Aimee K. Ryan

Pitx2 determines left-right asymmetry of internal organs in vertebrates

The handedness of visceral organs is conserved among vertebrates and is regulated by asymmetric signals relayed by molecules such as Shh, Nodal and activin. The gene Pitx2 is expressed in the left lateral plate mesoderm and, subsequently, in the left heart and gut of mouse, chick and Xenopus embryos. Misexpression of Shh and Nodal induces Pitx2 expression, whereas inhibition of activin signalling blocks it. Misexpression of Pitx2 alters the relative position of organs and the direction of body rotation in chick and Xenopus embryos. Changes in Pitx2 expression are evident in mouse mutants with laterality defects. Thus, Pitx2 seems to serve as a critical downstream transcription target that mediates left–right asymmetry in vertebrates.

A CDK-Independent Function of Mammalian Cks1: Targeting of SCFSkp2 to the CDK Inhibitor p27Kip1

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.

POU Domain Factors in Neural Development

Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types. Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors. Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development. The POU domain family of transcription factors was defined following the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the C. elegans gene unc-86, shared a region of homology, known as the POU domain. The POU domain is a bipartite DNA binding domain, consisting of two highly conserved regions, tethered by a variable linker. The approximately 75 amino acid N-terminal region was called the POU-specific domain and the C-terminal 60 amino acid region, the POU-homeodomain. High-affinity site-specific DNA binding by POU domain transcription factors requires both the POU-specific and the POU-homeodomain. Resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer, respectively, confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins. Overall the crystal structure of a monomer of the Oct-1 POU domain bound to the octamer element was similar to that predicted by the NMR solution structures of the POU-specific domain and the POU-homeodomain in isolation, with the POU-specific domain consists of four alpha helices, with the second and third helices forming a structure similar to the helix-turn-helix motif of the lambda and 434 repressors; several of the DNA base contacts are also conserved. A homodimer of the Pit-1 POU domain was crystallized bound to a Pit-1 dimer DNA element that is closely related to a site in the proximal promoter of the prolactin gene. The structure of the Pit-1 POU domain on DNA is very similar to that of Oct-1, and the Pit-1 POU-homeodomain/DNA structure is strikingly similar to that of other homeodomains, including the Oct-1 POU-homeodomain. The DNA contacts made by the Pit-1 POU-specific domain are also similar to those of Oct-1 and conserved with many made by the prokaryotic repressors. In the Oct-1 crystal, the POU-specific domain recognizes a GCAT half-site, while the corresponding sequence recognized by the Pit-1 POU-specific domain, GTAT, is on the opposing strand. As a result, the orientation of the Pit-1 POU-specific domain relative to the POU-homeodomain is flipped, as compared to the Oct-1 crystal structure, indicating the remarkable flexibility of the POU-specific domain in adapting to variations in sequence within the site. Also in contrast to the Oct-1 monomer structure is the observation that the POU-specific and POU-homeodomain of each Pit-1 molecule make major groove contacts on the same face of the DNA, consistent with the constraints imposed by its 15 amino acid linker. As a result, the Pit-1 POU domain homodimer essentially surrounds its DNA binding site. In the Pit-1 POU domain homodimer the dimerization interface is formed between the C-terminal end of helix 3 of the POU-homeodomain of one Pit-1 molecule and the N-terminus of helix 1 and the loop between helices 3 and 4 of the POU-specific domain of the other Pit-1 molecule. In contrast to other homeodomain crystal structures, the C-terminus of helix 3 in the Pit-1 POU-homeo-domain has an extended structure. (ABSTRACT TRUNCATED)

A role for the POU-III transcription factor Brn-4 in the regulation of striatal neuron precursor differentiation.

Both insulin‐like growth factor‐I (IGF‐I) and brain‐derived neurotrophic factor (BDNF) induce the differentiation of post‐mitotic neuronal precursors, derived from embryonic day 14 (E14) mouse striatal multipotent stem cells. Here we ask whether this differentiation is mediated by a member of the POU‐III class of neural transcription factors. Exposure of stem cell progeny to either IGF‐I or BDNF resulted in a rapid upregulation of Brn‐4 mRNA and protein. Indirect immunocytochemistry with Brn‐4 antiserum showed that the protein was expressed in newly generated neurons. Other POU‐III genes, such as Brn–1 and Brn–2, did not exhibit this upregulation. Basic FGF, a mitogen for these neuronal precursors, did not stimulate Brn‐4 expression. In the E14 mouse striatum, Brn‐4‐immunoreactive cells formed a boundary between the nestin‐immunoreactive cells of the ventricular zone and the β‐tubulin‐immunoreactive neurons migrating into the mantle zone. Loss of Brn–4 function during the differentiation of stem cell‐derived or primary E14 striatal neuron precursors, by inclusion of antisense oligonucleotides, caused a reduction in the number of β‐tubulin‐immunoreactive neurons. These findings suggest that Brn‐4 mediates, at least in part, the actions of epigenetic signals that induce striatal neuron‐precursor differentiation.

Establishing a Left-Right Axis in the Embryo

Vertebrates exhibit evolutionarily conserved asymmetries in the pattern of internal organ placement that are essential for their normal physiological function. Left‐right asymmetries in organ situs are dependent upon the formation of an intact left‐right axis during embryogenesis. Recently many of the molecular components involved in the initiation and maintenance of the left‐right axis have been described. These molecules and their function in promoting left‐right asymmetries are reviewed.

Wocko, a neurological mutant generated in a transgenic mouse pedigree

Naturally occurring mutations involving the nervous system have provided virtually all of our current understanding of the genetic regulation of neural development (Caviness and Rakic, 1978). The difficulty of isolating the corresponding genes, however, has precluded a molecular analysis of these mutants. Insertional mutagenesis, induced by microinjection of DNA into fertilized ova to produce transgenic animals, provides a molecular tag that marks the site of the mutational event. In this article, we describe a transgenic neurological mutation, designated wocko (Wo), which disrupts the development of the inner ear. These mutant mice display a dominant behavioral phenotype that consists of circling, hyperactivity, and head tossing, reminiscent of the shaker/waltzer class of mutants, and they display a recessive homozygous sublethal phenotype. Anatomical analyses showed that both structural and neural components of the vestibular system were disrupted, while analyses of mutant fetuses showed that these morphological abnormalities were due to aberrant development. Although low levels of transgene expression were detected using a sensitive PCR assay, several nonmutant pedigrees that contain the same construct also expressed the transgene in the inner ear, suggesting that low levels of transgene expression alone were not responsible for the wocko phenotype. Because the integrated transgene provides a marker to clone the wocko mutation, the analysis of this mutant will give unique insight into the molecular genetics of inner ear development and into a broad class of neurological mutations that affect the inner ear.

POU-er-ful developmental regulators revisited

The POU domain transcription factors have been proposed to be important regulators of key developmental processes. The characterization of naturally occurring mutations and targeted gene deletions has demonstrated that many of these proteins function in terminal differentiation events, including lineage determination, migration, proliferation, survival, and maintenance of mature cell types. POU domain factors can exert critical functions in cell types in which they exhibit either transient or sustained expression patterns, and it is likely that their target genes encode receptors, guidance molecules, and neuropeptides that define differentiated phenotypes.