Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice

Abstract

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.

Figure 1. Behavioral abnormalities of heterozygous adult KI Alk^F1178L males

A. Monitoring of locomotor activity and rears during dark and light phases. Each dot represents the mean of animals on one hour periods. The difference between genotypes was significant during the first dark phase for locomotor activity (p<0.05) and the number of rears was also significantly decreased in HT males during the dark phases (p<0.05). B. Results of the light/dark test indicate an increased anxiety of HT mice. C. Concentration of alkaline phosphatase and chloride in HT and WT mice. Error bars correspond to SEM, p-value for t-test *: <0.05, **: <0.01, ***: <0.001.

Figure 2. High neonatal lethality of homozygous KI Alk^F1178L mice and growth delay of surviving homozygotes

A. Breedings between HT were performed to obtain HM. Results of genotyping at birth (n=502) and at weaning (n=158) are shown. At birth: WT: n=131, HT: n=248 and HM: n=123. At weaning: WT: n=55, HT: n=97 and HM: n=6. p-value for the chi-square test: 7.10⁻⁸ B. Representative pictures of litters at different ages. At birth, the 3 genotypes are indistinguishable (WT, n=2; HT, n=2; HM, n=3). At P3, the subset of HM that survive may be identified due to their reduced size and delay in skin pigmentation. At P25, the surviving HM still present with a reduced size. C. Survival curves indicating the time of death for HM. D. Mean weight of each genotype from birth to weaning. Error bars correspond to SEM. E. X-ray of back paw of one WT and one HM young adults. Epiphyseal plates are indicated by arrows and present in both animals. White bar: 1 cm.

Figure 3. Absence of abnormalities of the brainstem nuclei expressing Alk in HM KI Alk^F1178L mice

A. In situ hybridization on coronal sections (rostral to caudal) of brainstem of WT and HM embryos at E18.5 with an Alk probe. Black scale bar: 0.5 mm. B. Magnification images of the cranial nerve motor nuclei. IO: Inferior Olivary nucleus; XIIn: hypoglossal motor nucleus; VIIn: facial motor nucleus; Vn: trigeminal motor nucleus.

Figure 4. Analysis of basic physiological functions at birth reveals a strong decreased milk intake for homozygous KI Alk^F1178L mice

A. Apneas were recorded by whole-body plethysmography on 94 newborn mice (WT, n=27; HT, n=41; HM, n=26) under normoxia, hypercapnia and hypoxia. Means of apnea duration for all genotypes are shown in the different conditions. Each dot represents a period of 30 seconds. Error bars correspond to SEM. B. Normalized apneas duration during normoxia are shown. Each apneas duration for the 10 min of normoxia was normalized by the mean of the duration in the litter. p-value for t-test between WT and HM = 0.00055. C. Representative pictures of milk intake in the stomach of newborn mice scored as traces (left panel), full (middle panel) or distended (right panel). D. Repartition of different milk intake scores for each genotype (WT, n= 62; HT, n=103; HM, n=46), p-values for the chi-square tests ***: <0.001.