| Cdx regulates Dll1 in Multiple Lineages | BIOL 303 Term Paper | |

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BACKGROUND
The vertebrate Cdx genes are a group of ParaHox genes that encode homeodomain transcription factors responsible for the anterior-posterior patterning of the embryonic axis, intestine, axial elongation and somitogenesis in mammals. These genes are related to Drosophila caudal (cad)[1] and there are three murine homologues Cdx1, Cdx2 and Cdx4 [2]. The Cdx gene products impact anterior-posterior vertebral patterning not only by direct regulation of Hox gene expression, but are also targets of non-Hox such as Wnt, Shh, RA and Notch pathways among others[3] [2]. The authors focus upon the Notch pathway, especially Notch ligand Dll1 as it is involved in intestinal differentiation.
Cdx1 is responsible for the axial positional information and the null mutants have an abnormal axial patterning only and no overt intestinal phenotype [4, 5]. Cdx2 is important for trophoblast differentiation, axial patterning and extension, and morphological specification of gut endoderm [5]. Homozygous Cdx2 null mutants are peri-implantation lethal as the blastocyst fails to undergo uterine implantation [3]. The Cdx1-Cdx2 conditional mutants show an abnormal somite formation and an increase in the number of Goblet cells in the small intestine [2]. Both Cdx1 and Cdx2 are important for the maintenance of the intestinal epithelium in adults.
Though there is considerably more information available about the different pathways that require Cdx function, the mechanisms behind it have not completely been understood. Hence, the authors wish to determine the Cdx-dependant mechanisms in the somites and the small intestine that control this phenotype seen in the Cdx1-Cdx2 mutants [2].
SUMMARY
The authors used Cdx1-/-, Cdx2f/f, Dll1f/f, actin-Cre ErT and villin-Cre ERT mice to produce heterozygote mutants [2]. The mutant transgenic mice consist of a complex mixture of specific mutations instead of just one mutation. Hence to avoid making incorrect and invalid conclusions, it is highly imperative that only the genes in question are being compared. Backcrossing ensures the genetic background is comparable. But by using non-transgenic littermates as non-mutant controls, the authors ensure that the environmental background in terms of cages, age, sex, etc. is also similar. An added advantage is that the mice do not require full backcrossing since the difference in the genes will be neutralised when the littermates are compared [6][Q1].
To overcome the embryo-lethality of homozygous Cdx2 null mutants and the functional overlap between Cdx members, the authors induce intestine specific inactivation of a floxed Cdx2 allele [2]. Floxing a gene is described as the placement of our DNA sequence of the gene of interest between two lox P sites. It allows the gene to be deleted, translocated or inverted, and produce organ-specific knockouts. Tamoxifen-inducible Cre recombinase with the villin promoter catalyses the recombination between the lox P sites in the small intestine alone, and knockout Cdx2 [3, 7][Q2]. Embryos were then collected at E6.5-E9.5 to study somitogenesis and at E18.5 to study Goblet cell differentiation in the gastrointestinal tract [2].
The authors then used differential staining with Periodic-Acid Schiff (PAS) or Alcian Blue to determine the Goblet cell number. The results showed that the Cdx2 single mutants and Cdx1-Cdx2 double mutants had similar number of Goblet cells which was higher than the number in the littermate controls and Cdx1 single mutants. Hence, Cdx2 alone suppressed Goblet cell differentiation [2].
The loss of Cdx2 on lineage decision events in the small intestine was investigated by a semi-quantitative RT-PCR and qPCR along with in situ hybridization (ISH). The expression levels of Math1, IFABP, TFF3 and Dll1 were quantified using the 2-ΔΔCt method. β-actin, a house keeping gene was used as a positive control and to normalize the data. The level of Math1 was significantly higher and it was upregulated (from ISH) in the small intestine Cdx2 mutants, which is due to reduction of Hes1 levels as seen. A reduction in the Dll1 levels was also seen in the single and double mutants. From the results, the authors inferred that the Cdx2 induces Goblet cell differentiation through regulation of Dll1 expression [2].
Cdx mutant embryos were examined for Dll1 expression as Dll1 mutants and Cdx mutants have similar somite defects. The Dll1 expression in Cdx1-Cdx2 mutants was normal when compared to controls at E7.5. However at E8.5, there was a significant reduction in Dll1 expression relative to controls. Thus, the results demonstrated that Cdx1 and Cdx2 are vital to maintain, and not the initiation of Dll1 transcripts [2].
From the results above, the authors hypothesized that Dll1 may be a direct Cdx target. They did a Transcriptional Element Search System (TESS) analysis which identified two putative Cdx response elements (CDREs) at the 5` proximal sequences of Dll1. An electrophoretic mobility shift assay (EMSA) revealed that there was a specific and direct interaction between these CDREs and Cdx members in vitro [2].
To determine if Cdx1 and Cdx2 occupied the Dll1 promoter in vivo at the motifs identified, the authors performed chromatin immunoprecipitation (ChIP) using Cdx1 and Cdx2 antibodies on the chromatin from E8.5 embryos / intestinal tract of E18.5 fetus. ChIP analysis is an immunoprecipitation technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions [8]. They observed that within the vicinity of the putative CDREs, there was a high concentration of both Cdx1 and Cdx2. This shows that the interaction of Cdx1 and Cdx2 in the Dll1 promoter region is specific [2]. [Q4]
To evaluate the genetic interaction between Cdx1-Cdx2 and Dll1 mutant alleles, a non-allelic non-complementation screen was done. This type of complementation occurs when mutations that lie in different genes, fail to complement each other and result in a mutant phenotype. This screen shows the interaction between the mutations that is a result of the functional connection/physical interaction between different gene products [9, 10].[Q5]
The assessment of the effect of Cdx and Dll1 interaction on the Goblet cell differentiation showed Cdx1+/-Cdx2+/-Dll1+/- compound heterozygotes displayed an increase in the number of Goblet cells relative to wild-type and Cdx1+/-Cdx2+/- controls [2].[Q5]
To determine the interaction of the mutant alleles in somitogenesis, the authors performed a whole mount ISH analysis on embryos collected at E9.5. The probes used were against Mox1, Uncx4.1 and Paraxis [2]. Mox1 plays an important role in the molecular signaling system that regulates somite development and is strongly expressed in the initial epithelial stage [11]. Irregularly shaped somites express the somatic marker gene Mox1 which can then be used to confirm presence of somites in mutant embryos [12]. Paraxis is responsible for the formation and regulation of epithelial somites along with maintaining somite polarity that is independent of Notch signalling [13-15]. Since both Cdx and Dll11 mutants show similar disruption in somitogenesis, somite markers such as Mox1 and Paraxis are used to determine the level of differentiation and segmentation of the somites ; and show genetic interaction between Cdx and Dll1 [11, 14, 15].[Q3]
There were no apparent somite defects seen in Cdx1+/- littermate controls and Cdx1+/-Cdx2+/- embryos, and they showed wild-type Mox1 and Paraxis staining. However, Cdx1+/-Cdx2+/-Dll1+/- compound heterozygotes exhibited indistinct somite borders and abnormal somite polarity (due to reduced expression of Mox1) [2]. Hence, from these findings the authors concluded that Cdx and Dll1 operate in a common genetic pathway in two distinct lineages and the Notch ligand Dll1 is downstream of Cdx. [Q5]

CRITIQUE
Choosing the appropriate controls for an experiment is very important as it plays a large role in establishing valid and conclusive results. The use of littermates as non-mutant controls does not only cover environmental variation but it also is less cumbersome. However, since fewer rounds of backcrossing are done, it is prudent to increase the number of mice used in the experiments, as the variability will increase [6]. It is also important to use wild-type controls along with littermate controls as there might be epigenetic modification of offspring by transfer of Cre recombinase in oocytes [16].
In floxing of the Cdx and Dll1 gene, one of the drawbacks with the use of Cre recombinase system is that it is not reversible. There are also potential side-effects seen due to the use of Tamoxifen [16].
In the experiment to quantify the number of Goblet cells via ISH, it would be better to use more than 3 animals per genotype as results can be easily misinterpreted in staining experiments. Increasing the number of animals would also strengthen the result and account for variability [1].
The use of specific antibodies for ChIP analysis is also one of the strong points of this paper. The major advantage of this technique is increased antibody specificity as target proteins naturally interact. There is also better chromatin and protein recovery efficiency [17].
The experimental approach was highly methodical and it followed sound logical reasoning. The strength of the paper lies in the correct usage of appropriate controls, along with extensive research and understanding of existing literature to make valid conclusions.
The authors then go on to further study Cdx1 and Cdx2 in the small intestine and found that though both the genes are functionally specific, they are transcriptionally specific too. The authors concluded that further characterization of Cdx1 and Cdx2 target genes and binding partners will be needed to better understand their molecular mechanisms of action [5].
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References

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