Double deletion of the active zone proteins CAST/ELKS in the mouse forebrain causes high mortality of newborn pups.

Presynaptic active zone cytomatrix proteins are essential elements of neurotransmitter release machinery that govern neural transmission. Among active zone proteins, cytomatrix at the active zone-associated structural protein (CAST) is known to regulate active zone size in retinal photoreceptors and neurotransmitter release by recruiting Ca2+ channels at various synapses. However, the role of ELKS-a protein from the same family as CAST-and the synergistic roles of CAST/ELKS have not been thoroughly investigated, particularly with regard to mouse behavior. Here, we generated ELKS conditional KO in mouse forebrain synapses by crossing ELKS flox mice with a CaMKII promoter-induced Cre line. Results showed that CAST is dominant at these synapses and that ELKS can support CAST function, but is less effective in the ELKS single KO. Pups of CAST/ELKS double KO in the forebrain were born in Mendelian rations but resulted in eventual death right after the birth. Anatomically, the forebrain neuronal compositions of CAST KO and CAST/ELKS double KO mice were indistinguishable, and the sensory neural network from whiskers on the face was identified as barrelette-like patches in the spinal trigeminal nucleus. Therefore, depletion of CAST and ELKS disrupts neurotransmission from sensory to motor networks, which can lead to deficits in exploration and failure to suckle.

Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome.

Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant to being combed flat. Until now, both simplex and familial UHS-affected case subjects with autosomal-dominant as well as -recessive inheritance have been reported. However, none of these case subjects were linked to a molecular genetic cause. Here, we report the identification of UHS-causative mutations located in the three genes PADI3 (peptidylarginine deiminase 3), TGM3 (transglutaminase 3), and TCHH (trichohyalin) in a total of 11 children. All of these individuals carry homozygous or compound heterozygous mutations in one of these three genes, indicating an autosomal-recessive inheritance pattern in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic causes of UHS and shed light on its pathophysiology and hair physiology in general.

c-Maf and MafB transcription factors are differentially expressed in Huxley's and Henle's layers of the inner root sheath of the hair follicle and regulate cuticle formation.

BACKGROUND: The hair follicle of mammalian skin consists of a group of concentric epithelial cell layers. The inner root sheath (IRS), which surrounds the hardening hair shaft beneath the skin surface, is subdivided into three layers, termed the cuticle of the IRS, Huxley's layer, and Henle's layer. The IRS forms a follicular wall in the hair canal and helps guide the developing hair shaft. c-Maf and MafB, members of the Maf family of transcription factors, play important roles in the developmental processes of various tissues and in cell type-specific gene expression. OBJECTIVE: The aim of this study is to reveal the pattern of expression and functional roles of c-Maf and MafB in the hair follicle. METHODS: We determined the precise location of c-Maf and MafB expression using immunofluorescent staining of mouse skin sections with layer-specific markers. We also analyzed whiskers of c-maf- and mafB-null mice (c-maf(-/-) and mafB(-/-), respectively) using scanning electron microscopy. RESULTS: c-Maf and MafB were differentially expressed in the Huxley's and Henle's layers of the IRS. Scanning electron microscopic analysis showed irregular cuticle patterning of whiskers of c-maf(-/-) and mafB(-/-) mice. The cuticles of mafB(-/-) mice were also thinner than those of wild-type mice. CONCLUSION: c-Maf and MafB are expressed in the IRS layers in a lineage-restricted manner and are involved in hair morphogenesis.

Defining BMP functions in the hair follicle by conditional ablation of BMP receptor IA.

Using conditional gene targeting in mice, we show that BMP receptor IA is essential for the differentiation of progenitor cells of the inner root sheath and hair shaft. Without BMPRIA activation, GATA-3 is down-regulated and its regulated control of IRS differentiation is compromised. In contrast, Lef1 is up-regulated, but its regulated control of hair differentiation is still blocked, and BMPRIA-null follicles fail to activate Lef1/beta-catenin-regulated genes, including keratin genes. Wnt-mediated transcriptional activation can be restored by transfecting BMPRIA-null keratinocytes with a constitutively activated beta-catenin. This places the block downstream from Lef1 expression but upstream from beta-catenin stabilization. Because mice lacking the BMP inhibitor Noggin fail to express Lef1, our findings support a model, whereby a sequential inhibition and then activation of BMPRIA is necessary to define a band of hair progenitor cells, which possess enough Lef1 and stabilized beta-catenin to activate the hair specific keratin genes and generate the hair shaft.

Curly vibrissae, a new mutation in the Wistar-derived rat.

A new mutation was identified in the PD (Preaxial Duplication) strain of rats, the main manifestations of which were curly and sparse vibrissae with retarded outer hair growth. As the main characteristic of this mutant rat is abnormally curled appearance of the vibrissae, "curly vibrissae" is proposed as the name of this mutant gene, and "cv" as its symbol. Genetic analyses reveal that the mutant characteristics are inherited as autosomal recessive traits and the cv gene is independent from the pd gene that carried by the original PD colony. The cv/cv homozygous rats have a small number of short and/or curly vibrissae around the nose. The vibrissae on the cheek and/or above the eyes are also short and curled; however, no vibrissa appears on the lower mandible. Although hair growth seems to be retarded, the outer hairs showed nearly normal length by 10 weeks of age. The outer hairs of matured cv/cv rats appear silky and translucent. The adult mutant rats often exhibit loss of hair on the head and/or back. Lactating females usually lose their abdominal hair. Both sexes of cv/cv homozygotes have normal reproductive ability. No internal malformations accompany vibrissa and hair abnormalities.

Expression of enhancing factor/phospholipase A2 in skin results in abnormal epidermis and increased sensitivity to chemical carcinogenesis.

Enhancing factor (EF), a growth factor modulator, is the mouse homologue of human secretory group II phospholipase A(2). EF exhibits growth-promoting activity in vitro, in the presence of epidermal growth factor, and also brings about phenotypic transformation of normal cells. In order to ascertain the role of EF in vivo, a human keratin-14 promoter was used to drive the expression of EF ectopically to squamous epithelial cells. The founder mouse and its progeny showed abnormal whiskers and a scaly, beaded tail. In these mice, keratinization pattern of the epidermis was disturbed and parakeratosis and acanthosis were noted. The transgenic mice, TgK14-EF, expressed EF in the suprabasal layers of tail epidermis as well as in the epithelial cells of hair follicle and sebaceous glands of skin. Expression of EF along with hyperplasia was also observed in other squamous epithelia such as buccal mucosa, tongue and oesophagus. TgK14-EF mice homozygous for the transgene showed delayed and scanty hair growth although the mice were healthy and fertile. The hemizygous TgK14-EF mice were sensitive to a two-stage chemical carcinogenesis and developed a higher number of papillomas than their normal littermates over the course of the experiment. The conversion rate of papilloma to carcinoma was two fold higher in the transgenic mice.

Deletion of the GATA domain of TRPS1 causes an absence of facial hair and provides new insights into the bone disorder in inherited tricho-rhino-phalangeal syndromes.

GATA transcription factors mediate cell differentiation in diverse tissues, and their dysfunction is associated with certain congenital human disorders. The six classical vertebrate GATA proteins, GATA-1 to GATA-6, are highly homologous, bear two tandem zinc fingers of the C(4) (GATA) type, and activate transcription. TRPS1, the only other vertebrate protein with the GATA motif, is a large, multitype zinc finger protein that harbors a single DNA-binding GATA domain and represses transcription. Monoallelic TRPS1 mutations cause two dominantly inherited human developmental disorders of the hair, face, and digits, tricho-rhino-phalangeal syndrome (TRPS) types I (MIM 190350) and III (MIM 190351); missense GATA domain mutations account for the more severe type III form. Here we report that heterozygous mice with deletions of the TRPS1 GATA domain (TRPS1(+/Deltagt)) display facial anomalies that overlap with findings for TRPS, whereas TRPS1(Deltagt/Deltagt) mice additionally reveal a complete absence of vibrissae. Unexpectedly, TRPS1(Deltagt/Deltagt) mice die of neonatal respiratory failure resulting from abnormalities of the thoracic spine and ribs. Heterozygotes also develop thoracic kyphoscoliosis with age and reveal structural deficits in cortical and trabecular bones. These findings directly implicate the GATA type zinc finger of TRPS1 in regulation of bone and hair development and suggest that skeletal abnormalities emphasized in descriptions of TRPS are only the extreme manifestations of a generalized bone dysplasia.

Whisker-related neural patterns develop normally despite severe whisker defects in Msx2 knockout mice.

In mice, whiskers on the snout form a highly specialized tactile organ with exquisitely patterned neural representations in the brain. Targeted deletion of the Msx2 gene leads to severe craniofacial defects, and stubby, curly whiskers. We examined the whisker pad histology, innervation, and whisker-related pattern formation along the trigeminal pathway in Msx2 -/- mice. Although the whiskers are severely deformed, whisker follicle structure, pattern and density of innervation, as well as central neural patterns in the brainstem, thalamus, and cortex appeared normal. We conclude that whisker-related neural patterns can form in the absence of normal whiskers, as long as whisker follicle innervation is intact.

trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice.

To investigate the nerve growth factor requirement of developing oro-facial somatosensory afferents, we have studied the survival of sensory fibers subserving nociception, mechanoreception or proprioception in receptor tyrosine kinase (trkA) knockout mice using immunohistochemistry. trkA receptor null mutant mice lack nerve fibers in tooth pulp, including sympathetic fibers, and showed only sparse innervation of the periodontal ligament. Ruffini endings were formed definitively in the periodontal ligament of the trkA knockout mice, although calcitonin gene-related peptide- and substance P-immunoreactive fibers were reduced in number or had disappeared completely. trkA gene deletion had also no obvious effect on the formation of Meissner corpuscles in the palate. In the vibrissal follicle, however, some mechanoreceptive afferents were sensitive for trkA gene deletion, confirming a previous report [Fundin et al. (1997) Dev. Biol. 190, 94-116]. Moreover, calretinin-positive fibers innervating longitudinal lanceolate endings were completely lost in trkA knockout mice, as were the calretinin-containing parent cells in the trigeminal ganglion.These results indicate that trkA is indispensable for developing nociceptive neurons innervating oral tissues, but not for developing mechanoreceptive neurons innervating oral tissues (Ruffini endings and Meissner corpuscles), and that calretinin-containing, trkA dependent neurons in the trigeminal ganglion normally participate in mechanoreception through longitudinal lanceolate endings of the vibrissal follicle.

Further genetic analysis of two autosomal dominant mouse eye defects, Ccw and Pax6(coop).

PURPOSE: The work forms part of a major project to study the genetics of mouse cataract mutants found during the course of mutagenesis experiments. The long-term aim is to find the underlying gene mutation in each cataract mutant. Here we report further studies of the mutant cataract and curly whiskers (Ccw), previously mapped to Chromosome 4, and also investigations of the corneal opacity (Coop) mutant, which is shown to involve a mutation in the Pax6 gene. METHODS: For Ccw, the methods included mapping relative to microsatellite markers and histological studies. For the Coop mutant, breeding methods were used to show that Coop was allelic with Pax6. The Pax6 coding region in the mutant was then sequenced. RESULTS: The Ccw locus was mapped to approximately position 45cM on the consensus map of Chr 4. Histologically, progressive degeneration of the lens was seen. In the Coop mutant, a base-pair change C->T was found at position 1033 in the Pax6 gene, which created a stop codon leading to premature termination of translation, and to a truncated Pax6 protein. CONCLUSIONS: The phenotype in Ccw/+ heterozygotes involves a new type of lens degeneration in the mouse. On the basis of the phenotype and the locus position, no candidate gene has yet been identified. The Pax6coop mutant differs in phenotype from known null alleles of Pax6, implying that it is a hypomorph.

Whiskers amiss, a new vibrissae and hair mutation near the Krt1 cluster on mouse chromosome 11.

Whiskers amiss (wam) is a new spontaneous recessive mutation in the SELH/Bc strain of mice that causes a phenotype of askew, sometimes kinked or curled, breakable whiskers and disheveled-appearing body hair, apparently owing to disoriented guard hairs. Heterozygotes on three genetic backgrounds are indistinguishable from normal. Using informative SSLPs in the F2 generation after crosses to two normal strains, we have mapped wam to the region of the type I keratin cluster on Chromosome (Chr) 11, within an approximately 6-cM segment according to the current Mouse Genome Database (MGD) map position of flanking SSLPs. Although several other hair mutations also map to the Krt1 region (Re, Rim3, Bdai, Bsk), none has a hair and whisker phenotype similar to that of wam, and, because all are transmitted as dominants, interpretable complementation tests could not be done. Scabbing and tissue loss occur on the rims of the pinnae and tail tip in some aging wam homozygotes, suggesting that wam may be an animal model of a genetic ectodermal disorder. The SELH/Bc strain background appears to have an unusually high rate of spontaneous mutation; wam is the sixth mutation to be described.

Medullary thyroid carcinomas in transgenic mice expressing a Polyoma carboxyl-terminal truncated middle-T and wild type small-T antigens.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

Defective whisker follicles and altered brainstem patterns in activin and follistatin knockout mice.

Whisker pad innervation and whisker-specific pattern formation were examined in mice lacking the gene for activin betaA or for follistatin. Both strains of mice die within 24 h after birth. A normal array of whisker follicles is present in the snout of either phenotype. However, activin betaA-deficient mice lack whiskers, and in follistatin-deficient mice the whiskers are thin and curled. We examined the effects of aberrant, albeit innervated, follicles on the formation of whisker-specific patterns (barrelettes) in the trigeminal brainstem. Activin betaA knockout mice lack barrelettes, although the trigeminal afferent topography is not compromised. Physiological recordings suggest that trigeminal ganglion cells in these mice are less responsive to stimulation of whisker follicles. Barrelettes in follistatin-deficient mice are not as well developed as in controls, but can be discerned in some cases. These results are consistent with the notion that formation of barrelettes depends on neural activity initiated by the whiskers.

Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.

Integrin expression is normally confined to the basal layer of the epidermis, but when epidermal homeostasis is perturbed, the receptors are also expressed by suprabasal, differentiating keratinocytes. We have used the involucrin promoter to express functional human integrin subunits alpha 2, alpha 5, and beta 1 in the suprabasal epidermal layers of transgenic mice. In mice expressing alpha 5 or beta 1 alone or alpha 2 beta 1 or alpha 5 beta 1 heterodimers, there were hair and whisker abnormalities and a failure of eyelid fusion. In addition, mice expressing beta 1 alone or in combination with alpha 2 or alpha 5 exhibited epidermal hyper-proliferation, perturbed keratinocyte differentiation, and skin inflammation, all of which are features of a common human skin disease, psoriasis.

Hemifacial deficiency induced by a shift in dominance of the mouse mutation far: a possible genetic model for hemifacial microsomia.

Hemifacial deficiency appeared in 10% of juvenile mice when BALB/cGaBc mice carrying the recessive lethal mutation far were crossed with ICR/Bc. The hemifacial deficiency increased to 15-20% after one backcross to ICR/Bc and then remained at that level for 11 additional generations of backcrossing of far into ICR/Bc. Neither the ICR/Bc strain nor BALB/cGaBc (+/far) produces hemifacial deficiency. Genetic and anatomical studies of adults and fetuses showed that the hemifacial deficiency was due to +/far in the ICR/Bc strain genome; that is, far becomes an incomplete dominant in the ICR/Bc strain background. The hemifacial deficiency (38% of +/far) is probably caused by premature synostosis of the maxilla and premaxilla, observable on day 16 of gestation. An additional 20% of +/far in ICR/Bc have cleft palate and die at birth. Most +/far in both strains have a hidden anomaly, bilateral splitting of the maxillary branch of the trigeminal nerve. far/far homozygotes of both strain backgrounds have a syndrome of severe bilateral deficiency of the derivatives of the maxillary prominence. In human pedigrees, where the equivalents of the dominance modifiers in BALB/cGaBc and ICR/Bc would segregate within families, it would be difficult to recognize that sporadic hemifacial deficiency and severe bilateral maxillary deficiency were due to the same gene. We suggest that human bilateral and unilateral abnormalities of tissue derived from the first branchial arch should be analyzed with the awareness that, in mice, at least, the two kinds of syndrome are due to the same mutant gene.

Selective breeding for variations in patterns of mystacial vibrissae of mice. Bilaterally symmetrical strains derived from ICR stock.

The establishment of certain patterns of mystacial vibrissae in mice has been the aim of an extensive breeding program carried on in this laboratory since 1977. In a companion paper we have reported on variations in this pattern in an outbred population of ICR mice. Starting with 21 ICR animals we bred, mostly by brother-sister mating, for 13 bilaterally symmetric patterns of mystacial vibrissae characterized by the presence (or absence) of supernumerary whiskers (SWs). The strains are classified as follows: I, a mouse strain with the standard pattern; II, eight strains bred for the occurrence of SWs at a given site or sites; and III, four mouse strains bred for a maximal number of SWs in different regions of the whiskerpad. Commonly, SWs occur in regions that coincide with the zones of mergence between the three facial processes except for two class II strains in which we bred for SWs in the "straddler" row of vibrissae, and for one class III strain, in which we cultivated the tendency (that appeared late in our program) to have SWs at the crest of a facial process. For classes I and II we analyzed the results for about 18 generations in terms of "improvement," meaning an increase in the percentages of animals with the desired phenotype together with a decreased frequency of undesired SWs. For class III, success in breeding meant the increase of the mean number of the desired SWs. All results led to the same conclusion: there is a genetic basis for the occurrence of SWs. The side preference of a particular SW is not strain dependent. It disappears in those class I and II strains in which almost 100% of animals obtained the desired phenotype. The increase in number of SWs in one zone of mergence does not depend on the presence of SWs in the other. Where tested, we almost always found a representation of an SW in a topologically equivalent location within the "barrelfield" area of the somatosensory cerebral cortex. Except for some diseases early in the breeding program, and some side effects of inbreeding that were eliminated, the population was without obvious defects. Where tested, there was no correlation between the occurrence of SWs and sex. The observed variations in pattern of mystacial vibrissae and their genetic background led us to propose a morphogenetic model for the formation of the pattern of mystacial vibrissae.

Hairless cats in Great Britain.

Ten hairless kittens are known to have been born in Britain since 1978. Pedigree study supports the hypothesis of a monogenic, recessive mode of inheritance proposed in previous reports. A review of the literature suggests the possibility of at least two mutations giving rise to hairless cats, one of which has normal whiskers and the other attenuated whiskers. For these, the gene symbols hi, and hr, respectively, have been proposed.