PECAM - is used for demonstrating the presence of endothelial cells in tissue sections. K-N: used phosphorylated histone H3 (red)
O : counted positive cells in cortical region of each gonad and normalized to
XY heterozygous mutant .:. Mutual interdependence, positive feed-forward loop between Fgf9 and Sox9
Fgf9 transcripts are found near gonad surface
Wnt4 transcripts found near gonad mesonephric boundary A loss of either sox9 or fgf9
would decrease the chances of
the cell undergoing the
testis pathway. Fgf9 is initially expressed in both gonads but becomes male specific after the expression of SRY in male "directional" cells fgf9 is not required for initial sox9 levels but it is necessary for its maintenance Look at this pathway's conservation in other species; Flies, Rats, Monkeys, etc. Look at the specific biochemical interaction/ mechanistic pathway between Wnt4 and Fgf9
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