Lack of discreet colocalization of epithelial apoptosis to the atretic precursor in the colon of the Fibroblast growth factor receptor 2IIIb mouse and staining consistent with cellular movement suggest a revised model of atresia formation.

BACKGROUND: Colonic atresias in the Fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) mouse model have been attributed to increased epithelial apoptosis and decreased epithelial proliferation at embryonic day (E) 10.5. We therefore hypothesized that these processes would colocalize to the distal colon where atresias occur (atretic precursor) and would be excluded or minimized from the proximal colon and small intestine. RESULTS: We observed a global increase in intestinal epithelial apoptosis in Fgfr2IIIb -/- intestines from E9.5 to E10.5 that did not colocalize to the atretic precursor. Additionally, epithelial proliferations rates in Fgfr2IIIb -/- intestines were statistically indistinguishable to that of controls at E10.5 and E11.5. At E11.5 distal colonic epithelial cells in mutants failed to assume the expected pseudostratified columnar architecture and the continuity of the adjacent basal lamina was disrupted. Individual E-cadherin-positive cells were observed in the colonic mesenchyme. CONCLUSIONS: Our observations suggest that alterations in proliferation and apoptosis alone are insufficient to account for intestinal atresias and that these defects may arise from both a failure of distal colonic epithelial cells to develop normally and local disruptions in basal lamina architecture.

Utility and limits of Hprt-Cre technology in generating mutant mouse embryos.

BACKGROUND: Hprt-Cre doubles the prevalence of homozygous null embryos per litter versus heterozygous breedings without decreasing litter size. Resulting mutant embryos are genotypically and phenotypically equivalent between strategies. We set out to confirm the effectiveness of this approach with other alleles and hypothesized that it would increase efficiency in generating compound mutants. MATERIALS AND METHODS: Null mutants for Cyp26b1, Pitx2, and Shh were generated with Hprt-Cre from conditional alleles as were double and triple allelic combinations of Fgfr2IIIb, Raldh2, and Cyp26b1. Embryos were genotyped and phenotyped by whole mount photography, histology, and immunohistochemistry. RESULTS: Fifty percent of Hprt-Cre litters were homozygous null for Cyp26b1 (15/29) and Pitx2 (75/143), with phenotypic and genotypic equivalence to mutants from standard heterozygous breedings. In multi-allele breedings, mutant embryos constituted half of litters without significant embryo loss. In contrast, Shh breedings yielded a smaller ratio of embryos carrying two recombined alleles (6 of 16), with a significant litter size reduction because of early embryonic lethality (16 live embryos from 38 deciduae). CONCLUSIONS: Hprt-Cre can be used to efficiently generate large numbers of mutant embryos with a number of alleles. Compound mutant generation was equally efficient. However, efficiency is reduced for genes whose protein product potentially interacts with the Hprt pathway (e.g., Shh).

Exogenous Sonic hedgehog protein does not rescue cultured intestine from atresia formation.

BACKGROUND: The mechanism of intestinal atresia formation remains undefined. Atresia in fibroblast growth factor receptor 2IIIb (Fgfr2IIIb(-/-)) mutant mouse embryos is preceded by endodermal apoptosis and involution of the surrounding mesoderm. We have observed that involution of the atretic segment is preceded by the downregulation of Sonic hedgehog (SHH) in the endoderm, which is a critical organizer of the intestinal mesoderm. We hypothesized that supplementation of Fgfr2IIIb(-/-) intestinal tracts with exogenous SHH protein before atresia formation would prevent involution of the mesoderm and rescue normal intestinal development. METHODS: In situ hybridization was performed on control and Fgfr2IIIb(-/-) intestinal tracts for Shh or forkhead box protein F1 (FoxF1) between embryonic (E) day 11.5 and E12.0. Control and Fgfr2IIIb(-/-) intestinal tracts were harvested at E10.5 and cultured in media supplemented with fibroblast growth factor (FGF) 10 + SHH, or FGF10 with a SHH-coated bead. In situ hybridization was performed at E12.5 for Foxf1. RESULTS: SHH and Foxf1 expression were downregulated during intestinal atresia formation. Media containing exogenous FGF10 + SHH did not prevent colonic atresia formation (involution). A SHH protein point source bead did induce Foxf1 expression in controls and mutants. CONCLUSIONS: Shh and Foxf1 expression are disrupted in atresia formation of distal colon, thereby serving as potential markers of atretic events. Application of exogenous SHH (in media supplement or as a point source bead) is sufficient to induce Foxf1 expression, but insufficient to rescue development of distal colonic mesoderm in Fgfr2IIIb(-/-) mutant embryos. Shh signal disruption is not the critical mechanism by which loss of Fgfr2IIIb function results in atresia formation.

Unique combination of anatomy and physiology in cells of the rat paralaminar thalamic nuclei adjacent to the medial geniculate body.

The medial geniculate body (MGB) has three major subdivisions, ventral (MGV), dorsal (MGD), and medial (MGM). MGM is linked with paralaminar nuclei that are situated medial and ventral to MGV/MGD. Paralaminar nuclei have unique inputs and outputs compared with MGV and MGD and have been linked to circuitry underlying some important functional roles. We recorded intracellularly from cells in the paralaminar nuclei in vitro. We found that they possess an unusual combination of anatomical and physiological features compared with those reported for "standard" thalamic neurons seen in the MGV/MGD and elsewhere in the thalamus. Compared with MGV/MGD neurons, anatomically, 1) paralaminar cell dendrites can be long, branch sparingly, and encompass a much larger area; 2) their dendrites may be smooth but can have well defined spines; and 3) their axons can have collaterals that branch locally within the same or nearby paralaminar nuclei. When compared with MGV/MGD neurons, physiologically, 1) their spikes are larger in amplitude and can be shorter in duration; 2) their spikes can have dual afterhyperpolarizations with fast and slow components; and 3) they can have a reduction or complete absence of the low-threshold, voltage-sensitive calcium conductance that reduces or eliminates the voltage-dependent burst response. We also recorded from cells in the parafascicular nucleus, a nucleus of the posterior intralaminar nuclear group, because they have unusual anatomical features that are similar to those of some of our paralaminar cells. As with the labeled paralaminar cells, parafascicular cells had physiological features distinguishing them from typical thalamic neurons.

Cortical and collicular inputs to cells in the rat paralaminar thalamic nuclei adjacent to the medial geniculate body.

The paralaminar nuclei, including the medial division of the medial geniculate nucleus, surround the auditory thalamus medially and ventrally. This multimodal area receives convergent inputs from auditory, visual, and somatosensory structures and sends divergent outputs to cortical layer 1, amygdala, basal ganglia, and elsewhere. Studies implicate this region in the modulation of cortical 40-Hz oscillations, cortical information binding, and the conditioned fear response. We recently showed that the basic anatomy and intrinsic physiology of paralaminar cells are unlike that of neurons elsewhere in sensory thalamus. Here we evaluate the synaptic inputs to paralaminar cells from the inferior and superior colliculi and the cortex. Combined physiological and anatomical evidence indicates that paralaminar cells receive both excitatory and inhibitory inputs from both colliculi and excitatory cortical inputs. Excitatory inputs from all three sources typically generate small summating EPSPs composed of AMPA and NMDA components and terminate primarily on smaller dendrites and occasionally on dendritic spines. The cortical input shows strong paired-pulse facilitation (PPF), whereas both collicular inputs show weak PPF or paired-pulse depression (PPD). EPSPs of cells with no low-threshold calcium conductance do not evoke a burst response when the cell is hyperpolarized. Longer-latency EPSPs were seen and our evidence indicates that these arise from axon collateral inputs of other synaptically activated paralaminar cells. The inhibitory collicular inputs are GABAergic, activate GABA(A) receptors, and terminate on dendrites. Their activation can greatly alter EPSP-generated spike number and timing.