Not all valid argument forms are truth-functional. Some use universal quantifiers (ie, every) and some use existential quantifiers (ie, some). However, it is imperative that a universal quantification is not ambiguous, as in “everything is not blue” — is everything non-blue, or is it that some things non-blue?

An especially powerful valid deductive argument form is reductio ad absurdum. If you are unsure how to directly argue P, you may use reductio ad absurdum. First, assume it is not the case that P; second, include known truths amongst your premises; lastly, draw a conclusion that is a contradiction. It is impossible for a valid argument with true premises to result in a contradiction. Ergo, the premise P must not be true.

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Complex gene regulation propels vertebrate neurogenesis — and cell polarity also plays a critical role. This paper explores the relevance of the polarity protein PAR-1 and its regulatory kinase aPKC to neurogenesis, as well as the importance of the ubiquitin ligase Mind bomb (Mib). Mib is imperative for Notch ligand activity, and interactions between Mib and Par-1 are characterized.

What is the experimental system/techniques?

To verify that PAR-1 and aKPC are vital to neurogenesis, they were over- and under-expressed (via mRNA and morpholino injection, respectively). Direct examination and in situ hybridization for neuronal markers were two techniques used to quantify primary neuron populations. Markers included N-tubulin and Hox11L2 (for primary neurons), Sox2 and Sox3 (for neuronal progenitors). Phospho-histone-3 staining was used to measure mitosis.

To test whether differences between wild-type, abundant or ablated PAR-1 and apKC levels affect neuronal differentiation directly or via cytoskeletal disruption, the C17.2 strain of cells was used. This strain lacks epithelial morphology yet can differentiate into neurons — thus mutant differentiation in these cells must be independent of their cytoskeletal (in)activity. If PAR-1 and aPKC are unable to disrupt or enhance neuronal differentiation in these cells, then their own activity must involve cytoskeletal intervention.

The Notch pathway is a major regulator of neurogenesis. Perhaps PAR-1 and aPKC are mediated by the Notch pathway. To test this, a luciferase reporter with multimerized CSL-binding sites was used in C17.2 cells cotransfected with various aPKC and PAR-1 construct. Immunoprecipitation, spectrometry, over- and under-expression and other techniques were all used to probe the interaction of PAR-1 with Mib. Dil1 signaling activity depends on the ubiquitination of its intracellular domain by Mib — thus, fusing Dil1 to an ubiquitin would rescue it from the effects of PAR-1.

With earlier experiments revealing that PAR-1 phosphorylates Mib, researchers set out to identify Mib’s phosphorylation sites relevant to this pathway. Various Mib proteins were engineered with substitutions in putative serine/threonine phosphorylation motifs. Their levels were then assayed in presence or absence of PAR-1 in Xenopus embryo lysates.

What are the results and conclusions?

Over- and under-expression of PAR-1 and aKPC reveals that they influence neurogenesis in Xenopus embryos. Increased PAR-1 caused a surge in the number of primary neurons. Depletion of PAR-1 significantly reduced the number of primary neurons. Wnt signaling was unaffected, indicating that PAR-1 is not mediated by the Wnt pathway. Supportive of these findings, that aPKC increases decreased primary neuron quantities; aPKC decreases increased primary neuron quantities. Interestingly, populations of neuronal progenitors were not remarkably affected despite the presence or absence of PAR-1 activity causing wide rises and falls in primary neuron populations. This finding was supported by a corresponding consistence of mitotic activity despite activation or deactivation of PAR-1 activity. However N-tubuln activity was found to be remarkably increased within the bounds of its normal expression (as opposed to being expressed ectopically). Thus PAR-1 must promote neuronal differentiation yet not progenitor pool expansion. Increasing and decreasing PAR-1 and aPKC activity in C17.2 cells yielded parallel results – thus, their activity is intrinsic and does not rest upon a cytoskeletal pathway.

The luciferase reporter with multimerized CSL-binding sites was activated by aPKC and inhibited by PAR-1. Specifically, PAR-1 modulated Dil1 but not Notch ICD — PAR-1 repressed its activity while aPKC modestly enhanced it. Upstream of Dil1 is Mib. Mass spectrometry identified a mammalian PAR-1 homolog among proteins associated with Mib. Their physical interaction was verified by coprecipitation from cell lysates. An immune complex kinase assay revealed that PAR-1 phosphorylated Mib and a similar E3 ubiquitin ligase, identifying Mib as an in vitro substrate for PAR-1. In vivo, PAR-1 and homologs downregulated Mib levels in lysates from embryos and cultures. Mib mutated in its Eg ligase capability was unaffected by PAR-1, even when PAR-1 was overexpressed. Ablation of PAR-1 led to an increase in Mib protein levels. Treating with proteasome inhibitors cancelled the inhibitory effect of PAR-1 on Mib, suggesting that inhibition occurs via a proteasome pathway. Thus, PAR-1 likely phosphorylates Mib causing Mib’s proteasome-dependent degradation. Furthermore, Mib ubiquitinates Dil1; by fusing Dil1 to an ubiquitin and assaying neuronal development, it was shown that Dil1’s over-ubiquitination led to reduced neuronal growth and was impervious to overexpression of its inhibitor PAR-1. This shows that PAR-1 regulates Dil1 by phosphorylating E3-ubiquitin ligases such as Mib which in turn ubiquitinate Dil1.

Most Mlb mutants remained sensitive to PAR-1-mediated degradation, with the sites M2 and M8 each critical for this process; indeed, M2 and M8 mutations rendered the Mib mutants impervious to the effects of PAR-1. Thus, specific phosphorylation sites in Mib are required for PAR-1-dependent changes in Dil1 ubiquitination.

Vertebrate Crossveinless-2 (CV2) is a BMP4-dependent secreted protein that can potentiate or antagonize BMP4 signaling and is required for formation of crossveins in the Drosophila wing. What effects does CV2 depletion have in the cell? Since it is dependent on a robust BMP4 gradient, what is CV2’s function with respect to BMP4? Does it complex with BMP4? CV2 is indeed cleaved – but is it cleaved by tolloid proteases, as is Chordin? Does CV2 bind to purified Chordin with high affinity, and do CV2 and Chordin have similar in vivo activities?

What is the experimental system/techniques?

Cdx2 null mice die before gastrulation. Researchers thus engineered a conditional Cdx2-ablated mutant to study Cdx2’s role in the gut endoderm. Mice heterozygous for the conditional allele were crossed with wild-type mice; the heterozygotes were bed to form homozygous mutant F2 progeny. Cdx2 was equally ablated throughout the intestine.

Mutant duodenal epithelium proliferation was assayed by BrdU incorporation. Apoptosis was assayed via cleaved caspase-3 and also TUNEL staining. Mutant Cdx2 epithelial cells identity was directly observed by transmission electron microscopy of ultrastructural features. In addition, identity of these cells was probed by an immunohistochemical assay for keratin 13 and p63 (markers of keratinocytes, which TEM indicates is the identity of these mutant Cdx2 epithelial cells). For further study, markers of anterior foregut endoderm were assayed.

Gene expression profiling (via microarray) was performed using RNA samples extracted from total E18.5 control and mutant ileum as well as normal esophagus. Additional profiling was performed with PCR to examine the cell fate switch in the Cdx2-deficient posterior intestine, focusing on transcriptional regulators known to be crucial in regulating intestinal differentiation.
Cdx factors regulate Hox genes, which are expressed both within and outside the endoderm and affect gastrointestinal development. Analysis was performed on various Hox genes, including those whose early gut expression domains are well characterized. Further study was done, particularly on Wnt proteins and their targets.

What are the results and conclusions?

Cdx2 deficiency prevents colon formation and caused complete intestinal obstruction in Cdx2 mutants — the poor wretches. Despite live births, mutant pups died in postnatal day one (P1). Their posterior gut region was grossly abnormal. Wild-type controls had a colon and rectum at the terminus of their intestinal tract; mutants all lacked colons, with various other defects arising. Mutant duodenums were distended and translucent due to fluid retention caused by distal obstructed.

BrdU incorporation revealed an expanded proliferative compartment in the mutant duodenal epithelium. However, assays of apoptosis revealed a wild-type apoptotic rate. Expanded proliferation and a lack of enhanced cell death suggest that Cdx2 mutants have an epithelial proliferative pattern reminiscent of early embryonic stages prior to intestinal differentiation. Transmission electron microscopy revealed abundant tonofilaments in mutant posterior epithelial cells. Tonofilaments are typical of squamous epithelial cells — they are often seen in the desmosomal junctions of keratinocytes. Keratinocytes are typical of stratified esophageal epithelia but are extremely rare in the normal intestine. Immunohistochemical assays of keratin 13 and p63 — common in keratinocytes but rare in wild-type midgut and hindgut endoderm — revealed that the mutant epithelium was positive for these markers. Furthermore, the fact that an anterior foregut endoderm marker was detected in Cdx2-deficient ileum indicates it was indeed anteriorized and did not just undergo a significant developmental delay.

Mutant ileum was more transcriptionally similar to esophageal tissue than to normal ileum. Nearly all intestine-specific genes were downregulated in the mutant ileum. There were significant expression changes in 40% each of: genes signficantly expressed in E18.5 intestinal epithelium; and genes expressed more in the intestine than stomach. Also, genes expressed specifically in differentiated intestinal epithelium were all heavily downregulated. Genes involved in keratinocyte formation were significantly upregulated — including nine of those involved in esophageal formation. This strengthens the notion of the identity of the mutant epithelium.

Cdx2 mRNA obviously decreased — and so did several intestine-enriched transcription factors. Reverse-transcription and PCR revealed that this transcriptional modification occurred early in development. In fact, the expression of Math1 was reduced from E12.5 onward — this is significant as it is crucial for differentiation of intestinal secretory cell types. Conversely, foregut-enriched genes were ectopically activated in the mutant posterior intestine as early as E12.5. Additional genes were dramatically activated ectopically, and various more genes were repressed in their wild-type expression domains.

Hox9, expressed in the posterior hindgut, was underexpressed in Cdx2 mutants. In the early gut, Hoxc8, Hoxb9, Hoxc9, Hoxa13 and Hoxd13 mRNA levels were significantly lower in the mutant posterior intestine. However various Parahox genes maintained wild-type expression, indicating that not all factors along the AP axis of the gut were impacted by Cdx2 deficiency. Wnt proteins was upregulated due to anteriorization of the gut, causing subsequent upregulation of Wnt targets.

In conclusion, this paper finds that Cdx2 is necessary for posterior patterning of the gut along the AP axis. It controls many factors – prior studies postulated that it functions via of Hox factors. However, there are several Hox factors which are independent of Cdx2 yet still fall along the AP axis. Thus Cdx2 must function by targeting a litany of transcription factors, many of which are Hox factors. Cdx2 mutants are anteriorized, and can be rescued by Cdx1.