<i>aldh7a1</i> Regulates Eye and Limb Development in Zebrafish

<div><p>Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of <i>aldh7a1</i> as a gene necessary for normal eye development. We show that morpholino knockdown of <i>aldh7a1</i> in zebrafish causes uveal coloboma and misregulation of <i>nlz1</i>, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of <i>aldh7a1</i> leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The <i>aldh7a1</i> morphant phenotype is partially rescued by co-injection of <i>nlz1</i> mRNA suggesting that <i>nlz1</i> is functionally downstream of <i>aldh7a1</i> in regulating cell proliferation in the optic cup. These results support a role of <i>aldh7a1</i> in ocular development and skeletal abnormalities in zebrafish.</p></div

Expression pattern of <i>aldh7a1</i> in zebrafish.

<p>Whole-mount <i>in situ</i> hybridization of <i>aldh7a1</i> at (A) 24 hpf and (B) 48 hpf. L, lens; OF, optic fissure; PF, pectoral fin. Scale bar: 65 µm in A; 60 µm in B.</p

Expression pattern of genetic eye development markers in control and <i>aldh7a1</i> morphant embryos.

<p>(A) Expression of <i>nlz1</i> in optic fissure is down-regulated in (B) <i>aldh7a1</i> morphant fish. <i>vax2</i> and <i>pax2.1</i> do not seem to show significant change in expression between control MO (C,E) and <i>nlz1</i> morphant (D, F) fish. (G) Co-injection of <i>nlz1</i> mRNA resulted in partial rescue of <i>aldh7a1</i> MO phenotype, examined at 28 hpf, while co-injection of <i>vax2</i> mRNA showed no change. Scale bar: 65 µm.</p

Aldh7a1 is required for retinal cell proliferation.

<p>(A–B) Dividing cells in developing eye were labeled with phosophohistone-3 antibody (H3P) in (A) Control MO and (B) <i>alh7a1</i>morphant embryos at 28 hpf. (A) and (B) are projection images of z-stacks through the depth of the eye. (C) Average number of dividing cells per eye quantified for control MO (n = 6), Nlz1 MO (n = 7), <i>nlz1</i> mRNA (n = 6) rescued Statistical significance indicated above columns *P<0.05, **P<0.01, ***P<0.0001. Scale bar: 65 µm.</p

Aldh7a1 is important for optic fissure closure.

<p>(A–D) Injection of Aldh7a1 morpholino results in failure of optic fissure to close at 28 hpf and sustained at 5 dpf. (A) Eye of control morpholino (MO) injected embryo at 28 hpf, (B) Eye of <i>aldh7a1</i> morphant at 28 hpf; (C) Ventral view of eye in control MO embryo at 5 dpf, (D) Ventral view of eye in embryos injected with 7.5 ng Aldh7a1 MO at 5 dpf, black bars indicate edges of optic fissures; (E) Bar graph demonstrate distribution of eye phenotypes 0, I, II, and III at 28 hpf following 7.5 ng Aldh7a1 MO injection followed by partial rescue of phenotype upon co-injection of two doses of <i>aldh7a1</i> mRNA. All control MO injected embryos displayed “0” phenotype. (F) zn-5 staining of control MO injected embryos at 48 hpf compared to (G) <i>aldh7a1</i> morphant which displays optic nerve hypoplasia, arrow-heads indicate optic nerve. Scale bar: 65 µm in A,B; 125 µm in C,D; 75 µm in F,G.</p

Impaired leukocyte trafficking and skin inflammatory responses in hamsters lacking a functional circadian system

The immune system is under strong circadian control, and circadian desynchrony is a risk factor for metabolic disorders, inflammatory responses and cancer. Signaling pathways that maintain circadian rhythms (CRs) in immune function in vivo, and the mechanisms by which circadian desynchrony impairs immune function, remain to be fully-identified. These experiments tested the hypothesis that the hypothalamic circadian pacemaker in the suprachiasmatic nucleus (SCN) drives CRs in the immune system, using a non-invasive model of SCN circadian arrhythmia. Robust CRs in blood leukocyte trafficking, with a peak during the early light phase (ZT4) and nadir in the early dark phase (ZT18), were absent in arrhythmic hamsters, as were CRs in spleen clock gene (per1, bmal1) expression, indicating that a functional pacemaker in the SCN is required for the generation of CRs in leukocyte trafficking and for driving peripheral clocks in secondary lymphoid organs. Pinealectomy was without effect on CRs in leukocyte trafficking, but abolished CRs in spleen clock gene expression, indicating that nocturnal melatonin secretion is necessary for communicating circadian time information to the spleen. CRs in trafficking of antigen presenting cells (CD11c(+) dendritic cells) in the skin were abolished, and antigen-specific delayed-type hypersensitivity skin inflammatory responses were markedly impaired in arrhythmic hamsters. The SCN drives robust CRs in leukocyte trafficking and lymphoid clock gene expression; the latter of which is not expressed in the absence of melatonin. Robust entrainment of the circadian pacemaker provides a signal critical to diurnal rhythms in immunosurveilliance and optimal memory T-cell dependent immune responses