William W. Branford

Gsh-4 encodes a LIM-type homeodomain, is expressed in the developing central nervous system and is required for early postnatal survival.

We present an initial characterization of the murine Gsh‐4 gene which is shown to encode a LIM‐type homeodomain. Genes in this category are known to control late developmental cell‐type specification events in simpler organisms. Whole mount and serial section in situ hybridizations show transient Gsh‐4 expression in ventrolateral regions of the developing neural tube and hindbrain. Mice homozygous for a targeted mutation in Gsh‐4 suffer early postnatal death resulting from immature lungs which do not inflate. Prenatal administration of progesterone and glucocorticoid, to extend gestational term and accelerate maturation, resulted in lung inflation at birth. Nevertheless, the hormonally treated mutants generally failed to survive beyond an hour after birth, due to ineffective breathing efforts. It is concluded that Gsh‐4 plays a critical role in the development of respiratory control mechanisms and in the normal growth and maturation of the lung.

Evolutionary conservation and tissue-specific processing of Hoxa 11 antisense transcripts.

Abstract. We previously described the existence of abundant, processed, polyadenylated murine Hoxa 11 antisense transcripts. Of particular interest, in the developing limbs the antisense transcripts were observed to be present in a pattern complementary to that of the sense transcripts, suggesting a possible regulatory function (Hsieh-Li et al. 1995). We have analyzed the human HOXA 11 genomic locus, showing strong evolutionary conservation of regions potentially encoding antisense transcripts. Human HOXA 11 fetal kidney antisense cDNAs were identified and sequenced, demonstrating the evolutionary conservation of Hoxa 11 antisense transcription. As for the mouse, the human antisense RNAs were polyadenylated and showed several alternative processing patterns, but shared the sequences of a common 3′ exon. The evolutionary conservation of the opposite strand transcripts strongly suggests function. A significantly long open reading frame was observed, but mouse-human comparisons argued against true coding function. Murine kidney Hoxa 11 antisense transcription and processing was also examined, revealing tissue-specific differences between limb and kidney. A novel procedure, designated Race in Circles, was devised and used to define mouse limb antisense transcription start sites. Furthermore, comparisons of human, mouse, and chicken sense transcript Hoxa 11 homeobox nucleotide sequences and their respective encoded homeodomains indicate a very strong selective pressure in vertebrates against mutations that result in coding changes. Given the significant differences in amino acid sequences of the homeodomains of different Hox genes, this observation argues for individual homeodomain functional specificity.

Nodal signaling: CrypticLefty mechanism of antagonism decoded.

The secreted TGFbeta factor Lefty antagonizes Nodal signaling during vertebrate embryogenesis, but how it does so has been a mystery. Recent analyses have elucidated the molecular mechanisms underlying this function of Lefty.

Spx1, a novel X-linked homeobox gene expressed during spermatogenesis.

Spx1, a novel mouse homeobox gene, encodes a homeodomain characteristic of the paired-like class of homeobox genes and has been mapped to the distal end of the X chromosome. Northern blot hybridization of adult tissues detected high levels of a single Spx1 transcript in the testis. Further analysis by in situ hybridization revealed predominant Spx1 expression within the spermatogonia/preleptotene spermatocytes and round spermatids of spermatogenic stages IV-VII. These expression data suggest SPX1 may play a role in the regulation of spermatogenesis.

Disease-associated casein kinase I δ mutation may promote adenomatous polyps formation via a Wnt/β-catenin independent mechanism

The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKIδ and ϵ) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKIδ/ϵ might upregulate CKIδ/ϵ activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKIϵ and CKIδ regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKIδ missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKIδ(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKIϵ. Second, CKIδ(R324H) is more potent than wildtype CKIδ in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKIδ kinase activity in an in vitro kinase assay or Wnt/β‐catenin signal transduction as assessed by a β‐catenin reporter assay, it alters morphogenetic movements via a β‐catenin‐independent mechanism in early Xenopus development. This novel human CKIδ mutation may alter the physiological role and enhance the transforming ability of CKIδ through a Wnt/β‐catenin independent mechanism and thereby influence colonic adenoma development.

Malformations of the heart, kidney, palate, and skeleton in α-MHC-Hoxb-7 transgenic mice

Abstract To begin to define the genetic network involved in cardiogenesis, we generated mice bearing the α-myosin heavy chain (MHC)- Hoxb-7 transgene. We hypothesized that using the cardiac-specific α-MHC promoter, we can direct ectopic expression of Hoxb-7 in the heart and perturb its normal development. Both whole mount in situ hybridization and northern analyses showed that this α-MHC promoter resulted in transgene expression in the developing heart. Severe ventricular septal defects (VSD) were found in several mutant mice. Interestingly, transgenic mice were observed to have other malformations as well, including cleft palate, renal anomalies, and skeletal abnormalities in the craniocervical and costosternal regions. The kidney defect consisted of double ureter and pelvis. In summary, we have shown that a dominant gain-of-function mutation of Hoxb-7 using the murine α-MHC promoter results in perturbation of the genetic circuitry underlying multiple developmental processes, including cardiogenesis. Misexpression of Hoxb-7 during heart development may be involved in the pathogenesis of VSD.