Specific aim 1.

Previously I showed that Cdx4 acts as an activator in inducing Pax6 transcription as

overexpression of chimeric VP16Cdx4 is able to recapitulate the effect of Cdx4
overexpression and also Cdx4 caudal activation domain is required for Cdx4 activity.
The interaction is probably direct as overexpression of Cdx4 with crucial DNA
binding mutation lack the activity (Fig1. D).
I further showed that while Cdx4 acitvates Pax6 it inhibits a crucial downstream
target of Pax6, neurogenic factor Ngn2. The regulation of Ngn2 by Cdx4 and Pax6 is
probably stochastic as Cdx4 inhibits Pax6 dependent Ngn2 activation and Pax6 can
overcome this repression at higher concentration. Last time I showed this data
using in situ hybridization of Ngn2 mRNA probe. The committee suggested me to
repeat the experiment using Ngn2 antibody. Hence I have repeated the experiment
and analyzed it using Ngn2 antibody and have got similar results (Fig. 2)
I also investigated if Cdx4 regulates Notch signaling pathway in regulating
neurogenesis. For analysis used Hes5 as a marker for proliferation and Dll1 as
marker of differentiation. My data suggest that Cdx4 does indeed regulate Notch
signaling pathway although that regulation is probably indirect and depends on
Pax6 regulation (Fig. 3).
To investigate if Cdx4 also regulates pluripotency I analyzed the expression of
pluripotency cell markers Cash4 and Sax1 on manipulating Cdx4 activity. My data
suggest that Cdx4 inhibits Cash4 (data not shown) and Sax1 (Fig. 4) expression.
Since overexpression of VP16Cdx4 also inhibits Sax1 expression, it suggests that
the regulation is also indirect.
I also wanted to study the interaction between the genes involved in regulating
onset neurogenesis, specifically the role of pluripotency markers, Cash4 and Sax1,
in inhibiting differentiation and any feedback by Pax6 on the Cdx4 and Sax1. To
achieve this I overexpressed Cash4. My data suggest that while Cash4 is expressed
in the pluripotent cells it however doesnt repress Pax6 activation in those cells.
Cash4 also seems to have a positive interaction with Sax1 as ectopic expression of
Cash4 activated Sax1 expression. I didnt find any effect of Caash4 on Cdx4
regulation (data not shown). I am still in process of deciphering the role of Sax1 on
Cash4, Cdx4 and Pax6 regulation. Unfortunately I am not able to clone the chicken
Sax1 even after various attempts, hence I have decided to overexpress the mouse
version of the factor to see what can be the putative effect of Sax1.
With respect to role of Pax6 in prompting differentiation , my data suggest that
while Pax6 doesnt inhibit Sax1 expression, Pax6 dependent mechanism does
indeed restrict Sax1 antierior limit of expression. Pax6 also inhibits Cdx4 although
indirectly (Fig 6).

Altogether, my data from overexpression studies of Cdx4, Cash4 and Pax6 have
helped me generate a Gene Regulatory Network that might be involved in
regulating onset of neurogenesis in chicken spinal cord. (Fig 7)
Specific aim 2:
I hypothesized that Cdx4 directly regulates Pax6 transcription by binding to a
conserved intronic sequence in Pax6 first intron. To investigate this aim, I cloned the
conserved intronic segment from chicken Pax6 into a luciferase reporter construct. I
performed my luciferase assays in Neuro2a cells which have been previously used
by a research group to investigate role of Cdx2 in directly regulating Pax3. However
my analysis didnt result in expected data.
To address this concern, I am repeating these experiments in chicken as has been
done by other groups.
Specfic aim 3.