CAPÍTULO 2. ANALISIS, DISEÑO Y ELABORACIÓN DEL PROTOTIPO
2.4. C ONSTRUCCIÓN DEL PROTOTIPO
2.4.1. Diseño del Segundo Sprint
The results presented in this thesis indicate that SoxS activity is essential for normal mouse embryogenesis. Chimaeras generated with Sox3 disrupted or null alleles show retarded growth and die mid-way through gestation. The morphology and results from molecular marker analysis reveal defects in the developing nervous system, in agreement with this being the main site o f Sox3 expression. However, the most dramatic effects are seen in mesoderm. Although mesoderm derivatives are specified, certain tissues fail to become properly organised. This includes the condensation of pre-somitic mesoderm to somites and the fusion o f the allantois with the chorion. It is the failure o f the latter which results in embryonic lethality.
As mentioned in Chapter 5, a number o f mutations have been described which lead to phenotypes similar to those seen in chimaeras made with mutant Sox3 ES cells. These mutations are in genes that encode for a variety o f proteins, including extra cellular matrix molecules, cell adhesion molecules and proteins involved with intracellular signal transduction. All these classes o f molecules may be involved in the signal pathway from extra-cellular growth factors to gene activation. Antibodies to many o f these proteins are available and it will be o f interest to see if the level or distribution o f any o f these molecules is affected by the Sox3 mutations. There are at least three ways this could be done: First, to generate more chimaeras and section them in utero for analysis by immunohistochemistry. The level o f chimerism could easily be demonstrated if a mouse line that ubiquitously expresses LacZ (such as ROSA 26, Zambrowicz et a l., 1997) was used as a source o f host blastocysts.
Following staining for B-galactosidase activity, cells derived from the host embryo would stain blue, but tissue derived from the mutant ES cells would not.
Secondly, a similar approach o f immunohistochemistry could be applied to sections o f teratocarcinomas. The advantage with this approach is the investigation o f a tumour that is solely derived from the mutant Sox3 ES cells. Finally, mutant ES cells can be differentiated in vitro and fibroblasts etc immortalised and used to investigate whether the mutation alters cell adhesion or migration properties (Fassler et a l.,
1995).
A more general approach, aimed to uncover downstream targets o f Sox3, is to differentiate Sox3^ hemizygous and wild type ES cells and to generate a cD NA library from each cell type. Subtracting common cD NA clones o f the mutant library from the wild type library will lead to enrichment o f clones that are more frequently expressed in wild type cells. These cloned cDNAs will include potential downstream targets o f Sox3. Such an experiment is planned in collaboration with Marco Bianchi and lab. in Milan.
The phenotype o f Sox3~ was far from expected for the following reasons: 1) Sox3 is not expressed in mesodermal derivatives. 2) mutations in Soxl and Sox4 suggest that there may be redundancy within the Sox gene family. 3) the phenotype in men lacking S 0X 3 is much less severe. Reasons for the first two anomalies have already being discussed in Chapter 5. Reasons why the phenotype in humans is less severe include the possibility that mouse and human S0X 3 have different roles in
embryogenesis or perhaps in human embryos another SOX gene product is capable o f compensating for loss of S0X 3. Of the two closest members of this sub-family, SOXl and S0X 2, the former protein has higher sequence similarity to S0X 3. So if there was redundancy in protein function, perhaps it is only between SOXl and S 0X 3. If this were the case, human SOXl would have to be expressed in human embryogenesis earlier than its mouse counterpart, to provide for the role acquired by mouse Sox3 in early post-implantation development.
Other mutations designed to recapitulate human diseases have been shown not to recreate the human phenotype (reviewed in Erickson, 1996). Reasons for these discrepancies have been attributed to the differences between the biochemistry and development of mice and humans, as well as the genetic background of the mouse which will influence penetrance. In the chimaera experiments, the only way to change the genetic background would be to perform the targeting event in ES cells from other mouse strains. Perhaps the easiest way to investigate any background effects on the mutation will be to create a floxed allele o f Sox3 in ES cells and pass this mutation through the germline. Then homozygous
(9) or hemizygous (c3)
mice can be crossed onto a wide variety of genetic backgrounds and when the resulting progeny are crossed to a Cre recombinase transgenic mouse a null mutation will be formed in the Sox3^^^‘^ embryos.Such a conditional null mutation of Sox3 would also be invaluable for in studying the loss of gene function later in embryogenesis. Crossing Sox3^^°^^^‘ mice to transgenic mice expressing Cre under a suitable promoter could address the question of the role
o f Sox3 in neurogenesis in vivo. mice could be crossed to Soxl
heterozygous mice to investigate if the loss o f Sox3 in the neuroepithelium exacerbates the Soxl -/- phenotype. It is known from expression studies that these
Sox genes seem to be marking a pluripotent lineage in the developing nervous system, but the_g^stion of whether this sub-family o f Sox genes are essential for the developl^ent of the CNS is yet to be ascertained. Experiments to mis-express Soxl
in differentiating P19 cells appears to increase the number of neurons seen in the culture (Pevny et al., 1997). This could be interpreted as increasing the potential of these cells to become neuronal or maintaining / causing proliferation of the neural precursors. Whether Sox2 and Sox3 expression will causes analogous results in the
in vitro assay has yet to be investigated.
As discussed in Chapter 3 experiments are planned to see if Sox genes are involved in the regulation o f certain Hox genes. Recent work with a Drosophila Sox gene has suggested other targets for regulation by the Sox genes in the mouse (Russell & Ashburner, 1997). The Dichaete {D) mutation in Drosophila, has been shown to be caused by ectopic expression o f soxVOD. This mis-expression in the developing wing hinge corresponds to a down regulation o f wingless (wg) in the same cells (Russell & Ashburner, 1997). Furthermore, the D / p h e n o t y p e can be rescued by driving expression o f wg to the wing hinge. It was therefore concluded that the ectopic Sox70D in Dichaete mutants is either directly or indirectly preventing wg expression and this causes aberrant development of the wing hinge. There are 9 potential binding sites for the Sox70D protein in sequences just downstream of wg, so it is possible that ectopic Sox70D binds and represses wg transcription or Sox70D is
displacing another Sox gene product from the enhancer. Russell and Ashburner have reported the discovery o f a Sox gene called sox50D which is endogenously expressed in the wing hinge.
If Drosophila Sox proteins do regulate wg transcription, then this relationship may have been evolutionarily conserved. If this is the case, then SOX proteins would be involved in the regulation o f the mammalian homologues o f wg, the Wnt genes. Mammalian Wnt genes are expressed in the developing central nervous system in distinct regions along the dorso-ventral axis. For instance, Wnt-1, Wnt-3 and Wnt-3a
are all expressed in the dorsal midline o f neural tube. Wnt-4 is expressed in a larger domain o f the dorsal neural tube and floor-plate and Wnt-7a and Wnt-7b are expressed in a larger domain in the medial and ventral tube (Parr & McMahon, 1994). Several
Sox genes are expressed in the developing central nervous system (see table 1.1), it would therefore be interesting to ascertain if any o f these genes are critical for the regulation o f particular Wnt genes.
Sox genes and their protein products have many interesting properties. The genes appear to respond to early inducing signals and their expression correlates with the establishment o f specific cell fates. Their complex expression patterns and the nature o f the proteins themselves suggest they will serve many different functions during embryogenesis. This complexity is being borne out by mutant studies in several genes such as Soxl, Sox2, Sox4, S0X 9 and sox70D. The studies presented in this thesis have shown that Sox3 also plays an essential role in mouse early development. The investigation o f the mechanism o f how the Sox3 mutation effects development
will be particularly interesting as it may reveal how the briefest of transient gene expression in a specific tissue can be influence later development. These studies may also elucidate some important developmental differences between mice and men.
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404 ■1^ 217 147 - 123 - 110 - f 1 g t g a t c t t a t t t g c t a t t t c g t g t a c g c a t t t c g t t c g g g t g g t g g g g a a 51 g g g g t t a t t t t g t t c c a g g t t t t t t c a a a g t t t n g t t c t t a a t t g c t a a g 101 tonaa a a a t c A A T A A A t t t t a t a a c c a a t a t q c t Q a a Q t c c t q a t a a t t t 151 t n c a g g t c c g a a a a t t t g a a g t g c g g g t t g g g a a t g g g g a t a g t c t t a g t •201 t t t a t a a c t g a g t t t g c a g a c c t g g g c g g g g g c g g g g g g g g g a g c a t g a t 251 g t a a a a t g a t t t c c t t t a a g c t a t t g g a g a t c c a a g c t a c a a c c g c c c c a 301 a t a c t c t g a a g t g g g a a c t a g Figure A .lLeft: R Nase protection o f R N A sam ples (3/xg) from : adult liver (L ), ES cells treated with retinoic acid for 7 hours (E S), \0 .5 d p c em bryo heads (H ) and 11.5d/?c w hole embryos (E). Size o f full length probe is show n (•^). NB the protected fragments in ES,H and E lanes.
Top right: map o f riboprobe (transcribed from #45) in relation to Sox3 gene Open reading frame depicted by raised box, the HM G dom ain is shaded green.
Bottom right sequence o f clone #45. NB. the poly-adenylation signal (as indicated )
This experim ent proves that the w hole o f the transcribed sequence is present in 241.1
Figure A.2
P robe I Sox-3 4kb -, pgk-hygro |--- sk b , Targeting construct Kosx3 P robe T . I____________ cOC M utated Sox-3 1kbTop: Schematic diagram of the SoxS genomic locus. The transcribed region of the locus is represented by the orange/green shading and the start of transcription by the arrow. The open reading frame of Sox3 is contained in a single exon, designated by the raised box. The region encoding the HMG box, DNA binding domain is shaded in green.
Middle: Schematic diagram of the Sox3 targeting construct, K0SX3. Shaded in magenta is the hygro' gene driven by the pgk promoter which allows for positive selection for stable insertion of the construct using Hygromycin. The regions of homology either side of the hygrb gene are included (kb = kilobase pairs)
Schematic diagram of the intended mutated Sox3 locus following homologous
recombination with the targeting construct K0SX3. The HMG box is
disrupted by the insertion of the hygro' cassette. The position of the probe used in Southern analysis is indicated by a red box. NB. probe sequence is internal to any homologous recombination event.
Figure A 3
- panel A : Southern blot analysis o f clone ST125/40A. The Hindlll digest of ST125/40A DNA, clearly indicates the presence of two bands that hybridise to M PI.5 (< ). This indicates that either the locus has been rearranged or was incorrectly targeted.Figure A .4
- panel B : Three morphologically normal chimaeras (lld p c) made with clone 40A ES cells. The regions anterior to the fore-limb bud were used to test for fi-galactosidase activity. This clearly shows that the embryo on the right is significantly derived from ES cell. The regions posterior to the fore-limb bud were used in a whole mount in situ hybridisation to detect
Sox3 expression. This demonstrates that the chimaera on the right is still expressing high levels of Sox3 in the developing spinal cord. This proves that Stl25/40A ES cells do not possess a null mutation for Sox3. An identical result was obtained with clone ST 125/79.
Bglll
Bstell
Hindlll
SspI
wt 40A
wt 40A
wt 40A
wt 40A
kb
mm
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