The mouse Hoxd13(spdh) mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes.

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have studied the genetics of this condition, by using several engineered Hoxd alleles, as well as by looking at the expression of Hox and other marker genes. We show that the mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes.     

Imaginal disc-autonomous expression of a defect in sensory bristle patterning caused by the lethal(3)ecdysoneless1 (1(3)ecd1) mutation of Drosophila melanogaster

The temperature-sensitive mutation 1(3)ecd1 of Drosophila melanogaster is known to autonomously impair the ability of the larval prothoracic gland to produce the steroid molting hormone ecdysone in response to stimulation by the tropic neuropeptide prothoracicotropic hormone. It is shown that autonomous expression of the 1(3)ecd1 mutation in metamorphosing imaginal tissues disrupts the spatial pattern of sensory bristles. Transfer of homozygous mutant animals to the restrictive temperature at the time of pupariation resulted in the elimination of sensory microchaetae and macrochaetae. This effect was specific to the sensory bristles; nonsensory bristles were not eliminated, nor were other types of innervated cuticular sense organs. In the case of the dorsal thoracic macrochaetae, normal ecd gene function is required during an early period of bristle development (0-18 h after puparium formation at 20 degrees C). It is during this period that important determinative events take place in developing imaginal tissues that are responsible for the establishment of bristle progenitor cells. It is proposed that the ecd gene product may be required for the response of certain classes of cells to specific, regulatory signals.     

Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) regulates growth and patterning of the postnatal mouse cerebellum

COUP-TFII (also known as Nr2f2), a member of the nuclear orphan receptor superfamily, is expressed in several regions of the central nervous system (CNS), including the ventral thalamus, hypothalamus, midbrain, pons, and spinal cord. To address the function of COUP-TFII in the CNS, we generated conditional COUP-TFII knockout mice using a tissue-specific NSE-Cre recombinase. Ablation of COUP-TFII in the brain resulted in malformation of the lobule VI in the cerebellum and a decrease in differentiation of cerebellar neurons and cerebellar growth. The decrease in cerebellar growth in NSE^Cre/+/CII^F/F mice is due to reduced proliferation and increased apoptosis in granule cell precursors (GCPs). Additional studies demonstrated that insulin like growth factor 1 (IGF-1) expression was reduced in the cerebellum of NSE^Cre/+/CII^F/F mice, thereby leading to decreased Akt1 and GSK-3β activities, and the reduced expression of mTOR. Using ChIP assays, we demonstrated that COUP-TFII was recruited to the promoter region of IGF-1 in a Sp1-dependent manner. In addition, dendritic branching of Purkinje cells was decreased in the mutant mice. Thus, our results indicate that COUP-TFII regulates growth and maturation of the mouse postnatal cerebellum through modulation of IGF-1 expression.     

The mouse rib-vertebrae mutation disrupts anterior-posterior somite patterning and genetically interacts with a Delta1 null allele

Rib-vertebrae (rv) is an autosomal recessive mutation in mouse that affects the morphogenesis of the vertebral column. Axial skeleton defects vary along the anterior-posterior body axis, and include split vertebrae and neural arches, and fusions of adjacent segments. Here, we show that defective somite patterning underlies the vertebral malformations and altered Notch signaling may contribute to the phenotype. Somites in affected regions are irregular in size and shape, epithelial morphology is disrupted, and anterior-posterior somite patterning is abnormal, reminiscent of somite defects obtained in loss-of-function alleles of Notch signaling pathway components. Expression of Dll1, Dll3, Lfng and Notch1 is altered in rv mutant embryos, and rv and Dll1(lacZ), a null allele of the Notch ligand Delta1, genetically interact. Mice double heterozygous for rv and Dll1(lacZ), show vertebral defects, and one copy of Dll1(lacZ) on the homozygous rv background enhances the mutant phenotype and is lethal in the majority of cases. However, fine genetic mapping places rv into an interval on chromosome seven that does not contain a gene encoding a known component of the Notch signaling pathway.     

Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning

In the vertebrate limb, the posteriorly located zone of polarizing activity (ZPA) regulates digit identity through the morphogen Sonic Hedgehog (Shh). By genetically marking Shh-responding cells in mice, we have addressed whether the cumulative influence of positive Shh signaling over time and space reflects a linear gradient of Shh responsiveness and whether Shh could play additional roles in limb patterning. Our results show that all posterior limb mesenchyme cells, as well as the ectoderm, respond to Shh from the ZPA and become the bone, muscle, and skin of the posterior limb. Further, the readout of Shh activator function integrated over time and space does not display a stable and linear gradient along the A-P axis, as in a classical morphogen view. Finally, by fate mapping Shh-responding cells in Gli2 and Gli3 mutant limbs, we demonstrate that a specific level of positive Hh signaling is not required to specify digit identities.     

A model generating the pattern of cartilage skeletal elements in the embryonic chick limb.

The development of the vertebrate limb involves the production of a specific external form arising from cell division and other growth processes at the cellular level, and the origin within it of specific patterns of tissues arising by cellular differentiation, of which the pattern of cartilages which pre-figure the limb skeleton is the most striking.In this paper we propose a model for the differentiation (or the preceding determination) process that, using only localized cell to cell interactions, can approximate the cartilage pattern in any limb shape. The model requires cells to modify their metabolism irreversibly at critical threshold levels of a diffusible morphogen which may be made or destroyed by these cells. Restrictions inherent in the successful development of a total limb pattern using this system lead to the prediction that the process is confined to a distal band which has no significant interaction with more proximal regions but within this band the characteristic features of the anterior-posterior axis of the limb develop without additional interactions. Cartilage elements are initiated as single “cells” and expand centrifugally to their final size; these elements developing sequentially along the anterior-posterior axis, showing a distinct polarity of size. The model also predicts that equivalent cartilage elements in all vertebrate limbs will be roughly the same relative size at determination, the extensive range of adult structures arising by differential growth and fusion, possibly controlled by global aspects of the model.It must be emphasized that this model only satisfactorily simulates the anterior-posterior patterning of cartilage elements, the disto-proximal pattern being externally imposed.The final cartilage pattern is shown to be a function of (1) the developing shape of the limb, (2) the position of an initiator region that starts the patterning process and (3) the rate of production of the diffusible morphogen. Using parameters selected with as much realism as possible the model gives a good approximation to the pattern of c cartilages found in the normal chick limb; modifying the shape of the limb to that of the talpid³ mutant produces the characteristics features of the cartilage pattern found in that mutant and modifying the rate of morphogen production simulates patterns resembling those found in some ancestral vertebrate fossil forms.     

A rat homolog of the mouse deafness mutant jerker (je)

An autosomal recessive deafness mutant was discovered in our colony of Zucker (ZUC) rats. These mutants behave like shaker-waltzer deafness mutants, and their inner ear pathology classifies them among neuroepithelial degeneration type of deafness mutants. To determine whether this rat deafness mutation (−) defines a unique locus or one that has been previously described, we mapped its chromosomal location. F₂ progeny of (Pbrc:ZUC × BN/Crl) A/a B/b H/h+/− F₁ rats were scored for coat color and behavioral phenotypes. Segregation analysis indicated that the deafness locus might be loosely linked with B on rat Chromosome (Chr) 5 (RNO5). Therefore, 40 −/− rats were scored for BN and ZUC alleles at four additional loci, D5Mit11, D5Mit13, Oprd1, and Gnb1, known to map to RNO5 or its homolog, mouse Chr 4 (MMU4). Linkage analysis established the gene order (cM distance) as D5Mit11–(19.3)–B–(17.9)–D5Mit13–(19.2)–Oprd1–(21.5) − (1.2) Gnb1, placing the deafness locus on distal RNO5. The position of the deafness locus on RNO5 is similar to that ofjerker (je) on MMU4; the phenotypes and patterns of inheritance of the deafness mutation and je are also similar. It seems likely that the mutation affects the rat homolog of je. The rat deafness locus should, therefore, be named jerker and assigned the gene symbol Je. Received: 13 June 1995 / Accepted: 4 January 1996     

Osteochondrodystrophy (ocd): a new autosomal recessive mutation in the mouse

Osteochondrodystrophy (ocd) is a new autosomal recessive mouse mutation characterized by a short, slightly domed head, reduced body size, disproportionately shortened long bones of the legs, supination of the forefeet, and short thickened tail. Histologically, the epiphyses are thinner than normal. The columnar organization of the proliferative zone of cartilage is disorderly, with pleomorphic and occasionally necrotic chondrocytes. Osteochondrodystrophy has been mapped to a position near the centromere of mouse chromosome (Chr) 19.     

Doubleridge,a mouse mutant with defective compaction of the apical ectodermal ridge and normal dorsal-ventral patterning of the limb

doubleridge is a transgene-induced mutation characterized by polydactyly and syndactyly of the forelimbs. The transgene insertion maps to the proximal region of chromosome 19. During embryonic development of the mutant forelimb, delayed elevation and compaction of the apical ectodermal ridge (AER) produces a ridge that is abnormally broad and flat. Fgf8 expression persists in the ventral forelimb ectoderm of the mutant until E10.5. Strong expression of Fgf8 and other markers at the borders of the AER at E11.5 gives the appearance of a double ridge. At E11.5, apoptotic cells are distributed across the broadened ridge, but at E13.5, there is reduced apoptosis in the interdigital regions. The Shh expression domain is widely spaced at the posterior margin of the AER. The doubleridge AER is morphologically similar to that of En1 null mice, but the expression of En1 and Wnt7a is properly restricted in doubleridge, and the dorsal and ventral structures are correctly determined. doubleridge thus exhibits an unusual limb phenotype combining abnormal compaction of the AER with normal dorsal/ventral patterning.     

Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK)

We have previously shown that the Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, we created mice deficient in NIK by homologous recombination at the Nik gene. Nik(-/-) mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik(-/-) embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik(-/- )embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrated that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant function in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice.     

The recombinant limb as a model for the study of limb patterning, and its application to muscle development

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning. Received: 31 August 1998 / Accepted: 29 September 1998     

The role of gap junctions in patterning of the chick limb bud

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.