Kylie N. Cane

Fur seal adaptations to lactation: insights into mammary gland function

The fur seal (Arctocephalus spp. and Callorhinus spp., members of the pinniped family) is a mammal with the unusual capability to modulate its lactation cycle by turning milk production on and off without the typical mammalian regression and involution of the mammary gland. Lactation has evolved from constraints arising from the spatial and temporal separation of infant nursing and maternal foraging as the mother gives birth and feeds the pup on land while acquisition of nutrients for milk production occurs at sea. The lactation cycle begins with the female fur seal undergoing a perinatal fast of approximately 1 wk, after which time she departs the breeding colony to forage at sea. For the remainder of the long lactation period (116-540 days), the mother alternates between short periods ashore suckling the young with longer periods of up to 4 wk of foraging at sea. Milk production continues while foraging at sea, but at less than 20% the rate of production on land. Fur seals produce one of the richest milk reported, with a very high lipid content contributing up to 85% of total energy. This feature serves as an adaptation to the youngs need to produce an insulating blubber layer against heat loss and to serve as an energy store when the mother is away foraging at sea. This atypical pattern of lactation means mothers have long periods with no suckling stimulus and can transfer high-energy milk rapidly while on land to minimize time away from foraging grounds. The absence of suckling stimulus and milk removal during foraging does not result in the onset of involution with associated apoptosis of mammary secretory cells and a subsequent progressive breakdown of the cellular structure of the mammary gland. The mechanisms controlling lactation in the fur seal mammary gland have been investigated using molecular and cellular techniques. These findings have shed light on the processes by which the unique features of lactation in the fur seal are regulated.

Development of the autonomic nervous system: A comparative view

In this review we summarize current understanding of the development of autonomic neurons in vertebrates. The mechanisms controlling the development of sympathetic and enteric neurons have been studied in considerable detail in laboratory mammals, chick and zebrafish, and there are also limited data about the development of sympathetic and enteric neurons in amphibians. Little is known about the development of parasympathetic neurons apart from the ciliary ganglion in chicks. Although there are considerable gaps in our knowledge, some of the mechanisms controlling sympathetic and enteric neuron development appear to be conserved between mammals, avians and zebrafish. For example, some of the transcriptional regulators involved in the development of sympathetic neurons are conserved between mammals, avians and zebrafish, and the requirement for Ret signalling in the development of enteric neurons is conserved between mammals (including humans), avians and zebrafish. However, there are also differences between species in the migratory pathways followed by sympathetic and enteric neuron precursors and in the requirements for some signalling pathways.

Generating diversity: Mechanisms regulating the differentiation of autonomic neuron phenotypes

Sympathetic and parasympathetic postganglionic neurons innervate a wide range of target tissues. The subpopulation of neurons innervating each target tissue can express unique combinations of neurotransmitters, neuropeptides, ion channels and receptors, which together comprise the chemical phenotype of the neurons. The target-specific chemical phenotype shown by autonomic postganglionic neurons arises during development. In this review, we examine the different mechanisms that generate such a diversity of neuronal phenotypes from the pool of apparently homogenous neural crest progenitor cells that form the sympathetic ganglia. There is evidence that the final chemical phenotype of autonomic postganglionic neurons is generated by both signals at the level of the cell body that trigger cell-autonomous programs, as well as signals from the target tissues they innervate.

Proliferation and Cell Cycle Dynamics in the Developing Stellate Ganglion

Cell proliferation during nervous system development is poorly understood outside the mouse neocortex. We measured cell cycle dynamics in the embryonic mouse sympathetic stellate ganglion, where neuroblasts continue to proliferate following neuronal differentiation. At embryonic day (E) 9.5, when neural crest-derived cells were migrating and coalescing into the ganglion primordium, all cells were cycling, cell cycle length was only 10.6 h, and S-phase comprised over 65% of the cell cycle; these values are similar to those previously reported for embryonic stem cells. At E10.5, Sox10+ cells lengthened their cell cycle to 38 h and reduced the length of S-phase. As cells started to express the neuronal markers Tuj1 and tyrosine hydroxylase (TH) at E10.5, they exited the cell cycle. At E11.5, when >80% of cells in the ganglion were Tuj1+/TH+ neuroblasts, all cells were again cycling. Neuroblast cell cycle length did not change significantly after E11.5, and 98% of Sox10−/TH+ cells had exited the cell cycle by E18.5. The cell cycle length of Sox10+/TH− cells increased during late embryonic development, and ∼25% were still cycling at E18.5. Loss of Ret increased neuroblast cell cycle length at E16.5 and decreased the number of neuroblasts at E18.5. A mathematical model generated from our data successfully predicted the relative change in proportions of neuroblasts and non-neuroblasts in wild-type mice. Our results show that, like other neurons, sympathetic neuron differentiation is associated with exit from the cell cycle; sympathetic neurons are unusual in that they then re-enter the cell cycle before later permanently exiting.

The lactation cycle of the fur seal

The fur seal is a mammal with an unusual ability to turn its milk production on and off without significantly altering the gross morphology of the mammary gland. This atypical lactation cycle is due to the fact that maternal foraging and infant nursing are spatially and temporally separate (Bonner, 1984). Maternal care involves the suckling of offspring over a period of at least 4 months, but lactation can extend to more than 12 months. Following a perinatal fast of approximately 1 week, females depart the breeding colony to forage at sea and, for the remainder of lactation, alternate between short periods ashore suckling their young with longer periods of up to 4 weeks foraging at sea. Whilst foraging at sea, milk production in the fur seal mammary gland either ceases or is reduced (Arnould & Boyd, 1995b).

Different neural crest populations exhibit diverse proliferative behaviors.

The rate of proliferation of cells depends on the proportion of cycling cells and the frequency of cell division. Here, we describe in detail methods for quantifying the proliferative behavior of specific cell types in situ, and use the method to examine cell cycle dynamics in two neural crest derivatives—dorsal root ganglia (DRG) using frozen sections, and the enteric nervous system (ENS) using wholemount preparations. In DRG, our data reveal a significant increase in cell cycle length and a decrease in the number of cycling Sox10+ progenitor cells at E12.5–E13.5, which coincides with the commencement of glial cell generation. In the ENS, the vast majority of Sox10+ cells remain proliferative during embryonic development, with only relatively minor changes in cell cycle parameters. Previous studies have identified proliferating cells expressing neuronal markers in the developing ENS; our data suggest that most cells undergoing neuronal differentiation in the developing gut commence expression of neuronal markers during G2 phase of their last division. Combined with previous studies, our findings show that different populations of neural crest‐derived cells show tissue‐specific patterns of proliferation.

Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells

Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine‐regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial

The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP.