Claire M. Schreiner

Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures.

In most cells, the ubiquitously expressed Na+/H+exchanger isoform 1 (NHE1) is thought to be a primary regulator of pH homeostasis, cell volume regulation, and the proliferative response to growth factor stimulation. To study the function of NHE1 during embryogenesis when these cellular processes are very active, we targeted the Nhe1 gene by replacing the sequence encoding transmembrane domains 6 and 7 with the neomycin resistance gene. NHE activity assays on isolated acinar cells indicated that the targeted allele is functionally null. Although the absence of NHE1 is compatible with embryogenesis, Nhe1 homozygous mutants (-/-) exhibit a decreased rate of postnatal growth that is first evident at 2 wk of age. At this time, Nhe1 -/- animals also begin to exhibit ataxia and epileptic-like seizures. Approximately 67% of the -/- mutants die before weaning. Postmortem examinations frequently revealed an accumulation of a waxy particulate material inside the ears, around the eyes and chin, and on the ventral surface of the paws. Histological analysis of adult tissues revealed a thickening of the lamina propria and a slightly atrophic glandular mucosa in the stomach.

R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis.

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2Tg). The predicted protein encoded by Rspo2Tg neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2Tg/Tg mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2Tg/Tg lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2Tg and Lrp6- alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.

Sp8 is crucial for limb outgrowth and neuropore closure

In this report we describe the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation.

Endogenous electric current is associated with normal development of the vertebrate limb

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5–2 μA/cm2) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self‐referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed — the direction of Na+ uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04–10.8 μA/cm2. Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 μM amiloride, a Na+ channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large (∼100 μA/cm2) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied “countercurrent” pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5–3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.

Ectopic expression of Hox-2.3 induces craniofacial and skeletal malformations in transgenic mice

To better understand the role of the Hox-2.3 murine homeobox gene during development, a dominant gain-of-function mutation was generated. The developmental malformations that resulted when the chicken beta-actin promoter was used to direct widespread expression of the Hox-2.3 gene in transgenic mice included early postnatal death as well as craniofacial abnormalities, including open eyes and cleft palate. Ventricular septal defects were also observed in the hearts of three transgenic mice. Skeletal malformations were seen in the bones of the craniocervical transition, with the occipital, basisphenoid, and atlas bones deficient or misshapen. Interestingly, one mutant exhibited an extra pair of ribs as well as alterations in cervical vertebrae identities. Some of the malformations observed in Hox-2.3 gain-of-function mutants overlap with those seen in Hox-1.1 and Hox-2.2 misexpression mutants which suggests functional similarities between paralogous homeobox genes. The results of these experiments are consistent with a role for Hox-2.3 in specifying positional information during development.

The loss of ventral ectoderm identity correlates with the inability to form an AER in the legless hindlimb bud.

We have characterized the early stages of murine hindlimb morphogenesis in the legless (lgl)mutant and non-mutant littermates. Initially the entire ventral ectoderm expresses many genetic markers characteristic of the AER (en-1, fgf-8, msx-2, dlx-2, cd44, and cx-43). Subsequently, the expression domain of most of these genes is restricted to the thickened ectoderm of the disto-ventral limb margin prior to forming an AER. In lgl, the expression of these genes is initiated but not maintained and the disto-ventral marginal ectoderm does not thicken. In contrast, Wnt7a expression is initiated and maintained in the dorsal ectoderm. The limb mesenchyme of lgl and non-mutant embryos initially expresses lmx-1b and fgf-10 uniformly. As the ventro-distal marginal ectoderm thickens, lmx-1b is progressively dorsally restricted in non-mutants but continues to be expressed ventrally in lgl hindlimb buds. These data suggest that establishment of a dorso-ventral ectodermal interface is not sufficient for AER formation and that restriction of lmx-1b to the dorsal mesenchyme is coordinately linked to AER formation.

Disrupting the establishment of polarizing activity by teratogen exposure

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.

Valproate-induced limb malformations in mice associated with reduction of intracellular pH.

Valproic acid (VPA) is a commonly used antiepileptic agent that recently has been found useful in the treatment of affective disorders and prophylaxis of migraine. VPA induces congenital malformations, especially spina bifida, in the offspring of women treated with this agent during early pregnancy. The mechanism by which VPA induces abnormal development remains unknown despite many studies in experimental animals in which VPA causes malformations similar to those seen in human infants. Because of its chemical structure as a weak organic acid and its capability to induce postaxial forelimb ectrodactyly in C57BL/6 mice, we postulated that VPA acts to perturb limb morphogenesis by reducing embryonic intracellular pH (pHi). We administered VPA, 200 to 400 mg/kg, to C57BL/6 mice on day 9 of gestation. A dose-dependent incidence of postaxial forelimb ectrodactyly was observed. Forelimb bud pHi was estimated by computer-assisted image analysis from the transplacental distribution of 14C-DMO. At the highest doses, 300 and 400 mg/kg, a decrease of pHi of 0.2 to 0.3 pH units was observed uniformly throughout the limb bud 1 h after VPA treatment. None of these changes were seen after treatment with 2-en VPA, a nonteratogenic analog of VPA. Furthermore, the capability of VPA to induce postaxial forelimb ectrodactyly was greatly enhanced by coadministration of agents that inhibit pHi regulatory processes. These data support the hypothesis that VPA-induced postaxial ectrodactyly in murine fetuses can be attributed to reduction in limb bud pHi.

Carbonic anhydrase distribution in rodent embryos and its relationship to acetazolamide teratogenesis.

The carbonic anhydrase inhibitor, acetazolamide, leads to a unique distal postaxial right forelimb deformity in rats and CBA/J mice, but SWV mice are completely resistant. Using Hanssons histochemical method, the distribution of carbonic anhydrase and its inhibition by acetazolamide in rat, CBA/J mouse, and SWV mouse embryos were compared. Carbonic anhydrase activity was demonstrable in many tissues of sensitive rat and CBA/J mouse embryos and in resistant SWV mouse embryos. The forelimb buds of resistant and sensitive embryos possess carbonic anhydrase activity in the area between the ectoderm and adjacent mesenchyma with no localization of enzyme activity corresponding to the malformation seen in acetazolamide teratogenesis. This suggests that carbonic anhydrase in the forelimbs is not the primary site of action for acetazolamide. A distinctive staining pattern of nucleated erythrocytes in resistant embryos indicated the presence of a low activity form of carbonic anhydrase in nearly half of the erythrocytes. A five-to tenfold greater amount of acetazolamide was needed to completely inhibit carbonic anhydrase activity in nucleated erythrocytes from resistant embryos than in those from sensitive embryos. The existence of a low activity form of carbonic anhydrase in SWV embryo erythrocytes may be the basis of resistance to acetazolamide teratogenesis.